Potassium channels in intestinal epithelial cells and their pharmacological modulation: a systematic review

Abstract

Several potassium channels (KCs) have been described throughout the gastrointestinal tract. Notwithstanding, their contribution to both physiologic and pathophysiologic conditions, as inflammatory bowel disease (IBD), remains underexplored. Therefore, we aim to systematically review, for the first time, the evidence on the characteristics and modulation of KCs in intestinal epithelial cells (IECs). PubMed, Scopus, and Web of Science were searched to identify studies focusing on KCs and their modulation in IECs. The included studies were assessed using a reporting inclusiveness checklist. From the 745 identified records, 73 met the inclusion criteria; their reporting inclusiveness was moderate-high. Some studies described the physiological role of KCs, while others explored their importance in pathological settings. Globally, in IBD animal models, apical K_Ca1.1 channels, responsible for luminal secretion, were upregulated. In human colonocytes, basolateral K_Ca3.1 channels were downregulated. The pharmacological inhibition of K_2P and K_v influenced intestinal barrier function, promoting inflammation. Evidence suggests a strong association between KCs expression and secretory mechanisms in human and animal IECs. Further research is warranted to explore the usefulness of KC pharmacological modulation as a therapeutic target.

INTRODUCTION

Potassium channels (KCs) are ubiquitous in cells and play a significant role in several physiological functions such as: resting membrane potential, action potential, and neurotransmitter release. These channels are responsible for K⁺ ion efflux in and out of excitable and nonexcitable cells. All KCs are formed by a pore-forming domain, responsible for K⁺ ions transport, and by a regulatory domain that senses diverse stimuli (1). KCs are constituted by 2, 4, or 6 transmembrane domains (TMs) (1–4). Based on gene family, structure and functional characteristics, KCs are grouped into four families: 1) calcium- and sodium-activated potassium channels (K_Ca/Na) (6/7 TMs); 2) inwardly rectifying potassium channels (K_ir) (2 TMs); 3) two-pore-domain potassium channels (K_2P) (4 TMs); and 4) voltage-gated potassium channels (K_v) (6 TMs) (1–7). A standardized nomenclature for KCs has been proposed by the Nomenclature Committee of the Union of Basic and Clinical Pharmacology (NC-IUPHAR) (5, 8); the nomenclature and characteristics of the KCs found in the gastrointestinal tract are summarized in Table 1.

Table 1.

Nomenclature and characteristics of the potassium channels in gastrointestinal tract

Name (IUPHAR)	Gene Name (HGNC)	Alternative Name	Associated Subunits	Description	Tissue/Cells	Channel Modulators
						Activators	Inhibitors	Blockers
Calcium-activated K+ channels
KCa1.1	KCNMA1	BK channel, maxi-K+ channel, Slo, Slo1	KCNMB1–4, BK-β	Large conductance, voltage gated, calcium activated	Colonic epithelium, gastric smooth muscle	NS004, NS1619, 17β-estradiol	Paxilline (mouse)	ChTX, IbTX, TEA, penitrem A, quinidine
KCa2.1	KCNN1	SK1, SKCa1	Calmodulin tightly complexed to COOH terminus	Small conductance, calcium activated	Gastric tumor, IEC-6	EBIO, NS309	Apamin, UCL1684	TEA
KCa2.2	KCNN2	SK2, SKCa2	Calmodulin tightly complexed to COOH terminus, protein kinase CK2, and protein phosphatase 2A	Small conductance, calcium activated	Colonic mucosa	EBIO, NS309	Apamin, UCL1684	TEA
KCa2.3	KCNN3	SK3, SKCa3, hKCa3	Calmodulin tightly complexed to COOH terminus	Small conductance, calcium activated	Colon	EBIO, NS309	Apamin, UCL1684	TEA
KCa3.1	KCNN4	SK41, IK1, Gardos channel, KCa4, IKCa1	Calmodulin tightly complexed to COOH terminus	Intermediate conductance, calcium activated	Colonic crypt cells, Paneth cells	EBIO, NS309, SKA-121	None	ChTX, TRAM-34, senicapoc, clotrimazole
Inwardly rectifying K+ channels
Kir6.1	KCNJ8	uKATP-1	SUR1, SUR2A, SUR2B	Inward-rectifier current, ATP dependent	Small and large intestinal epithelium	Nicorandil and diazoxide, pinacidil (mouse), cromokalim, minoxidil	Glibenclamide, tolbutamide	None
Kir6.2	KCNJ11	BIR	SUR1, SUR2A, SUR2B	Inward-rectifier current, ATP dependent	Small intestine	Cromokalim and diazoxide (mouse) nicorandil, minoxidil, pinacidil	Glibenclamide, tolbutamide	None
Kir7.1	KCNJ13	Kir1.4, IRK13	None reported	Inward-rectifier current	Small intestine	None	None	Ba2+, Cs+, VU590, ML418
Two-pore domain K+ channels
K2P2.1	KCNK2	TREK-1, TPKC1	Not established	Open rectifier or voltage dependent	Intestine	Arachidonic acid, chloroform, halothane, isoflurane, GI‐530159, BL-1249	Norfluoxetine	None
K2P3.1	KCNK3	TASK-1, TBAK-1, OAT-1	p11 (annexin II subunit)	Open rectifier	Small intestine, colon	Halothane	None	Anandamide, R-(+)-methanandamie, arachidonic acid, Ba2+
K2P4.1	KCNK4	TRAAK	Not established	Open rectifier	Small intestine	Arachidonic acid, riluzole	None	Ba2+
K2P5.1	KCNK5	TASK-2	Not established	Open rectifier	Small and large intestinal epithelium	Halothane	None	Quinidine, clofilium (mouse)
K2P9.1	KCNK9	TASK-3	Not established	Open rectifier	Colonic epithelium	Halothane	Anandamide, R-(+)-methanandamide	None
K2P12.1	KCNK12	THIK-2	Not established	Not established	Small intestine, colon	None	None	None
Voltage-gated K+ channels
Kv1.1	KCNA1	HuK (I), MBK1, MK1, RCK1, RBK1, HBK1	KVβ1, KVβ2, PSD95, SAP97, SNAP25	Voltage gated, delayed rectifier	IEC-6	None	None	4-AP (rat), TEA (mouse), margatoxin
Kv1.2	KCNA2	HuK (IV), MK2, BK2, RCK5, RAK, BGK5, XSha2, NGK1, HBK5	Kv1.1, Kv1.4, Kvβ1, Kvβ2	Voltage gated, delayed rectifier	IEC-6	None	None	Noxiustoxin, margatoxin, α-dendrotoxin
Kv1.3	KCNA3	MK3, MBK3, RCK3, hPCN3, HuK (III), HLK3, RGK5, KV3, HGK5, n-channel	KVβ, hDlg, β1-integrin, KchaP	Voltage gated, delayed rectifier	Small intestine, IEC-6	None	None	Noxiustoxin and TEA (mouse), maurotoxin, margatoxin, correolide
Kv1.4	KCNA4	HuK (II), hPCN2, HK1, RCK4, RHK1, RK4, RK8, MK4	KVβ, PSD95, SAP97, SAP90, α-actinin-2, KChaP, ơ-receptor	Voltage gated, A-type, fast inactivating	Small intestine	None	None	Fampridine (4-AP)
Kv1.5	KCNA5	HCK1, HK2, HpCN1, KV1, RK3, RMK2, fHK, HuK (II)	KVβ1, KVβ2, KCNA3B	Voltage gated, delayed rectifier	Colon	None	None	Fampridine (4-AP)
Kv1.6	KCNA6	HBK2, MK1.6, RCK2, KV2	Kvβ1, Kvβ2, Caspr2	Voltage gated, delayed rectifier	Colon	None	None	TEA, α-dendrotoxin, ChTX
Kv2.1	KCNB1	hDRK1, DRK1, Shab	Kv5.1, Kv6.1–6.4, Kv8.1–8.2, and Kv9.1-9.3	Voltage gated, delayed rectifier	Small intestine, colon	Linoleic acid	None	TEA (rat)
Kv2.2	KCNB2	CDRK	Kv5.1, Kv6.1–6.4, Kv8.1–8.2, and Kv9.1-9.3	Voltage gated, delayed rectifier	Colonic mucosa, gastrointestinal smooth muscle	None	None	Fampridine (4-AP), TEA
Kv4.1	KCND1	mShal	KChIP 1-4, DP66, DPP10	Voltage gated	Colon	None	None	Fampridine (4-AP)
Kv4.3	KCND3	KCND3L, KCND3S, KSHIVB	KChIP 1-4, DP66, DPP10, MinK, MiRPs	Voltage gated	IEC-6	None	None	Fampridine (4-AP), bupivacaine, nicotine
Kv6.3	KCNG3	Kv10.1	Coassembles with Kv2.1	Voltage gated modifier	Small intestine	None	None	None
Kv6.4	KCNG4	None	Coassembles with Kv2.1	Voltage gated modifier	Small intestine, colon	None	None	None
Kv7.1	KCNQ1	KVLQT1	KCNE1 (minK/IsK), KCNE3 [minK-related peptide 2 (MiRP2)]	Voltage gated, slow delayed rectifier, K+ recycling at basolateral membrane of intestinal crypt cells (with KCNE3)	Small intestine, colon, intestinal crypt cells and surface epithelium	None	XE991, linopirdine (mouse)	Chromanol 293B
Kv7.2	KCNQ2	KVLQT2, KQT2	KCNQ3, KCNE2	Voltage gated	Small intestine	Retigabine	XE991, linopirdine	TEA
Kv7.4	KCNQ4	KvLQT4	KCNQ4	Voltage gated	Colonic mucosa	Retigabine	XE991, linopirdine	TEA
Kv10.1	KCNH1	eag1a, eag1b, KCNH1a, KCNH1b, ether-à-go-go	Hyperkinetic (Hk), CaM, Slob, epsin, KCR1 (KC regulator)	Voltage gated	Colon, tumor cells from different tissues	None	None	Astemizole
Kv11.1	KCNH2	erg1, HERG, ether-à-go-go-related gene	minK (KCNE1), MiRP1 (KCNE2)	Voltage gated	Small intestine, colorectal cancer cells	RPR260243	E4031	Astemizole. terfenadine, disopyramide, dofetilide, ibutilide

See Refs. 2, 3, 5, 8–18.. 4-AP, 4-aminopyridine; BL-1249, N-[2-(2H-tetrazol-5-yl)phenyl]-5,6,7,8-tetrahydronaphthalen-1-amine; ChTX, charybdotoxin; IbTX, iberiotoxin; EBIO, 1-ethyl-2-benzimidazolinone; GI‐530159, 4-[4-[2-[4-(4-aminophenoxy)phenyl]-1,1,1,3,3,3-hexafluoropropan-2-yl]phenoxy]aniline; HGNC, HUGO Gene Nomenclature Committee; IEC-6, rat epithelial cell line; IUPHAR, International Union of Basic and Clinical Pharmacology; NS004, 1-(5-chloro-2-hydroxyphenyl)-5-(trifluoromethyl)-2,3-dihydro-1H-1,3-benzodiazol-2-one; NS1619, 1-[2-hydroxy-5-(trifluoromethyl)phenyl]-5-(trifluoromethyl)-2,3-dihydro-1H-1,3-benzodiazol-2-one; NS309, 6,7-dichloro-1H-indole-2,3-dione-3-oxime; SKA-121, 5-methylnaphtho[2,1-d]oxazol-2-amine; TEA, tetraethylammonium; TRAM-34, 1-[(2-chlorophenyl)diphenylmethyl]-1H-pyrazole; UCL1684, 6,12,19,20,25,26-hexahycro-5,27:13,18:21,24-trietheno-11,7-methano-7H-dibenzo; XE991, 10,10-bis(pyridin-4-ylmethyl)anthracen-9-one; VU590, 7,13-bis(4-nitrobenzyl)-1,4,10-trioxa-7,13-diazacyclopentadecane, dihydrochloride.

It is acknowledged that KCs play a significant role in the development and progression of several human diseases (4, 7, 19). This has already been evidenced in inflammatory bowel disease (IBD), a multifactorial complex entity that includes Crohn's disease (CD) and ulcerative colitis (UC). At the intestinal level, homeostasis is maintained through the opening of apical K⁺ channels that promote K⁺ secretion, and basolateral K⁺ channels, creating the driving force for Cl⁻ secretion, via apical Cl⁻ channels (3, 7, 20–26). This process leads to a K⁺ diffusion potential that promotes the maintenance of a negative membrane potential (20, 26). The reduction of electrolyte absorption has been reported to be more important for the intestinal electrolytic disequilibrium than the increase in secretion. Higher K⁺ excretion and lower sodium absorption may contribute to this intestinal ionic imbalance and consequently cause diarrhea (7, 20, 22, 27, 28). Altered expression and/or function of epithelial ion channels and transporters is considered the main cause of electrolyte retention in the intestinal lumen of IBD patients, leading to diarrhea, a hallmark symptom of this condition (29). However, it is known that the modulation of KCs contributes to indirect regulation of sodium absorption in the intestinal epithelial cells (IECs) (7, 28, 30).

The modulation of KCs by chemical molecules has been the most used pharmacological tool for the investigation of the function of these channels (31, 32). Chemicals modulating of KC function fall into three general categories: metal ions, organic small molecules, and venom-derived peptides (Table 1). These chemical components allow to activate, inhibit, or block the channel activity. KC openers/activators and inhibitors act by allosteric modulation; while KC blockers act on the conducting pore, causing a direct interruption of the ion conduction pathway (32). The importance of epithelial KCs, and underlying intracellular pathways, remains largely unexplored (30). Indeed, among other settings, KC modulation rises as a sound research pathway to understand, in depth, the pathogenesis of diarrhea in IBD patients. In this context, this systematic review aimed to analyze the published evidence on the characteristics of KCs and their modulation mechanisms in IECs. After a summary of the characterization of KCs, their pharmacological modulation in IECs will be described. By doing so, we intend to revisit the pathogenesis of diarrhea in IBD and to identify potential therapeutic targets that allow the development of better treatments for IBD-associated diarrhea.

MATERIALS AND METHODS

Search Strategy

Literature search was performed from inception to September 2020, using three electronic databases: PubMed [https://www.ncbi.nlm.nih.gov/pubmed], Scopus [https://www.elsevier.com/solutions/scopus] and Web of Science [https://www.isiwebofknowledge.com]. The keywords and Medical Subject Headings (MeSH) terms used were as follows: (potassium channels OR potassium channel modulation) AND (intestinal epithelial cells) AND (intestines OR epithelial OR epithelium OR epithelial cells OR enterocytes). The reference sections of the selected studies and review articles were manually reviewed to ensure that all pertinent articles were included. The strategy followed for studies’ selection is presented in Fig. 1.

Figure 1.

Flow diagram of data collection and study selection. IEC, intestinal epithelial cells.

Eligibility Criteria

The inclusion criteria were 1) studies regarding KCs in IECs; and 2) studies concerning modulation of KCs. No limitations in terms of publication dates or language were applied. We excluded studies that 1) were review articles, reports, editorials, book chapters, or conference papers; 2) did not study KCs; 3) were not performed in IECs; 4) did not study KCs modulation on IECs; and 5) were not performed in mammals.

Study Selection and Data Collection Process

Studies were screened and selected by two authors following the recommendations of the Preferred Reporting Items for Systematic Reviews and Meta-Analysis Guidelines (33). In a first phase, titles and abstracts were carefully analyzed. Whenever a particular study failed to meet the inclusion criteria, it was excluded. In a second phase, the full text of all the remaining potentially relevant studies was analyzed, and the inclusion and exclusion criteria were applied again. The lists of full articles that met inclusion criteria were compared by each author, and disagreements were resolved by discussion and consensus. The data collected from each study were: type of KCs, type of cell or tissue used, related methods for KCs analysis, and main findings reported in the article. The characterization of the studies was described based on their performance in human or animal intestinal epithelial cells.

Quality Assessment

Due to the high potential of bias in the setting of clinical studies, several quality checklists for their assessment are available. On the other hand, as far as the authors know, no checklists or scores assess basic science studies. However, even though the experimental conditions are much more controlled in basic science setting, with inherent lower risk of bias (34), the attempts to evaluate prior literature in a rigorous and quantitative manner are very important. Indeed, besides allowing the critical assessment of the existing evidence, such assessment may guide researchers to improve studies’ design and, thereby, the quality of the evidence. In this study, we developed a quality checklist with 14 criteria (see Table 4) following the recommendations of Armstrong and Green (35) and the requirements defined by the Science in Risk Assessment and Policy (SciRAP) and by the Organization for Economic Co-operation and Development. Each criterion of the checklist was scored as follows: information not available in the paper (0 points); limited information provided (1 point); and complete information regarding that aspect (2 points). The checklist was independently applied by two authors, and discrepancies were solved by consensus. No specific set of information need to obtain a score of “complete information” was explicitly defined within each criterion but left to the discretion of the two reviewing authors. For each paper, the scores of all items were added and divided by the maximum score (28 points) to obtain paper’s overall quality score.

Table 4.

Assessment of the reporting inclusiveness of the included studies, using a tool developed by the authors, scored as 0 points (information not available in the paper); 1 point (limited information provided); and 2 points (complete information regarding that aspect)

Reporting Assessment	Al-Hazza et al. (36)	Alzamora et al. (37)	Antico et al. (38)	Ayabe et al. (39)	Banks et al. (40)	Barmeyer et al. (41)	Basalingappa et al. (42)	Bowley et al. (43)	Burckhardt and Gögelein (44)	Butterfield et al. (45)
Criteria
Problem definition
1. Scientific background and explanation of rationale	2	2	2	2	2	2	2	2	1	1
Purpose and hypothesis
2. Definition of the specific objectives or hypotheses	0	1	1	1	2	2	2	2	1	1
3. Definition of the endpoints to study	1	2	2	1	1	2	2	1	1	1
Study design
4. Accurate description of the laboratory methodologies (easy to understand and described in enough detail to allow replication), definition of the test compounds, experimental conditions, and other important information; use of validated methods	2	2	2	2	2	2	2	2	2	2
5. Ethical review permissions, when applicable	2	2	1	2	1	2	2	2	0	2
6. Description of the statistical methods, when adequate	2	2	2	0	2	2	0	2	1	2
Data collection
7. Obtain valid data and ensure that it is reliable	2	2	2	2	2	2	2	2	2	2
8. Evaluation by independent observers; blinding; evidence of independent repetitions	2	0	0	0	0	1	0	0	0	0
Analyzing data and manuscript drafting
9. Cite relevant scientific papers when presenting evidence	1	2	2	2	2	2	2	2	2	1
10. Accessible and transparent presentation of data throughout the paper (including the appropriate measures of precision/variance)	2	2	2	2	2	2	2	2	2	2
11. Critical discussion of the results; comparison with relevant research on the field	2	2	2	2	2	2	2	2	2	2
12. Draw consistent conclusions based on the evidence presented in the paper	2	2	2	2	2	2	2	2	1	2
13. State the contribution to cumulative scientific knowledge and the practical implications of the findings	2	1	1	1	1	1	2	1	1	1
14. Disclose conflicts of interest and declaring funding sources	2	1	2	1	1	2	2	0	0	0
Overall score	1.71	1.64	1.64	1.43	1.57	1.86	1.71	1.57	1.14	1.36
Standard deviation	0.61	0.63	0.63	0.76	0.65	0.36	0.73	0.76	0.77	0.74
Overall score/maximum score	86%	82%	82%	71%	79%	93%	86%	79%	57%	68%

Reporting Assessment	Daneshmand et al. (46)	Dedek and Waldegger (47)	Demolombe et al. (48)	Diener et al. (49)	Ding et al. (50)	Duan et al. (51)	Freeman et al. (52)	Furness et al. (53)	Grishin et al. (54)	Grotjohann et al. (55)
Criteria
Problem definition
1. Scientific background and explanation of rationale	2	2	1	2	2	2	1	2	2	2
Purpose and hypothesis
2. Definition of the specific objectives or hypotheses	2	1	1	2	1	2	1	1	2	2
3. Definition of the endpoints to study	2	2	1	1	2	1	2	1	2	1
Study design
4. Accurate description of the laboratory methodologies (easy to understand and described in enough detail to allow replication), definition of the test compounds, experimental conditions, and other important information; use of validated methods	2	2	2	2	2	1	2	2	1	2
5. Ethical review permissions, when applicable	2	0	0	0	2	1	2	2	2	2
6. Description of the statistical methods, when adequate	2	0	0	2	1	2	2	0	0	2
Data collection
7. Obtain valid data and ensure that it is reliable	2	2	2	2	2	2	2	2	2	2
8. Evaluation by independent observers; blinding; evidence of independent repetitions	2	1	0	2	2	1	1	0	1	2
Analyzing data and manuscript drafting
9. Cite relevant scientific papers when presenting evidence	2	2	2	1	2	2	2	2	2	2
10. Accessible and transparent presentation of data throughout the paper (including the appropriate measures of precision/variance)	2	2	2	2	2	2	2	2	2	2
11. Critical discussion of the results; comparison with relevant research on the field	2	2	2	1	2	2	2	2	2	2
12. Draw consistent conclusions based on the evidence presented in the paper	2	2	2	1	2	2	2	2	2	2
13. State the contribution to cumulative scientific knowledge and the practical implications of the findings	1	1	1	1	2	2	2	2	2	1
14. Disclose conflicts of interest and declaring funding sources	0	0	1	1	0	2	2	2	2	1
Overall score	1.79	1.36	1.21	1.43	1.71	1.71	1.79	1.57	1.71	1.79
Standard deviation	0.58	0.84	0.80	0.65	0.61	0.47	0.43	0.76	0.61	0.43
Overall score/maximum score	89%	68%	61%	71%	86%	86%	89%	79%	86%	89%

Reporting Assessment	Grunnet et al. (56)	Grunnet et al. (57)	Halm et al. (58)	Hay-Schmidt et al. (59)	Heinke et al. (60)	Hirota and McKay (61)	Huang et al. (62)	Inagaki et al. (63)	Joiner et al. (64)	Jons et al. (65)	Klaerke et al. (67)
Criteria
Problem definition
1. Scientific background and explanation of rationale	2	2	2	2	2	2	2	2	2	2	2
Purpose and hypothesis
2. Definition of the specific objectives or hypotheses	2	2	1	2	1	1	1	2	1	1	2
3. Definition of the endpoints to study	2	2	1	1	0	2	2	2	2	1	1
Study design
4. Accurate description of the laboratory methodologies (easy to understand and described in enough detail to allow replication), definition of the test compounds, experimental conditions, and other important information; use of validated methods	2	2	2	1	1	2	2	2	2	2	1
5. Ethical review permissions, when applicable	0	0	2	0	1	2	2	2	0	0	0
6. Description of the statistical methods, when adequate	1	1	1	0	2	2	2	2	1	0	2
Data collection
7. Obtain valid data and ensure that it is reliable	2	2	2	2	2	2	2	2	2	2	2
8. Evaluation by independent observers; blinding; evidence of independent repetitions	1	2	1	0	2	0	2	2	1	0	0
Analyzing data and manuscript drafting
9. Cite relevant scientific papers when presenting evidence	2	2	2	2	1	1	2	2	1	2	2
10. Accessible and transparent presentation of data throughout the paper (including the appropriate measures of precision/variance)	2	2	1	2	2	2	2	2	2	2	1
11. Critical discussion of the results; comparison with relevant research on the field	2	2	2	2	2	2	2	2	2	2	2
12. Draw consistent conclusions based on the evidence presented in the paper	2	2	2	2	2	2	2	2	2	2	2
13. State the contribution to cumulative scientific knowledge and the practical implications of the findings	2	1	1	1	1	2	2	2	1	2	2
14. Disclose conflicts of interest and declaring funding sources	1	0	1	1	1	1	2	2	1	1	0
Overall score	1.64	1.57	1.50	1.29	1.43	1.64	1.93	2.00	1.43	1.36	1.36
Standard deviation	0.63	0.76	0.52	0.83	0.65	0.63	0.27	0.00	0.65	0.84	0.84
Overall score/maximum score	82%	79%	75%	64%	71%	82%	96%	100%	71%	68%	68%

Reporting Assessment	Kanthesh et al. (66)	Kovacs et al. (68)	Kunzelmann et al. (69)	Linley et al. (70)	Loganathan et al. (71)	Lomax et al. (72)	Lomax et al. (73)	Lotz et al. (74)	Magalhaes et al. (28)	Marie et al. (75)
Criteria
Problem definition
1. Scientific background and explanation of rationale	2	2	1	2	1	1	1	2	2	1
Purpose and hypothesis
2. Definition of the specific objectives or hypotheses	2	2	1	1	1	1	2	2	1	2
3. Definition of the endpoints to study	2	1	1	1	1	1	1	1	2	1
Study design
4. Accurate description of the laboratory methodologies (easy to understand and described in enough detail to allow replication), definition of the test compounds, experimental conditions, and other important information; use of validated methods	2	2	2	2	2	2	1	2	2	2
5. Ethical review permissions, when applicable	2	2	0	2	2	0	2	2	2	2
6. Description of the statistical methods, when adequate	2	1	1	1	2	2	2	0	2	2
Data collection
7. Obtain valid data and ensure that it is reliable	2	2	2	2	2	2	2	2	2	2
8. Evaluation by independent observers; blinding; evidence of independent repetitions	2	1	0	0	2	1	1	1	0	2
Analyzing data and manuscript drafting
9. Cite relevant scientific papers when presenting evidence	2	2	2	2	2	2	2	2	2	2
10. Accessible and transparent presentation of data throughout the paper (including the appropriate measures of precision/variance)	2	2	2	2	2	1	1	2	2	2
11. Critical discussion of the results; comparison with relevant research on the field	2	2	2	2	2	2	2	2	2	2
12. Draw consistent conclusions based on the evidence presented in the paper	2	2	1	2	2	1	1	2	2	2
13. State the contribution to cumulative scientific knowledge and the practical implications of the findings	2	2	1	2	2	1	1	2	2	2
14. Disclose conflicts of interest and declaring funding sources	2	2	1	2	2	1	1	0	2	2
Overall score	2.00	1.79	1.21	1.64	1.79	1.29	1.43	1.57	1.79	1.86
Standard deviation	0.00	0.43	0.70	0.63	0.43	0.61	0.51	0.76	0.58	0.36
Overall score/maximum score	100%	89%	61%	82%	89%	64%	71%	79%	89%	93%

Reporting Assessment	McDaniel et al. (76)	McNamara et al. (77)	Mei et al. (78)	Montero et al. (79)	Nakamura et al. (80)	Ousingsawat et al. (81)	Paul et al. (82)	Perry and Sandle (83)	Pillozzi et al. (84)	Pouokam et al. (86)
Criteria
Problem definition
1. Scientific background and explanation of rationale	2	2	2	1	2	2	1	2	2	2
Purpose and hypothesis
2. Definition of the specific objectives or hypotheses	2	2	1	1	2	2	1	2	1	2
3. Definition of the endpoints to study	2	2	2	1	2	2	2	1	2	2
Study design
4. Accurate description of the laboratory methodologies (easy to understand and described in enough detail to allow replication), definition of the test compounds, experimental conditions, and other important information; use of validated methods	2	2	2	2	2	2	2	2	2	1
5. Ethical review permissions, when applicable	1	2	2	0	2	2	2	2	2	1
6. Description of the statistical methods, when adequate	2	2	2	2	0	2	1	2	2	2
Data collection
7. Obtain valid data and ensure that it is reliable	2	2	2	1	2	2	2	2	2	2
8. Evaluation by independent observers; blinding; evidence of independent repetitions	0	1	0	0	0	0	2	2	2	2
Analyzing data and manuscript drafting
9. Cite relevant scientific papers when presenting evidence	1	2	1	1	2	2	2	2	2	1
10. Accessible and transparent presentation of data throughout the paper (including the appropriate measures of precision/variance)	2	2	2	2	2	2	2	2	2	2
11. Critical discussion of the results; comparison with relevant research on the field	1	2	2	2	1	1	2	2	2	1
12. Draw consistent conclusions based on the evidence presented in the paper	1	1	2	1	1	1	2	1	2	1
13. State the contribution to cumulative scientific knowledge and the practical implications of the findings	1	1	1	1	1	2	1	2	2	1
14. Disclose conflicts of interest and declaring funding sources	1	0	2	0	0	2	1	1	2	1
Overall score	1.43	1.64	1.64	1.07	1.36	1.71	1.64	1.79	1.93	1.50
Standard deviation	0.65	0.63	0.63	0.73	0.84	0.61	0.50	0.43	0.27	0.52
Overall score/maximum score	71%	82%	82%	54%	68%	86%	82%	89%	96%	75%

Reporting Assessment	Potier et al. (85)	Rao et al. (87)	Sandle et al. (88)	Sausbier et al. (89)	Schultheiss and Diener (24)	Silver et al. (90)	Singh et al. (92)	Simms et al. (91)	Song et al. (93)	Sørensen et al. (94)	Sorensen et al. (95)
Criteria
Problem definition
1. Scientific background and explanation of rationale	2	2	1	2	2	2	2	2	2	2	2
Purpose and hypothesis
2. Definition of the specific objectives or hypotheses	2	2	2	1	2	2	1	2	2	2	1
3. Definition of the endpoints to study	2	2	2	1	2	2	2	2	2	2	1
Study design
4. Accurate description of the laboratory methodologies (easy to understand and described in enough detail to allow replication), definition of the test compounds, experimental conditions and other important information; use of validated methods	2	2	2	2	2	2	2	2	2	2	2
5. Ethical review permissions, when applicable	2	2	2	2	0	2	2	2	2	2	2
6. Description of the statistical methods, when adequate	2	2	1	2	2	2	2	2	2	2	2
Data collection
7. Obtain valid data and ensure that it is reliable	2	2	2	2	2	2	2	2	2	2	2
8. Evaluation by independent observers; blinding; evidence of independent repetitions	0	0	1	2	2	2	2	2	2	2	0
Analyzing data and manuscript drafting
9. Cite relevant scientific papers when presenting evidence	2	2	2	1	2	2	2	2	2	2	2
10. Accessible and transparent presentation of data throughout the paper (including the appropriate measures of precision/variance)	2	2	2	2	2	2	2	2	2	2	2
11. Critical discussion of the results; comparison with relevant research on the field	2	2	2	2	2	2	2	2	2	2	1
12. Draw consistent conclusions based on the evidence presented in the paper	2	2	2	2	1	2	2	2	2	2	1
13. State the contribution to cumulative scientific knowledge and the practical implications of the findings	2	1	1	2	1	2	1	2	1	1	1
14. Disclose conflicts of interest and declaring funding sources	1	1	1	1	1	1	2	2	1	1	2
Overall score	1.79	1.71	1.64	1.71	1.64	1.93	1.86	2.00	1.86	1.86	1.50
Standard deviation	0.58	0.61	0.50	0.47	0.63	0.27	0.36	0.00	0.36	0.36	0.65
Overall score/maximum score	89%	86%	82%	86%	82%	96%	93%	100%	93%	93%	75%

Reporting Assessment	Sorensen et al. (96)	Thompson-Vestet al. (97)	Turnheim et al. (98)	Valero et al. (99)	Wachter and Turnheim (100)	Wang et al. (101)	Wang et al. (102)	Wiener et al. (103)	Zeng et al. (104)	Zhang et al. (105)	Zundler et al. (106)	Average Score
Criteria
Problem definition
1. Scientific background and explanation of rationale	2	2	2	2	2	2	2	2	2	2	2	1.84
Purpose and hypothesis
2. Definition of the specific objectives or hypotheses	1	2	1	1	1	2	2	1	2	1	2	1.51
3. Definition of the endpoints to study	1	1	1	2	1	2	1	1	2	2	2	1.52
Study design
4. Accurate description of the laboratory methodologies (easy to understand and described in enough detail to allow replication), definition of the test compounds, experimental conditions, and other important information; use of validated methods	2	2	2	2	2	2	1	2	2	2	2	1.89
5. Ethical review permissions, when applicable	2	2	0	2	0	2	1	0	2	2	2	1.44
6. Description of the statistical methods, when adequate	2	0	1	2	2	2	2	1	2	2	2	1.52
Data collection
7. Obtain valid data and ensure that it is reliable	2	2	2	2	2	2	2	2	2	2	2	1.99
8. Evaluation by independent observers; blinding; evidence of independent repetitions	0	0	0	2	0	1	2	0	2	1	2	0.96
Analyzing data and manuscript drafting
9. Cite relevant scientific papers when presenting evidence	2	2	2	2	2	2	2	2	2	2	2	1.85
10. Accessible and transparent presentation of data throughout the paper (including the appropriate measures of precision/variance)	2	2	2	2	2	2	2	2	2	2	2	1.95
11. Critical discussion of the results; comparison with relevant research on the field	2	2	1	2	2	2	2	2	2	2	2	1.90
12. Draw consistent conclusions based on the evidence presented in the paper	2	2	1	2	2	2	1	2	2	2	2	1.78
13. State the contribution to cumulative scientific knowledge and the practical implications of the findings	1	1	1	1	1	1	1	2	2	1	2	1.41
14. Disclose conflicts of interest and declaring funding sources	1	1	1	2	1	1	1	1	1	2	2	1.18
Overall score	1.57	1.50	1.21	1.86	1.43	1.79	1.57	1.43	1.93	1.79	2.00	1.62
Standard deviation	0.65	0.76	0.70	0.36	0.76	0.43	0.51	0.76	0.27	0.43	0.00	0.31
Overall score/maximum score	79%	75%	61%	93%	71%	89%	79%	71%	96%	89%	100%	81%

0 = Information not available; 1 = limited information; 2 = complete information.

RESULTS

Search and Study Selection

A total of 720 results were identified through electronic database searching; 108 duplicates were excluded. Twenty-five additional records were identified through manual search (Fig. 1). Based on titles and abstract, 637 records were screened, and 497 studies were excluded for not matching the inclusion criteria; 140 articles were eligible for full text analysis (Fig. 1). From these, 67 were excluded for not studying KC modulation in IECs, and 4 for not having available full text (Fig. 1). The remaining 73 studies (24, 28, 36–106) were included in this systematic review.

Potassium Channels in the Gastrointestinal Tract

Table 1 summarizes the characteristics of the KCs present in the gastrointestinal tract: 1) name, according to the Nomenclature Committee of the Union of Basic and Clinical Pharmacology (NC-IUPHAR); 2) gene name, proposed by HUGO (Human Genome Organization) Gene Nomenclature Committee; 3) alternative or unofficial name; 4) associated subunits; 5) description of the functional characteristics; 6) tissue and/or cells where each KC was found; and 7) channel modulators (activators/openers, inhibitors, and blockers). The KCs reported to exist in the intestinal epithelium are as follows: calcium-activated KCs (K_Ca1.1, K_Ca2.1, K_Ca2.2, K_Ca2.3, and K_Ca3.1), inwardly rectifying KCs (K_ir6.1, K_ir6.2, and K_ir7.1), two-pore domain KCs (K_2P2.1, K_2P3.1, K_2P4.1, K_2P5.1, K_2P9.1, and K_2P12.1), and voltage-gated KCs (K_v1.1, K_v1.2, K_v1.3, K_v1.4, K_v1.5, K_v1.6, K_v2.1, K_v2.2, K_v4.1, K_v4.3, K_v6.3, K_v6.4, K_v7.1, K_v7.2, K_v7.4, K_v10.1, and K_v11.1).

Description of the Studies

The data extracted from the 73 included studies was divided in two tables, one comprising data on KCs of human IECs (Table 2) and another including studies in animal IECs (Table 3). The selected studies were published between 1988 and 2019. Regarding the characterization of the KCs, nine studies did not specify the type of KC (40, 44, 49, 54, 55, 60, 72, 79, 82), seven did not specify the particular type of calcium-activated KC (24, 51, 73–75, 77, 103), two did not specify the voltage-gated KC (76, 87), and two did not specify the type of inwardly rectifying KC (46, 77), while another did not specify either the voltage-gated KC or the ATP-modulated KC (28).

Table 2.

Characterization of the studies in human intestinal epithelial cells

Reference	K+ Channel: Type	Cell/Tissue	K+ Channel: Related Methods	Main Findings
Nakamura et al. (80)	Kir7.1	Human small intestine cDNA sequence	NB, WB, IHC	Kir7.1 are expressed in epithelial cells of small intestine.
Lomax et al. (73)	KCa	Human colonic crypts from healthy mucosa	Patch clampKCa blockers: TEA, quinidine, Ba2+	Two different Ca2+-dependent K+ channels were identified in human colonic crypt cells: the high and low conductance K+ channels.
McNamara et al. (77)	KCa/KATP	Distal colonic mucosa biopsies (nondiseased) from patients undergoing resection for carcinoma	ISCK+ activator: estradiolK+ inhibitor: tolbutamideK+ blocker: TPeA	Estradiol increased KATP-dependent K+ transport and inhibited K+ transport through KCa channels in basolateral membrane of human colonocytes.
Bowley et al. (43)	KCa3.1	Sigmoid colonic mucosa biopsies from healthy patients	RT-PCR	KCa3.1 mRNA is expressed in the basolateral membrane of human colonic crypt cells.
Wang et al. (101)	KCa3.1	Intestine 407 cell line	Patch clamp, RT-PCRKCa blockers: Clt, ChTX, TEA, IbTxKCa inhibitor: apamin	KCa3.1 mRNA is expressed in intestinal epithelial cells. KCa3.1 channels are required for regulatory volume decrease process in intestinal epithelial cells.
Banks et al. (40)	N/S	Human ileal mucosa, T84 cell line	ISC and 86Rb effluxK+ blocker: ChTXK+ inhibitor: apamin	ACh induced basolateral K+ efflux, and this effect was reduced by ChTX plus apamin. In IEC, the synergistic interaction between ACh and CT is dependent on opening of basolateral K+ channels by ACh and apical Cl− channels by CT.
Lotz et al. (74)	KCa	T84 and Caco-2 cell lines	Wound-healing assays, fluorescence microscopyKCa activator: EBIO,KCa blockers: Clt, ChTX, Ba2+	Pharmacologic K+ channels inhibition by Clt, ChTX and Ba2+ accelerates wound healing in cell monolayers. EBIO had no effect on the repair rate.
Kovacs et al. (68)	K2P9.1	Gastrointestinal mucosa from human tissues	IHC, WB	K2P9.1 channels are expressed in the colonic epithelium and could also be observed in malignant gastrointestinal tumors. These channels might be involved in the K+ secretion of the epithelial cells.
Ding et al. (50)	Kv10.1	HT-29 and LoVo cell lines, colorectal biopsies from adenoma, cancer, noncancerous matched tissues	IHC, RT-PCR	mRNA levels of Kv10.1 was expressed in colorectal cancer samples and in HT29 and LoVo colonic cancer cell lines. Specific inhibition of Kv10.1 expression might be a therapeutic target for colorectal cancer.
Ousingsawat et al. (81)	Kv10.1	Colon biopsies from sigma diverticulitis, UC, colorectal adenocarcinoma, cecum carcinoma, colonic neoplasm, and normal patients	FISH, RT-PCR	Kv10.1 protein are expressed in human carcinomas and diverticulitis, whereas Kv10.1 mRNA expression was not found in the colon of normal patients (see Table 3, row 31).
Sandle et al. (88)	KCa1.1	Proximal and sigmoid colon mucosa biopsies from UC and healthy patients	WB, RT-PCR, IHC	In normal colon, the KCa1.1 channels are expressed in the apical membrane of surface and upper crypt cells. In turn, in colonic mucosa of UC patients KCa1.1 channels are extended along the entire crypt axis.
Perry and Sandle (83)	KCa1.1	Sigmoid colonic mucosa from normal patients	Patch clampSomatostatin (antidiarrheal peptide)	Somatostatin (antidiarrheal peptide) inhibited apical KCa1.1 channels in human colonocytes (see Table 3, row 34).
Simms et al. (91)	KCa3.1	Ileal mucosal biopsies from CD and healthy patients	RT-PCR, IHC	KCa3.1 channels are strongly expressed in Paneth cells. Ileal mucosa biopsies from CD patients with NOD2 disease-predisposing mutations (R702W, G908R, 1007fs) had reduced levels of KCa3.1 mRNA than control samples.
Loganathan et al. (71)	KCa3.1	Sigmoid colon mucosa biopsies from healthy patients	Patch clampKCa3.1 blockers: TRAM-34, Clt	Chemical hypoxia increased the permeability in intestinal epithelia through activation of the basolateral KCa3.1 channels, but TRAM-34 and clotrimazole blocked this activation.
Al-Hazza et al. (36)	KCa3.1	Sigmoid colon mucosa biopsies from active UC, quiescent UC, active CD, and normal patients	Patch clamp, IHC, qPCR	Basolateral KCa3.1 channels were distributed along the surface-crypt axis. KCa3.1 channel expression and activity were reduced in active UC, but KCa3.1 mRNA levels were similar to controls. This decrease in basolateral KCa3.1 channel led to epithelial cell depolarization in UC.
Paul et al. (82)	N/S	T84 and HT-29 cell lines	I SC	EGF inhibited carbachol-stimulated basolateral K+ currents. In contrast, the IFN-γ reversed this inhibitory effect on basolateral K+ conductance in intestinal epithelial cells.
Linley et al. (70)	KCa1.1	Sigmoid colonic biopsies from healthy patients	Patch clamp, immunostainingKCa blockers: penitrem A, Clt	KCa1.1 channels were inhibited by the penitrem A but insensitive to Clt. K+ secretion in human colonic epithelium was originated from a smaller population of goblet cells expressing apical KCa1.1 channels.
Marie et al. (75)	KCa	HT-29 cell line, colonic biopsy from patients with acute E. histolytica colitis	FluxOR K+ channel assay (K+ channel activation)KCa inhibitor: paxillineKCa blocker: Clt	Inhibition of KCa channels was highly effective in preventing amebic cytotoxicity in IECs. Clt reduced and paxilline blocked amebic killing of IECs.
Huang et al. (62)	K2P2.1	T84 cell line	Gene silencing of Trek1, RT-PCR, WB	K2P2.1 are expressed in IEC. The knockdown of K2P2.1 gene induced epithelial barrier disruption.
Zundler et al. (106)	KCa3.1	HT-29 cell line and IEC from surgical specimens and endoscopic biopsies of UC and CD patients	WB, RT-PCR K+ blockers: IbTx, ChTX, Clt, Ba2+K+ activator: 1-EBIO	mRNA levels of KCa3.1 were higher in epithelium from UC or CD patients. Clt inhibited KCa3.1 increasing wound healing response after mechanical injury on HT-29 cells. 1-EBIO activated KCa3.1 and retarding wound closure.Modulation of specific K+ channels regulates intestinal epithelial restitution (see Table 3, row 49).
Magalhaes et al. (28)	KATP, Kv	HT-29 cell line	Spectrofluorometry (K+ channel activity)K+ blockers: BaCl2 (unspecific), noxiustoxin (Kv) and AMP-PNP (KATP)K+ opener: pinacidil (KATP)	BaCl2 completely abolished MDP (specific ligand recognizing NOD2) effects on membrane potential, AMP-PNP partially reverse the effect, whereas noxiustoxin did not have a significant effect. Activation of NOD2 appears being responsible to hyperpolarize IECs, partially through the opening of KATP, but not Kv, channels.
Pillozzi et al. (84)	KCa3.1, Kv11.1	HCT-116 and HCT-8 cell lines	Patch clamp, RT-PCR, WBKCa3.1 activator: Riluzole, SKA-31KCa3.1 blocker: TRAM-34Kv11.1 inhibitor: E4031	KCa3.1 and Kv11.1 channels were higher expressed in Cisplatin-resistant colorectal cancer cells. Inhibition of Kv11.1 currents leads to upregulation of functional KCa3.1 channels, leading to synergistic activity with the proapoptotic effects of cisplatin and inhibiting proliferation.
Duan et al. (51)	KCa	T84 cell line	ISC (basolateral K+ conductance)KCa blockers: Clt and senicapoc	ErbB TKI-induced diarrhea mechanism involves amplification of basolateral K+ in IECs. Afatinib increased the activity of KCa, which were inhibited by Clt and senicapoc.Basolateral K+ channels inhibition (possibly KCa) might be a therapeutic target for diarrhea caused by ErbB TKIs (see Table 3, row 51).

ACh, acetylcholine; AMP-PNP, adenylyl-imidodiphosphate; CD, Crohn´s disease; Clt, clotrimazole; CT, cholera toxin; ChTX, charybdotoxin; EBIO, 1-ethyl-2-benzimidazolinone; ErbB TKI, small molecule ErbB tyrosine kinase inhibitor (TKIs); FISH, Fluorescence in situ hybridization; IECs, intestinal epithelial cells; IHC, immunohistochemistry; I_SC, short-circuit current; MDP, muramyldipeptide; mRNA, messenger ribonucleic acid; NB, Northern blot; NOD2, nucleotide-binding oligomerization domain-containing protein 2; N/S, nonspecified; qPCR, quantitative polymerase chain reaction; RT-PCR, reverse transcription polymerase chain reaction; UC, ulcerative colitis; WB, Western blot.

Table 3.

Characterization of the studies in animal intestinal epithelial cells

Reference	K+ Channel: Type	Cell/Tissue	K+ Channel: Related Methods	Main Findings
Montero et al. (79)	N/S	Midintestine of chicken	86Rb+ flux assay (channel activity)K+ efflux modifiers: Ba2+, quinine, verapamil, A23187, 3,4-DAP, apamin	K+ current was inhibited by Ba2+, quinine, and verapamil; it was increased by A23187, and it was unaffected by 3,4-DAP. Apamin, which has no effect on K+ currents, abolished the effect of A23187. These findings propose that K+ efflux appears to occur through Ca2+-activated K+ channels in enterocytes.
Turnheim et al. (98)	KCa1.1	Distal colon of healthy rabbits	Patch clampK+ blockers: LQV, Ba2+, quinine, trifluoperazine	KCa1.1 channel is present in basolateral membranes of colonocytes. LQV, Ba2+, quinine, and trifluoperazine inhibited this channel.
Wiener et al. (103)	KCa	Distal colon of healthy rabbits	86Rb+ flux assayKCa blocker: ChTxKCa inhibitor: apamin	KCa channels are present in basolateral membrane of surface and crypt cells of colon epithelium. KCa channels in surface cell vesicles were inhibited by apamin and in the crypt cell membrane fraction were inhibited by ChTx.
Burckhardt and Gögelein (44)	N/S	Distal colon of healthy rats	Patch clampK+ blockers: TEA, Ba2+, glibenclamide, ChTx, apamin, risotilide	Basolateral K+ conductance of small and maxi K+ channels was blocked by TEA and Ba2+.The small K+ channel might be responsible for maintaining the cell membrane resting potential in isolated crypts from rat distal colon.
Klaerke et al. (67)	KCa1.1	Distal colon of healthy rabbits	Patch clampKCa1.1 blocker: ChTx	ChTx blocked the KCa1.1 channel from the basolateral membrane of the colon surface cells.
Lomax et al. (72)	N/S	Colon of healthy and aldosterone-treated rats	ISCK+ blockers: TEA	Aldosterone-induced apical K+ conductance, predominantly in surface colonocytes and minor in crypts of colonic epithelium.
Diener et al. (49)	N/S	Distal colon of healthy rats	86Rb+ flux assay, patch clamp K+ blockers: chromanol 293B, TEA, quinine	293B blocked basolateral K+ conductance and induced a membrane depolarization.Intracellular cyclic AMP was responsible for regulation of K+ transport. There was a decrease in basolateral versus apical K+ conductance, leading to an enhanced efflux of K+ to the mucosal compartment and a reduced efflux to the serosal compartment.
Wachter and Turnheim (100)	KCa1.1	Distal colon of healthy rabbits	Patch clamp	Mg2+ inhibited basolateral Ca2+-activated K+ conductance in epithelial cells.
Butterfield et al. (45)	KCa1.1	Colonic surface cells and intact crypts of dietary K+-loaded rats	Patch clampK+ blockers: quinidine, TEA, Ba2+	Chronic dietary K+ loading increases the abundance of large conductance K+ channels in the apical membrane of colonic surface cells. Quinidine, TEA, or Ba2+ blocked these channels.
Schultheiss and Diener (24)	KCa	Proximal and distal colon of healthy rats	ISCK+ blockers: TEA, quinine	Carbachol increased Ca2+-activated basolateral and apical K+ conductance in distal and proximal colon. This increase led to K+ secretion.An increase in the intracellular cAMP concentration inhibited total basolateral K+ conductance, leading to the inhibition of K+ absorption.
Grotjohann et al. (55)	N/S	Distal colon of healthy rats	86Rb+ flux assay, ISCK+ blockers: TEA	Aldosterone-induced K+ secretion was restricted to surface epithelium, whereas cAMP-mediated K+ secretion was present equally in crypts and surface epithelium.
Heinke et al. (60)	N/S	Proximal and distal colon of rats	ISC, 86Rb+ flux assayK+ blockers: TEA, quinine, Ba2+	Carbachol increased the apicalK+ uptake and stimulates K+ efflux to the serosal compartment in distal colonic epithelium. In the proximal colon was responsible for basolateral K+ uptake and an increased efflux to the luminal side.
Grunnet et al. (56)	KCa1.1	Distal colon of healthy rabbits	Ba2+-sensitive 86Rb+ flux assay (channel activity)	KCa1.1 channels are the predominant channels in distal colon epithelium membranes of rabbits.
Wang et al. (102)	Kv1.1	IEC-6 (rat epithelial cell line)	RT-PCR, WBKv blocker: 4-AP	Kv channel expression was involved in the stimulation of cell migration by polyamines in IEC-6 cells. Blockade of Kv1.1 channel with 4-AP reduced normal cell migration.
Dedek and Waldegger (47)	Kv7.1 (KCNQ1/KCNE3)	Small intestine and colon of mice	NB, immunofluorescence	KCNQ1 and KCNE3 are colocalized in the basolateral membrane of crypt cells of the colon and the small intestine. KCNE3 expression was highest in colon and rather low in the small intestine.
Demolombe et al. (48)	Kv7.1	Small intestine of mice	In situ hybridization, RT-PCR	Kv7.1 mRNA expression is widely distributed in the small intestine, in the absence of its regulator IsK. The high level of Kv7.1 expression in epithelial tissues was consistent with its potential role in K+ secretion and recycling.
Kunzelmann et al. (69)	Kv7.1	Distal colon of dexamethasone-treated rats	ISC, patch clamp, RT-PCR	Kv7.1 are expressed in both epithelial cells derived from crypts and surface epithelium. This K+ channel works on K+ recycling and maintaining electrolyte secretion in the colonic epithelium.
McDaniel et al. (76)	Kv	IEC-6 (rat epithelial cell line)Fenfluramine- and 4-AP-treated rats	Patch clamp, RT-PCRKv blocker: 4-AP	Multiple Kv channel α-subunits (Kv1.1, Kv1.2, Kv1.3, Kv1.4, Kv1.5, Kv2.1, Kv4.3 and Kv9.3) and Kv channel β-subunits (Kvβ1.2, Kvβ2.1, and Kvβ3) were expressed in IEC-6 cells. 4-AP reduced whole cell Kv currents and caused membrane depolarization.
Ayabe et al. (39)	KCa3.1	Paneth cells of mouse small intestinal epithelium	RT-PCR, WB	mKCa3.1 channels are highly restricted expressed in Paneth cells. Highly selective blockers of the KCa3.1 modulate Paneth cell secretory response by decreasing secretion of antimicrobial peptides in response to bacterial exposure.
Rao et al. (87)	Kv	IEC-6 (rat epithelial cell line) and IEC-Cdx2L1 (Cdx2-transfected IEC-6 cells)	RT-PCR, WB, patch clamp,DFMO (a specific inhibitor for ODC: key enzyme for polyamine synthesis)	mRNAs and proteins levels of Kv1.1 and Kv1.5 channels were increased in differentiated IEC-Cdx2L1 cells and were associated with an increase in voltage-gated K+ currents and membrane hyperpolarization. The expression of Kv1.1 and Kv1.5 channels in differentiated IEC-Cdx2L1 cells was inhibited by polyamine depletion with DFMO.
Furness et al. (53)	KCa3.1	Intestinal segments of rats	IHC	KCa channels are present in basolateral and apical membranes of colonic enterocytes.
Grunnet et al. (57)	Kv1.3	Distal colon of healthy rats	IHC	Kv1.3 channels are present in the basolateral membrane of the crypt cells.
Hay-Schmidt et al. (59)	KCa1.1	Distal colon of healthy rats and rabbits	IHC	In colonic epithelium, the KCa1.1 channels are expressed in the apical as well as in the basolateral membranes of surface cells.
Joiner et al. (64)	KCa (SK, IK, and BK)	Colonic epithelial cells of healthy and dietary K+-depleted rats	86Rb+ flux assay, NB, WB	BKCa, SK2Ca, and IKCa channel transcripts were present in rat proximal colon, but only IKCa mRNA was downregulated in dietary K+-depleted rats.
Grishin et al. (54)	N/S	IEC-6 (rat epithelial cell line)	Measurement of intracellular K+, 86Rb+ efflux assay, RT-PCRK+ blockers: TEA, 4-AP, stromatoxin, chromanol 293B, 48F10	Apoptosis in IEC-6 enterocytes was associated with intracellular K+ efflux via K+ channels (possibly Kv2.1 or Kv7.1). After treatment with 48F10, a specific pharmacological inhibitor of K+ channels, apoptosis did not occur, or was significantly reduced in these cells.
Halm et al. (58)	KCa3.1	Colon of guinea pigs and rats	ISC, immunofluorescenceKCa activator: 1-EBIOK+ blocker: TRAM-34	KCa3.1 channels are localized in distal colonic mucosa.The K+ secretion was inhibited at a high concentration of TRAM-34, suggesting the involvement of multiple K+ conductive pathways in secretion.
Jons et al. (65)	Kir6.1	Small intestine of healthy rats	Radioactivity measurement (liquid scintillation counting)KATP activator: diazoxideKATP inhibitor: tolbutamide	Kir6.1-SUR2A complexes are directly involved in the regulation of intestinalpermeability. Diazoxide decreased tight junction permeability and tolbutamide reversed this effect.
Sausbier et al. (89)	KCa1.1	Distal colon of BK wild-type (BK+/+) and knockout (BK−/−) mice	IHC, RT-PCR	KCa1.1 channels are localize to the luminal membrane of colonic crypts and are required for K+ secretion in distal colonic mucosa.
Thompson-Vest et al. (97)	KCa3.1	Small intestine and colon of healthy rats	WB, RT-PCR	KCa3.1 channels are expressed in the small intestine and colon.
Freeman et al. (52)	Kv1	IEC-6 (rat epithelial cell line)	Spectrophotometric measurement with pH-sensitive dye, WB, IP	NSAIDs reduced the expression of functional delayed rectifier channels on the cell surface membrane by decreasing the total expression of Kv1.4 and Kv1.4 and Kv1.6 channel subunits coassociation.
Ousingsawat et al. (81)	Kv10.1	Proximal and distal colons of healthy mice and DMH-, MNU-, and DSS-treated mice	ISC, WB, RT-PCRKv blocker: 4-AP Eag blocker: astemizole	Application of both 4-AP and astemizole inhibited ISC in proximal and distal colon of DMH-treated mice. The activation of Kv and Kv10.1 channels (expression of Kv10.1 protein and mRNA) was induced by DMH and MNU treatment, whereas no changes were observed in the inflamed colon (DSS) (see Table 2, row 10).
Sorensen et al. (94)	KCa1.1	Distal colon of NMRI wild-type mice treated with high K+ diet and standard diet	ISC, RT-PCR, IHCKCa1.1 blocker: IbTX	KCa1.1 are localized in the luminal membrane of colonic crypts. mRNA levels of α- and β2-subunits were elevated in the colonic crypts of high K+ diet mice. The aldosterone-induced K+ secretion occurred via luminal KCa1.1 channels.
Hirota et al. (61)	KCa3.1	Colon and ileum of DSS-treated mice	ISC, WB, RT-PCRKCa activator: DCEBIOKCa blocker: clotrimazole	Activity of the KCa3.1 channel was reduced in colonic epithelium from DSS-treated mice. In turn, there was no significant difference in the expression of the KCa3.1 channel protein between the colon crypts of control mice and those of DSS-treated mice.
Perry and Sandle (83)	KCa1.1	Distal colon of high K+ diet and standard diet rats	Patch clamp	cAMP-dependent PKA had a stimulatory effect and somatostatin (antidiarrheal peptide) an inhibitory effect on apical KCa1.1 channels localized in surface colonocytes (see Table 2, row 12).
Barmeyer et al. (41)	KCa3.1	Colonic mucosa of healthy rats and IEC-6 (rat intestinal epithelial cell line)	86Rb+ efflux assay, WB, RT-qPCR, immunofluorescence	KCa3.1 isoforms are expressed in apical (Kcnn4c) and basolateral (Kcnn4b) membranes of colonic epithelial cells. Apical KCa3.1 channels might be responsible for K+ secretion and basolateral KCa3.1 channels for anion secretion.
Potier et al. (85)	KCa2.3	Apc+/+ and ApcMin/+ (mouse colon epithelial cell line with Apc mutation)	Patch clamp, WB, immunofluorescence stainingKCa2.3 inhibitor: apamin	KCa2.3 channel activity was suppressed in ApcMin/+ cells (no apamin-sensitive current). KCa2.3 protein expression was decreased in ApcMin/+ cells when compared to Apc+/+ cells.
Sorensen et al. (25)	KCa1.1	Distal colon of BK wild-type (BK+/+) and knockout(BK−/−) mice; CFTR wild-type (CFTR+/+) and knockout (CFTR−/−) mice	RT-PCR, ISCEpinephrine (agonist to promote cAMP-dependent K+ secretion)K+ blockers: IbTx, chromanol 293B, BaCl2	Addition of IbTx at the luminal side inhibited the major fraction of K+ currents and BaCl2 inhibited the residual current. Epinephrine-stimulated K+ secretion was inhibited in distal colonic mucosa of KCa1.1−/− mice. The KCa1.1 channel is responsible for Ca2+-activated colonic K+ secretion and might be upregulated by aldosterone.
Alzamora et al. (37)	Kv7.1 (KCNQ1:KCNE3)	Colonic crypts of rats	RT-PCR, Co-IP and WB, patch clamp, and basolateral ISC measurement (channel activity)	Kv7.1 (KCNQ1:KCNE3) channels are expressed on basolateral membrane of colonic crypts. Estrogen regulates the expression of KCNE1 and KCNE3 and was reduced in females throughout the estrous cycle.
Basalingappa et al. (42)	KCa3.1	IEC-18 (cell line derived from rat small intestine)	Patch clamp, RT-PCR, WBKCa3.1 blocker: TRAM-34	TRAM-34 was less effective in inhibiting KCa3.1 channels from apical membranes than from basolateral membranes. Kcnn4c (isoform expressed in apical membrane) can be a good candidate for a TRAM-34-resistant mediator of K+ secretion.
Daneshmand et al. (46)	KATP	Colon of TNBS treated	Macroscopic and microscopic assessmentKATP inhibitor: glibenclamideKATP opener: cromokalim	Lithium improved TNBS-induced macroscopic and histological features of colonic injury. KATP channels are involved in these protective effects, since glibenclamide, partially inhibited the ameliorative effects and cromakalim reversed them, decreasing TNBS-induced colonic injury.
Sorensen et al. (96)	KCa1.1	Distal colon of NMRI wild-type mice treated with high K+ diet and standard diet	qPCR, transepithelial voltage measurementKCa1.1 blocker: IbTx	The KCa1.1 α-subunit mRNA expression was more pronounced in crypt cells than expression in colonic epithelial surface cells. Luminal IbTx addition led to a reduction of colonic K+ secretion. KCa1.1-mediated K+ secretion mainly occurs via the apical membrane of the crypt cells.
Singh et al. (92)	KCa3.1 and KCa1.1	Distal colon of rats	ISC, WB, RT-PCRKCa blockers: Ba2+, ChTx, TEA, IbTx, TRAM-34	KCa3.1 and KCa1.1 channels are present on apical membranes of rat distal colon. Colonic mucosa exposure to aldosterone featured active K+ secretion via these channels.
Zhang et al. (105)	KCa1.1	Colonic mucosa of guinea pig	ISC, RT-PCR, WBKCa1.1 blockers: IbTx, ChTxKCa1.1 inhibitor: paxilline	IbTx and paxilline inhibited the ISC associated with K+ secretion. KCa1.1 are essential for this effect in apical membrane.
Antico et al. (38)	KCa1.1	Distal colon of rats	Immunofluorescence microscopyKCa1.1 blockers: IbTx, Clt, apamin	KCa1.1 channel activation plays a crucial role in the mechanisms of cell volume regulation and cell death in rat superficial colonocytes. High K+ efflux and IbTx inhibited cell volume recovery. Clt and apamin were ineffective on the regulatory volume decrease response.
Kanthesh et al. (66)	KCa1.1 (BK-α)KCa3.1	Colonic mucosa of DSS-treated mice	ISC, 86Rb+ flux assay, WB, RT-PCRK+ blocker: Ba2+KCa1.1 blocker: IbTxKCa3.1 blocker: TRAM-34	Apical KCa1.1 channel α-subunit expression and mRNA in colonic mucosa of DSS-treated mice were increased compared with controls. Apical and basolateral mRNA of KCa3.1 channels were significantly increased in DSS-treated colon, but no changes were observed in protein expression. The increased of active K+ secretion reflects upregulation of apical KCa1.1 channels.
Pouokam et al. (86)	Kir6.1 and Kir6.2	Distal colon of rats	IHC, RT-PCR	Protein expression and mRNA of the KATP channels subunits (Kir6.1, Kir6.2, SUR1, and SUR2B) was observed in basolateral membrane of colonic epithelium.
Song et al. (93)	Kv1.1	IEC-6 (rat epithelial cell line)	RT-PCR, WB, measurement of MP with DiBAC4AMK (plant extracts)	Kv1.1 mRNA and protein expression increased after AMK treatment in control and polyamine-deficient IEC-6 cells. Treatment with AMK stimulated the migration of IEC-6 cells through polyamine-Kv1.1 channel signaling pathway, which might promote the healing of intestinal injury.
Silver et al. (90)	Kv (Kv1.3, Kv1.4, Kv1.6)	IEC-6 (rat epithelial cell line) small intestine (duodenum, jejunum, and ileum) of indomethacin- or NS-398-treated and normal rats	RT-PCR, WB, measurement of Em with DiBAC4NSAIDs: indomethacin, NS-398	Treatment with indomethacin and NS-398 decreased protein expression of Kv1.3 channel in vitro (IEC-6) and in vivo (duodenum and ileum of rats) studies.Indomethacin and NS-398 treatment induced a significant suppression of Kv1.6 expression in small intestinal mucosa and decreased Kv1.4 protein expression in duodenum. Modulation of Kv channels by NSAIDs resulted in depolarization of Em, inhibition of cell migration, and wound healing and might contribute to GI toxicity.
Zundler et al. (106)	KCa3.1 and KCa1.1	IEC-18 (rat epithelial cell line)	WBKCa blockers: IbTx, ChTX, Clt, Ba2+KCa3.1 activator: 1-EBIO	Inhibition of KCa3.1 and KCa1.1 by Clt and IbTx, respectively, led to an increase in wound healing response after mechanical injury. The addition of Ba2+ had no effect on K+ modulation. Activation of KCa3.1 by 1-EBIO induced a slow-down wound healing response (see Table 2, row 20).
Zeng et al. (104)	Kv1.1	IEC-6 (rat epithelial cell line)	RT-PCR, WBKv blocker: 4-APRAPS and RAMPS (plant polysaccharide extracts)	Kv1.1 channel was inhibited by 4-AP that led to a decreased in Kv1.1 mRNA and protein expression, and then subsequently inhibited cell migration. RAPS and RAMPS reversed the inhibitory effects of 4-AP.
Duan et al. (51)	KCa	Afatinib-treated rats	ISCKCa blocker: Clt and senicapoc	K+ channel inhibitors, Clt and senicapoc, led to a decreased stool water content after afatinib administration (see Table 2, row 23).
Inagaki et al. (63)	Kv (Kv7.1, Kv7.2, Kv7.4)	Colonic mucosa of rats	ISC, RT-PCR, IHCKv7 inhibitor: XE991	XE991 inhibited the ISC associated with K+ secretion in the apical membrane of colonic epithelial cells. Kv7.2 and Kv7.4 were localized in the luminal membranes of surface and crypt cells, whereas the Kv7.1 was localized in basolateral membrane of crypt cells.
Mei et al. (78)	Kv1.3	Colon of DSS-treated and normal mice	RT-qPCR, WBKv1.3 blocker: PAP-1	Kv1.3 expression decreased in the DSS-induced colitis mice with PAP-1 injection.
Valero et al. (99)	KCa3.1	Duodenum and colon of KCa3.1+ and KCa3.1− mice treated with DOX and/or senicapoc	RT-PCR, IHCKCa blockers: senicapoc and TRAM-34	KCa3.1-overexpression mice changed intestinal functions by increasing the chyme accumulation and reducing spontaneous contractions. The suppression of KCa3.1 had no impact on intestinal function.

1-EBIO, 1-ethyl-2-benzimidazolinone; 3,4-DAP, 3,4-diaminopyridine; 4-AP, 4-aminopyridine; AMK, Atractylodes macrocephala koidz; Apc, adenomatous polyposis coli gene; cAMP, cyclic adenosine monophosphate; CFTR, cystic fibrosis transmembrane regulator; ChTx, charybdotoxin; Clt, clotrimazole; Co-IP, coimmunoprecipitation; cAMP, cyclic adenosine monophosphate; DCEBIO, 5,6-dichloro-1-ethyl-1,3-dihydro-2H-benzimidazol-2-one; DFMO, α-difluoromethylornithine; DiBAC4, Bis-(1,3-dibarbituric acid)-trimethine oxanol; DMH, dimethylhydrazine; DOX, doxycycline; DSS, dextran sulfate sodium; E_m, membrane potential; GI, gastrointestinal; IbTx, iberiotoxin; IHC, immunohistochemistry; IP, immunoprecipitation; I_SC, transepithelial short-circuit current; LQV, Leiurus quinquestriatus venom; MNU, N-methyl-N-nitrosourea; mRNA, messenger ribonucleic acid; NB, Northern blot; N/S, nonspecified; NSAIDs, nonsteroidal anti-inflammatory drugs; ODC, ornithine decarboxylase; PKA, protein kinase A; qRT-PCR, quantitative reverse transcription polymerase chain reaction; RA, radix astragali; RAM, Rhizoma atractylodis macrocephalae; RAMPS, polysaccharide extracts of atractylodes macrocephala; RAPS, polysaccharide extracts of astragalus membranaceus; RT-PCR, reverse transcription polymerase chain reaction; TEA, tetraethylammonium; TRAM-34, 1-[(2-chlorophenyl)diphenylmethyl]-1H-pyrazole; TNBS, 2,4,6-trinitrobenzene sulfonic acid; WB, Western blot.

Among the studies performed in human IECs (Table 2), 6 were only performed in cell lines derived from colon (T84, Caco-2, HT-29, HCT-116, and HCT-8) (28, 51, 62, 74, 82, 84), 11 resorted to tissue derived from human intestinal mucosa (36, 43, 68, 70, 71, 73, 77, 81, 83, 88, 91), and the remaining 4 used both cell lines and human intestinal tissue (40, 50, 75, 106). Regarding the studies performed in animal IECs (Table 3), 10 were performed only in cell lines (IEC-6 and IEC-18, both derived from small intestine of rats) (42, 52, 54, 76, 85, 87, 93, 102, 104, 106), 41 were performed in tissues derived from intestinal segments (24, 37–39, 44–49, 51, 53, 55–61, 63–67, 69, 72, 78, 79, 81, 83, 86, 89, 92, 94, 96–100, 103, 105), and 2 used both cell lines and intestinal tissue (41, 90).

Regarding the studies included, the expression and transcription of KCs were quantified by Western blot (37, 39, 41, 42, 52, 61, 62, 64, 66, 68, 78, 80, 81, 84, 85, 87, 88, 90, 92, 93, 97, 102, 104–106), Northern blot (47, 64, 80), and RT-PCR (36, 37, 39, 41–43, 48, 50, 54, 61–63, 66, 69, 76, 78, 81, 84, 86–97, 99, 101, 102, 104–106). For protein location, the authors resorted to immunohistochemistry (36, 50, 53, 57, 59, 63, 68, 80, 86, 88, 89, 91, 94, 99), immunofluorescence (38, 41, 47, 48, 58, 70, 81, 85), and immunoprecipitation techniques (37, 52).

The techniques used to evaluate channel activity were electrophysiological methods by short-circuit current (I_sc) measurement (24, 37, 40, 51, 55, 58, 60, 61, 63, 66, 69, 72, 77, 81, 82, 92, 94, 95, 105), patch-clamp recordings (36, 37, 42, 44, 45, 49, 67, 69–71, 73, 76, 83–85, 87, 98, 100, 101), ⁸⁶Rb⁺ efflux assay (40, 41, 49, 54–56, 60, 64, 66, 79, 103), spectrophotometry (28, 52, 75, 90, 93), or radioactivity measurement (65).

In this systematic review we propose a checklist to assess the reporting quality of the included studies. This instrument was applied by two authors and the final score was decided by consensus. The results, expressed as the percentage of the maximum possible score considering the 14 defined criteria, are presented in Table 4. The scores ranged from 54% [Montero et al. (79)] to 100% [Inagaki et al. (63), Kanthesh et al. (66), Simms et al. (91), and Zundler et al. (106)], and the mean quality score was 81% ± 0.31. Studies published earlier presented lower scores. In turn, the higher score parameters were 1) to “obtain valid data and ensure that it is reliable,” and 2) “accessible and transparent presentation of data throughout the paper” (Table 4). Further validation of this checklist will require its application in other research settings, with evaluation of the interobserver’s agreement, reliability, face and content validity, external validity, and perceived utility.

Expression and Pharmacologic Modulation of KCs

Table 2 summarizes the studies performed in human IECs regarding the expression and pharmacological modulation of KCs. The findings from animal studies are outlined in Table 3. The model of KCs secretion and absorption in the intestinal epithelial cell is schematically presented in Fig. 2.

Figure 2.

Schematic representation of potassium channel (KC) secretion and absorption in the intestinal epithelial cell. K⁺ secretion is mediated by the Na⁺-K⁺-ATPase and Na⁺-K⁺-2Cl^– cotransporter followed by the efflux of ion movement of apical KCs (K_Ca1.1). K⁺ absorption is mediated by the apical colonic H⁺-K⁺-ATPase followed by the efflux of basolateral KCs (K_Ca3.1, K_ir). In intestinal epithelial cells (IECs), the basolateral KCs are crucial in maintaining the driving force for transepithelial ionic transport. In the apical membrane, Na⁺ enters by the epithelial sodium channel (ENaC) and Na⁺/H⁺ exchanger and is exported via basolateral efflux of Na⁺-K⁺-ATPase. Apical colonic H⁺-K⁺-ATPase works essentially for K⁺ reabsorption.

Some studies reported the physiological role of KCs, mostly to secretion (24, 38, 41, 42, 44, 48, 49, 55, 58, 60, 63, 66, 68–70, 72, 77, 82, 83, 89, 92, 94, 95, 105), while others explored their importance in pathological settings. The type and location of the KCs presented high variability among both animal and human IECs. Indeed, while some studies reported channels located in the small bowel (39, 47, 48, 90, 91, 97), others mentioned colonic channels (36, 41, 43, 47, 50, 53, 57–59, 61, 63, 64, 66, 68, 69, 71, 78, 81, 86, 88, 89, 92, 94, 96, 97). The membrane location within each segment was also variable amid KC types. Regarding the localization of KCs in IECs, the most common research tool used was immunolocalization. Nevertheless, there are no standardized guidelines guiding interpretation and reporting the validity of antibodies. A validated antibody must be specific, selective and reproducible. The stringent validation of antibodies includes evaluation by techniques like Western blot, immunohistochemistry, immunofluorescence, immunoprecipitation, flow cytometry, and enzyme-linked immunosorbent assays; however, no consensus exists regarding the gold standard, hampering the specificity and reproducibility. In our pool, immunohistochemistry (36, 50, 53, 57, 59, 63, 68, 80, 86, 88, 89, 91, 94) and Western blot (37, 39, 42, 52, 61, 62, 64, 66, 68, 78, 80, 81, 84, 85, 87, 88, 90, 92, 93, 97, 102, 104–106) were most commonly applied to determine the KC localization. The validation of antibodies was performed by confirming the results with other immunolocalization assay, immunohistochemistry, immunofluorescence, or immunoprecipitation, which allowed to demonstrate that the antibody was also able to specifically recognize its target when used for those applications (37, 47, 68, 85). In studies in which only one technique was performed for the KC immunolocalization, the validation of the results was evaluated using two intestinal epithelial cell lines (50, 84, 106) or control samples for in vivo models (36, 63, 78, 81, 99).

Calcium-activated KCs.

Calcium-activated KCs are widely expressed in mammalian cells and are divided into three types: 1) K_Ca1.1 (large-conductance or BK); 2) K_Ca3.1 (intermediate-conductance or IK); and 3) K_Ca2.1, 2.2, and 2.3 (small-conductance or SK1, SK2, and SK3) (Table 1). For example, in mouse small intestinal crypts, K_Ca3.1 channels were expressed selectively in Paneth cells and their pharmacological inhibition modulated cell secretory response (39). On the other hand, in human ileal mucosa, K_Ca3.1 channels was strongly expressed in Paneth cells (91). The mucosal biopsies from CD patients with NOD2 disease-predisposing mutations had lower levels of K_Ca3.1 mRNA expression than control samples (91). Bowley and colleagues (43) described that K_Ca3.1 channels were expressed in the basolateral membrane of human colonic crypt cells. These results were later confirmed in a study in which it was shown that basolateral K_Ca3.1 channels were distributed along the surface of crypt-axis and that their levels were decreased in samples from active UC patients (both in human IECs) (36). Likewise, in colonic mucosa of UC patients, K_Ca1.1 channels were located along the entire crypt axis (88). In turn, in normal colon, the K_Ca1.1 channels were expressed in the apical membrane of surface and upper crypt cells (88). In mouse colonic mucosa, the K_Ca1.1 channels are localize to the luminal membrane of crypts and are required for K⁺ secretion (89). Later, Singh et al. (92) demonstrate that K_Ca1.1 and K_Ca3.1 channels are present on apical membranes of rat distal colon. Hirota and McKay (61) demonstrate that, in colon crypts from dextran sulfate sodium (DSS)-treated mice, there was no significant difference in the expression of the K_Ca3.1 channel compared to wild-type samples. In turn, K_Ca3.1 channel activity was reduced in colonic epithelium from DSS-treated mice (61). These results were later confirmed in a study in which it was shown that K_Ca3.1 channels had unaltered protein expression in DSS-treated colon (66). In contrast, apical and basolateral mRNA levels were significantly increased compared with controls (66). Intestinal epithelial restitution might be regulated through the pharmacological modulation of specific KCs (74, 106). In this context, Lotz et al. (74) reported that KC inhibition, with K_Ca blockers (clotrimazole, charybdotoxin, and barium), accelerated wound healing, in intestinal epithelial monolayers of the human colon carcinoma cell line T84. In turn, the activation of K_Ca with EBIO, a KC agonist, had no effect on wound repair rate. These results were later confirmed in a study in which it was shown that clotrimazole inhibited K_Ca3.1 increasing wound healing response in HT-29 cells (106). K_Ca3.1 can also be inhibited by TRAM-34, a specific K_Ca3.1 blocker more effective in the basolateral membrane than in the apical membrane (42). The effect of acetylcholine on IECs, through the induction of basolateral K⁺ efflux in IECs, was downregulated by KC blockers (charybdotoxin plus apamin). On the other hand, in the presence of cholera toxin, acetylcholine enhanced the secretory response of T84 cells (40). A study on the inhibition of human KCs has shown that genetic silencing and pharmacologic K_Ca inhibitors (clotrimazole and paxilline) were effective in reducing and blocking amebic killing of IECs (75). Duan and colleagues (51) observed that afatinib increased the activity of KCs, which, in turn, results in diarrhea by modification of intestinal fluid transport. K_Ca blockers (clotrimazole and senicapoc) reversed this effect, through the inhibition of basolateral KCs.

Inwardly rectifying KCs.

K_ATP channels are inwardly rectifying ATP-sensitive channels composed by an inwardly rectifying K_ir6 subunit that matches with sulphonylurea receptors (SUR) (5). Nakamura et al. (80) reported that K_ir7.1 channels are expressed in epithelial cells of small intestine. In rat colonic epithelium, protein expression and mRNA of the K_ir6.1 and K_ir6.2 channels subunits (SUR1 and SUR2B) were observed in basolateral membrane (86). Daneshmand et al. (46) demonstrated that in TNBS-induced colitis, K_ATP channels are involved protective effects of lithium on macroscopic and histological features of rat colonic injury.

In addition, our analysis revealed that altered activity of K_ATP channels may contribute to the activation of effects of pattern recognition receptors (PRRs), specialized in detecting pathogenic components and initiating immune response, on the membrane potential of IECs (28). Activation of cytosolic PRRs, like Nod-like receptors (NOD), appeared to be responsible for hyperpolarizing human colon carcinoma cell line (HT-29), partially trough the opening of K_ATP channels.

Two-pore domain KCs.

Two-pore domain KCs (K_2P) justify the high K⁺ permeability of the cells at rest, with a crucial role in defining their resting membrane potential and excitability (68). Huang et al. (62) demonstrated that K_2P2.1 channels are expressed in IECs. On the other hand, K_2P9.1 channels are expressed in human colonic epithelium and appear to be involved in the K⁺ secretion (68).

Voltage-gated KCs.

Voltage-gated KCs (K_v) respond to variations of membrane voltage, opening in response to membrane depolarization to facilitate K⁺ efflux (16). These channels, which are expressed in mammalian IECs, have a pore-forming α-subunit that includes tetramers of identical, or different, subunits (6, 37, 48, 52, 69, 76, 81, 87, 90, 93, 102, 104). The K_v7.1 (also denominated KCNQ1) α-subunit can associate with various KCNE β-subunits forming heteromultimeric channel with KCNE3 (17, 107–109). In the small intestine and colon of mice, the KCNQ1 and KCNE3 are colocalized in the basolateral membrane of crypt cells, but the KCNE3 expression was highest in colon than in the small intestine (47).

Grunnet et al. (57) demonstrated that K_v1.3 channels are present in the basolateral membrane of the crypt cells. On the other hand, the K_v7.2 and K_v7.4 channels are localized in luminal membranes of surface and crypt cells and seems to be involved in the K⁺ secretion in apical membrane of colonic epithelial cells (63).

Ding et al. (50) and Ousingsawat et al. (81) reported that K_v10.1 is expressed in colorectal carcinoma samples and colorectal cancer cell lines (HT-29 and LoVo). Pillozzi and colleagues (84) demonstrated that colorectal cancer cells expressed K_v11.1 and K_Ca3.1 channels and the inhibition of K_v11.1 channel led to an upregulation of functional K_Ca3.1 channels.

Freeman et al. (52) reported that nonsteroidal anti-inflammatory drugs (NSAIDs) reduced the expression of functional K_v channels on the cell surface membrane of a rat epithelial cell line (IEC-6). A study with indomethacin and NS-398 (NSAIDs) confirmed these results; both drugs led to an inhibition of K_v expression in IEC-6 cells and small intestinal epithelium of rats (90).

DISCUSSION

KCs are ubiquitous ionic channels that selectively transport K⁺ across mammalian cellular membranes (28). These channels play a key role in intestinal transport and homeostasis but are also involved in pathologic conditions, such as IBD diarrhea (30). In this particular situation, the secretory mechanisms of diarrhea may be related with the upregulation of apical KCs, while the basolateral channels appear to be downregulated in IECs (36, 105). In this context, the study of the characteristics and pharmacological modulation of KCs, in IECs lines and/or in experimental animal models of CD and UC, has attracted the attention of researchers. Several KCs have been reported in IECs, but their characteristics are still underexplored (28, 30).

The recent discovery of KCs modulating drugs was translated into important developments for the characterization of these channels. KCs modulators are mostly organic small molecules, peptides toxins, and ions that may be classified as activators, inhibitors or blockers (31, 32) (Table 1). These molecules change the activity of KCs by blocking the ion-conducting pore or by binding to the voltage sensor domain or auxiliary subunits (31). Nevertheless, blockers are not specific for a particular type of KCs and can affect other KCs and other ion channels (31).

Calcium-Activated KCs

The K_Ca1.1 channels are expressed in the basolateral membrane of rabbit colonocytes (56, 59, 67, 98, 100). Still, in rat (38, 45, 59, 92, 95, 96) and mouse colonocytes (66, 89), K_Ca1.1 channels are expressed in the apical membrane, mainly in the crypts. A study with guinea pigs reported that K_Ca1.1 channels are present in the surface and crypt regions of colonic epithelial cells (105). Thus, apical K_Ca1.1 channels seem to be upregulated in IECs of animals exposed to a high K⁺ intake, which contributes to IBD-diarrhea (83). In human colonic epithelium, K_Ca1.1 channels are located only in the apical membrane of goblet cells and are responsible for K⁺ secretion (70). On the other hand, in human colonocytes, the K_Ca1.1 channels are located in the apical membrane of surface and upper crypt cells, whereas in UC patients these channels are extended along the entire crypt axis (88). These channels may be inhibited by a wide range of chemical modulators including quinine (45, 98), trifluoperazine (98), TEA (45), chromanol 293B (95), paxilline (105), clotrimazole and apamin (38), toxins [charybdotoxin (103, 105), iberiotoxin (38, 95, 96, 105), and LQV (98)], and ions [Ba²⁺ (45, 98), BaCl₂ (95)]] (Tables 2 and 3).

K_Ca3.1 channels are considered the dominant basolateral KCs in human colonocytes, being distributed along the surface-crypt axis (36, 43). In human ileal epithelium these channels are located in Paneth cells (91). They are believed to play a role in intestinal pathophysiology, yet their association with IBD is not consensual. Indeed, while some studies have shown that K_Ca3.1 channels are downregulated in UC and CD (9, 36, 91), others observed higher K_Ca3.1 mRNA levels in the intestinal epithelium of IBD patients (106). These differences may be related to disease activity; in fact, K_Ca3.1 levels may be increased to promote wound healing (106). On the other hand, the inhibition of these channels by clotrimazole led to an enhanced wound healing response after mechanical injury in IECs (74, 106). Therefore, it has been hypothesized that specific KCs modulation regulates migration in intestinal epithelial restitution in distinct ways, depending on whether they are under inflammatory or noninflammatory conditions (106). Other studies reported that the inhibition of K_Ca3.1 channels of Paneth cells modulates their secretory response, reducing the release of antimicrobial peptides in response to bacterial exposure (39). A study with DSS-treated mice reported that mRNA levels of K_Ca3.1 channels were increased in the apical and basolateral membrane of colonocytes, but protein expression was unaffected (66), even though in healthy rats, K_Ca3.1 channels can be found in both basolateral (isoform Kcnn4b, responsible for K⁺ secretion) and apical (isoform Kcnn4c, responsible for anion secretion) membranes (9, 41, 53). However, the inhibition of K_Ca3.1 channels with TRAM-34 (specific K_Ca3.1 blocker) was shown to be more effective in the basolateral membrane than in the apical membrane (42).

The small-conductance Ca²⁺-activated KCs or K_Ca2.3 channels are regulated by intracellular calcium levels and can be pharmacologically blocked by apamin (85). K_Ca2.2 channels transcripts were detected in rat colonocytes (64). Potier et al. (85) suggested that the isoform K_Ca2.3/SK3 is regulated by the adenomatous polyposis coli (Apc) gene (classified as a tumor suppressor gene), with implications in the migration of intestinal epithelial cells. These studies raise the hypothesis that the pharmacological modulation of these channels may be a potential target on the treatment of colon cancers.

Inwardly Rectifying KCs

The K_ir6.1-SUR2A pair has been described in the rat small intestine epithelium and is directly involved in the regulation of intestinal permeability (65). In rat colonic epithelium, K_ir6.1 and K_ir6.2 channels subunits (SUR1 and SUR2B) are expressed in basolateral membrane (86). Nevertheless, K_ATP expression and modulation, in mammalian IECs was addressed by few studies. A study with TNBS-treated mice showed that the K_ATP channels are involved in improving the macroscopic and histological characteristics of the colonic injury (46). The inhibition of K_ATP channels by glibenclamide led to a partially inhibition of the ameliorative effects on TNBS-induced colonic injury and the activation by cromakalim reversed them (46). Magalhaes et al. (28) studied the impact of the activation of pattern recognition receptors (specialized in detecting pathogenic components and initiating immune response) on KC activity. Disproportionate signaling through these receptors has been suggested as a trigger for watery stools in IBD. K_ATP channels seem to be mediators in this pathway (28).

Two-Pore Domain KCs

K_2P9.1channels are expressed in human colonic epithelium and appear to be involved in the K⁺ secretion of IECs (68). K_2P2.1 channel is expressed in IECs and its inhibition leads to epithelial barrier disruption (62). In this context, it has been suggested that K_2P2.1 is crucial for the maintenance of intestinal epithelial barrier functions; as a consequence, the reduction of K_2P2.1 activity promotes intestinal epithelium inflammation (62).

Voltage-Gated KCs

According to various authors, K_v1.1 channel is involved in the mechanism by which polyamines determine cell migration after epithelium lesions (93, 102, 104). In fact, polyamines stimulate K_v1.1 channel expression in IECs, inducing the hyperpolarization of membrane and the restitution of intestinal injury (93). Moreover, the modulation of K_v1.1 channel with 4-aminopyridine decreased its expression with reduction of cell migration in IEC-6 (a rat epithelial cell line) (102, 104). The inhibition of the K_v channel expression by NSAIDs presented an impact in epithelial cell migration and intestinal mucosal restitution (52, 90). NSAIDs are involved in signaling pathways that induced gastrointestinal toxicity (52) that, in turn, induce changes in intestinal microflora, depolarize membrane potential, and inhibit cell migration and epithelial restitution (90). Two studies focusing on K_v10.1 channel confirmed the expression of mRNA levels in human colonic epithelium and in colonic cancer cell lines (50, 81). Thus the inhibition of K_v10.1 might be a possible marker and a therapeutic target for colonic cancer. The K_v7.1 channel is responsible for K⁺ secretion and recycling. Thus this channel may play an important role in maintaining electrical driving force for electrolyte secretion in IECs (48, 69). K_v7.1 channel can be associated with KCNE subunit and, as so, the pharmacological properties of the channel may change. In addition, coassembly of K_v7.1 with KCNE3 subunit produces a current with nearly instantaneous activation in colonic crypts (37). In mouse colon and small intestine, K_v7.1 and KCNE3 are colocalized in the basolateral membrane of crypt cells. Still, KCNE3 expression was highest in colon than small intestine (47). In turn, K_v7.2 and K_v7.4 channels, localized in luminal membranes of surface and crypt cells, appear to be involved in the K⁺ secretion in apical membrane of colonic epithelial cells (63). A single study demonstrated the existence of a synergy between KCs. When K_v11.1 channels inhibition occurs, there is a positive regulation of functional K_Ca3.1 channels, leading to synergistic activity and the consequent proapoptotic effect of cisplatin, a platinum-based drug commonly employed for cancer treatment - in colorectal cancer cells (84).

This systematic review aimed to synthetize, for the first time, the evidence regarding the modulation of KCs in IECs. Considering the lack of validated scores to assess the methodological quality of basic studies, we developed a checklist for this purpose (Table 4). Data from animal studies were separated from that obtained in human IECs, to facilitate the comparison of the findings; the different KCs categories were also analyzed separately.

This review has some limitations: 1) several articles were not found in electronic search, despite the effort to optimize the search strategy. We believe this may be due to problems in indexing services which, in most journals and search engines, are based in keywords. Indeed, some of the studies not retrieved in the electronic search did not contain keywords [as is the case of Grotjohann et al. (55) and Simms et al. (91)] while the others contained keywords that were too generalist or too specific or even a single keyword. This highlights the requirement for an adequate abstract’s construction and keyword choice, using two to three words for each and, whenever possible, the medical subject headings (MeSH) and subheadings; 2) the nomenclature and functional characterization of KCs are diverse, creating difficulties while summarizing the main findings; 3) most of the oldest studies did not specify the type of KCs and the methodology used; and 4) few studies addressed the role of intestinal epithelial KCs, limiting the translation of the findings to preclinical research, particularly in the setting of IBD.

Even though the knowledge on the expression and modulation of intestinal KCs has mushroomed in recent years, the signaling pathways and intracellular interactions are still unknown. Overall, the available studies suggest a strong relationship between KC expression and physiologic secretory mechanisms, both in human and animal IECs. Concerning disease settings, in IBD animal models’ apical K_Ca1.1 channels were upregulated, while human basolateral K_Ca3.1 channels were downregulated in IECs. The pharmacological inhibition of K_2P and K_v was associated to inflammation.

Given the involvement of KCs in mechanisms of IBD diarrhea, it would be of high interest to modulate KCs in IEC lines or experimental IBD animal models. In experimental ulcerative colitis models, K_Ca1.1 channel blockers or inhibitors should be tested to assess reduction of colon inflammation. In turn, for the intestinal epithelial K_ATP and K_v channels, one should test the effects of their activation in IECs. These findings would be extremely useful for the development of effective KCs modulators, which could be potential therapeutic targets. However, further studies must be conducted to confirm and clarify these findings, allowing to take advantage of KCs modulation in the management of pathophysiological conditions.

GRANTS

This work was performed under the financial support of Foundation for Science and Technology (FCT-Portugal) COMPETE/FEDER Grants PTDC/DTP-FTO/0735/2014 and UID/BIM/4308/2016). D.C. acknowledges FCT, Portugal Grant SFRH/BD/120445/2016.

DISCLOSURES

F. Magro served as speaker and received honoraria from Merck Sharp & Dohme, Abbvie, Vifor, Falk, Laboratórios Vitoria, Ferring, Hospira, and Biogen. None of the other authors has any conflicts of interest, financial or otherwise, to disclose.

AUTHOR CONTRIBUTIONS

D.C. and F.M. conceived and designed research; D.C. performed experiments; D.C. and M.M.E. analyzed data; D.C., M.M.E., F.R., and F.M. interpreted results of experiments; D.C. prepared figures; D.C. and M.M.E. drafted manuscript; D.C., M.M.E., F.R., and F.M. edited and revised manuscript; D.C., M.M.E., F.R., and F.M. approved final version of manuscript.