Basally activated nonselective cation currents regulate the resting membrane potential in human and monkey colonic smooth muscle

Abstract

Resting membrane potential (RMP) plays an important role in determining the basal excitability of gastrointestinal smooth muscle. The RMP in colonic muscles is significantly less negative than the equilibrium potential of K⁺, suggesting that it is regulated not only by K⁺ conductances but by inward conductances such as Na⁺ and/or Ca²⁺. We investigated the contribution of nonselective cation channels (NSCC) to the RMP in human and monkey colonic smooth muscle cells (SMC) using voltage- and current-clamp techniques. Qualitative reverse transcriptase-polymerase chain reaction was performed to examine potential molecular candidates for these channels among the transient receptor potential (TRP) channel superfamily. Spontaneous transient inward currents and holding currents were recorded in human and monkey SMC. Replacement of extracellular Na⁺ with equimolar tetraethylammonium or Ca²⁺ with Mn²⁺ inhibited basally activated nonselective cation currents. Trivalent cations inhibited these channels. Under current clamp, replacement of extracellular Na⁺ with N-methyl-d-glucamine or addition of trivalent cations caused hyperpolarization. Three unitary conductances of NSCC were observed in human and monkey colonic SMC. Molecular candidates for basally active NSCC were TRPC1, C3, C4, C7, M2, M4, M6, M7, V1, and V2 in human and monkey SMC. Comparison of the biophysical properties of these TRP channels with basally active NSCC (bI_NSCC) suggests that TRPM4 and specific TRPC heteromultimer combinations may underlie the three single-channel conductances of bI_NSCC. In conclusion, these findings suggest that basally activated NSCC contribute to the RMP in human and monkey colonic SMC and therefore may play an important role in determining basal excitability of colonic smooth muscle.

in the gastrointestinal (gi) tract, coordinated transit of luminal contents is regulated by a range of factors released from excitatory and inhibitory motor neurons as well as rhythmical electrical activity generated by specialized pacemaker cells known as interstitial cells of Cajal (ICC) (5, 13, 18, 25, 33). Signals from enteric neurons and ICC are transmitted to smooth muscle cells (SMC), where they are integrated into the final acts of excitation and contraction. Thus the specific ionic conductances that regulate resting membrane potential (RMP) in SMC play a critical role in determining the responsiveness of SMC to extrinsic excitatory and inhibitory signals and the basal pattern of contractile activity (25).

RMP of SMC in the colon is significantly less negative than the equilibrium potential for K⁺ (E_K) ions (23), suggesting that an opposing influx of Na⁺ and/or Ca²⁺ ions occurs, possibly through nonselective cation channels (NSCC). With RMP regulated to be positive to E_K, colonic SMC are more excitable because they lie close to the potential range for activation of voltage-dependent Ca²⁺ channels (34). Small depolarizing stimuli such as inputs from excitatory motor neurons can accomplish excitation-contraction coupling, and small hyperpolarizing stimuli, such as input from inhibitory motor neurons, can lead to inhibition of contraction. The level of the membrane potential, therefore, is fundamental to colonic motility.

Surprisingly few studies have examined the ionic mechanisms responsible for the relatively depolarized RMP (e.g., −60 to −40 mV) in colonic and other GI muscles. In vascular muscles, such as rabbit pulmonary artery, small background NSCC currents were suggested to shift RMP positive from E_K (3). Current-clamp recordings identified a background NSCC in rabbit coronary artery SMC that is activated by lysophosphatidylcholine and contributes to RMP (26). Basally active NSCC have also been suggested to contribute to RMPs in rabbit ear artery and urinary bladder SMC (1, 2, 27).

Human colonic smooth muscles have RMPs of ∼−50 mV (23). Here we tested the hypothesis that NSCC conductances contribute significantly to RMP in SMC. We used voltage- and current-clamp techniques to characterize NSCC in colonic SMC, and RT-PCR was performed on human and monkey colonic smooth muscles and SMC to identify molecular candidates for basally active NSCC.

MATERIALS AND METHODS

Human tissue.

Human colonic specimens were obtained under the guidelines and approval of the Ethical Committee of Samsung Medical Center (Seoul, Korea). Patients of both sexes (24 male and 9 female) ranging in age from 43 to 76 yr underwent surgery mainly for colon cancer. Under anesthesia, diseased colonic tissue was removed along with a small region of healthy tissue (ascending 8, transverse 3, descending 11, sigmoid 12). This healthy tissue was placed in cold Krebs-Ringer buffer (KRB) of the following composition (in mM): 118.5 NaCl, 4.7 KCl, 2.5 CaCl₂, 1.2 MgCl₂, 23.8 NaHCO₃, 1.2 KH₂PO₄, and 11.0 dextrose.

Animal tissue.

Cynomolgus monkey tissues were obtained from Charles River Preclinical Services (Sparks, NV). The protocol for euthanizing monkeys was approved by the Institutional Animal Care and Use Committee (IACUC) and assures compliance with the United States Department of Agriculture (USDA), Public Health Service (PHS) Office of Laboratory Animal Welfare (OLAW) Policy and the Animal Welfare act (Charles River Laboratories, Preclinical Services, Sparks, NV). Monkeys of either sex (36 monkeys, 2.5–7 yr) were sedated with Ketamine (10 mg/kg) via intramuscular injection to the quadriceps. They were then given Beuthanasia-D solution containing pentobarbital sodium and phenytoin sodium (200 mg/kg) via intravenous therapy followed by exsanguination. The proximal colon was removed, placed in cold KRB, and transported to our laboratory.

Preparation of isolated colonic SMC.

Segments of human and monkey colon were opened and pinned to the base of a dissecting dish containing fresh KRB. The mucosa and submucosa were removed. Freshly dispersed colonic SMC were prepared from colonic muscle strips using Ca²⁺-free Hank's solution containing (in mM): 125 NaCl, 5.36 KCl, 15.5 NaOH, 0.336 Na₂HPO₄, 0.44 KH₂PO₄, 10 glucose, 2.9 sucrose, and 11 Hepes, adjusted to pH 7.4 with Tris. Pieces of muscle were incubated for 50–55 min at 37°C in a Ca²⁺-free solution (2 ml) containing collagenase (4 mg/ml) (Worthington Biochemical, Lakewood, NJ), trypsin inhibitor (8 mg/ml), fatty acid-free bovine serum albumin (8 mg/ml), papain (2 mg/ml), and l-dithiothreitol (0.3 mg/ml; Sigma-Aldrich, MO). Tissue pieces were washed with Ca²⁺-free solution and agitated to produce a cell suspension. Dispersed SMC were stored at 4°C. Drops of the cell suspensions were placed on the bottom of a 300-μl chamber mounted on an inverted microscope and allowed to adhere to the bottom for 5 min before recording.

Voltage-clamp and current-clamp methods.

Whole cell voltage-clamp techniques and single-channel recordings (inside-out configuration) were used to record membrane currents from dissociated SMC. Membrane currents were amplified by an Axopatch 200B (Axon Instruments, Foster City, CA) and digitized with an analog-to-digital converter (Digidata 1322, Axon Instruments). Data were collected at 5 kHz, filtered at 2 kHz via Bessel filter, and digitized online with pCLAMP software (version 9, Axon Instruments). The data were analyzed with the use of pCLAMP software (version 10, Axon Instruments). Pipette resistances were 1–2 MΩ for whole cell and 3–5 MΩ for single-channel recordings. Conventional and perforated whole cell (amphotericin B) patch-clamp techniques were used for recording ionic currents under voltage clamp. The RMP was measured using the current-clamp configuration (I = 0). Experiments were performed at room temperature.

Solutions and reagents.

In conventional dialyzed whole cell experiments, the internal solution contained (in mM): 135 KCl, 1.0 EGTA, 0.424 CaCl₂, 0.1 Na₂GTP, 3 MgATP, 10 glucose, 2.5 creatine phosphate disodium and 10 HEPES. Calculated free Ca²⁺ was ∼100 nM by Maxchelator software (Stanford University). The external solution was (in mM): 135 NaCl, 5 KCl, 2 CaCl₂, 1.2 MgCl₂, 10 glucose, 10 HEPES and adjusted to pH 7.4 with Tris (Ca physiological salt solution, CaPSS). In some experiments, extracellular Ca²⁺ was replaced with equimolar Mn²⁺ (MnPSS). In Na⁺ replacement experiments, Na⁺ was replaced with either equimolar tetraethylammonium (TEA) or with N-methyl-d-glucamine (NMDG) under voltage-clamp conditions. In some experiments SMC were perfused in BaPSS [Ca²⁺ was replaced with Ba²⁺ (2 mM)]. To isolate inward currents, the pipette solution contained (in mM): 135 CsCl, 1.0 EGTA, 0.424 CaCl₂, 0.1 Na₂GTP, 3 MgATP, 10 glucose, 2.5 creatine phosphate disodium, and 10 HEPES. This solution was adjusted to pH 7.2 with Tris. For low-Cl⁻ solution (30 mM), CsCl was replaced with equimolar cesium aspartate (E_Cl was −40 mV). The calculated junction potential was 7 mV. For perforated patches, amphotericin B (60 mg/ml) was dissolved in DMSO, sonicated, and diluted in the pipette solution to give a final concentration of 270 μg/ml. For inside-out single-channel recordings, the pipette solution contained (in mM): 140 CsCl, 1 EGTA, 0.96 CaCl₂, 10 HEPES. Bath solution contained (in mM): 30 CsCl, 110 Cs-aspartate, 1 EGTA, 0.96 CaCl₂, and 10 HEPES. Solutions were adjusted to pH 7.4 and 7.2 with Tris, respectively. Certain experiments were used with symmetrical Cl⁻ (140/140 mM). The effects of gadolinium (Gd³⁺), lanthanum (La³⁺), nickel (Ni²⁺), and nicardipine were tested by bath perfusion. For current-clamp recordings (I = 0), SMC were exposed to CaPSS, and the internal solution contained (in mM): 135 KCl, 1.0 EGTA, 0.424 CaCl₂, 0.1 Na₂GTP, 3 MgATP, 10 glucose, 2.5 creatine phosphate disodium, and 10 HEPES. In current-clamp (I = 0) recordings, external Na⁺ was reduced to 5 mM with the remaining 130 mM Na⁺ replaced with NMDG. All chemicals used were purchased from Sigma.

RNA isolation, RT-PCR.

Colonic tissue and SMC were used for molecular studies. SMC were collected by applying suction to the pipette, resulting in aspiration of the cells into the pipette. Total RNA was isolated from human and monkey colonic tissue and SMC using TRIzol reagent (GIBCO, Gaithersburg, MD) according to the manufacturer's instructions. Eluted total RNA was treated with DNase I to remove genomic DNA, which might be isolated along with total RNAs. First-strand cDNA was prepared from the total RNA with a Superscript Reverse Transcriptase kit (GIBCO). One microgram of total RNA was reverse transcribed with 200 U of reverse transcriptase in a 20-μl reaction containing 25 ng of oligo(dT) primer, dNTPs each at 500 μM, 75 mM KCl, 3 mM MgCl₂, 10 mM DTT, and 50 mM Tris·HCl (pH 8.3). All primers, β2 macroglobulin (B2M), transient receptor potential (TRP) channel members from subfamilies C, M, and V, were designed from a region spanning at least two exons in reference to cDNAs in the National Center for Biotechnology Information (NCBI) (see Table 1). Each gene cDNA was amplified by RT-PCR with gene-specific primers. RT-PCR was carried out with AmpliTaq polymerase (PE Biosystems, Foster City, CA) in a 2300 thermal cycler (PE Biosystems), and PCR fragments were analyzed by gel electrophoresis (2% agarose). All amplicons were at the expected cDNA size, which showed that they were clean from genomic DNA contamination. RT-PCR analysis was performed on human and monkey tissue, and SMC were collected from four different individuals.

Table 1.

Oligonucleotides used in the TRP channel study

Name	Sequences (5′-3′)	Tm	Size of cDNA, bp	Gene	Specificity
TRPC1-2	CTGTGGATTATTGGGATGATTTGGTCA	57	138/3611	TRPC1	M/H/MK
TRPC1-2r	CACTTTGAGGGCAAAGGTTGCCAA	57		TRPC1	M/H/MK
TRPC2-1	CTCCTCCTGGCTGGGCTTG	58	159/770	TRPC2	M/H
TRPC2-1r	GGTGAAGCTGAGCATGCTGGTG	59		Trpc2	M/H
TRPC3-2	CAGCATTCTCAATCAGCCAACACG	57	157/13058	TRPC3	M/H/MK
TRPC3-2r	AAGTTCATAACGAAGGCTGGAGATATC	57		TRPC3	M/H/MK
TRPC4-1	CTACCTGATAGCTCCCAAAAGCC	57	152/152	TRPC4	M/H/MK
TRPC4-1r	GACCTTGCCTGTTCAAGTCTGAC	57		TRPC4	M/H/MK
TRPC5-3	CATCTCCCTGGTAGTGCTGCTG	59	134/23393	TRPC5	M/H/MK
TRPC5-3r	CACCTTCATCAAAGTAACTCATCCAGAG	58		TRPC5	M/H/MK
TRPC5-1	GGATTTTGCAATGAACTCCCTCTACC	58	146/5557	TRPC5	M/H
TRPC5-1r	ATGTTGGATATTGCGAAGAGTGCTTC	57		TRPC5	M/H
TRPC6-2	GTGGTCCTTGCTGTTGCCATTG	57	175/9469	TRPC6	M/H/MK
TRPC6-2r	CTTCAAATCTGTCAGCTGCATTCATGAC	58		TRPC6	M/H/MK
TRPC7-2	CTCATAATGAGAATCAAGATGTGCCTC	57	136/1167	TRPC7	M/H/MK
TRPC7-2r	CAGAATTCCTCATGCCAGCCTG	57		TRPC7	M/H/MK
hTRPM1-1	CGTGGATTGAAGCTCTTCCTTAGC	57	185/23243	TRPM1	H/MK
hTRPM1-1r	CTTTCATTGATTTCTTCCAACCTCATTGAC	58		TRPM1	H/MK
hTRPM2-1	CCTGCAGCAAGATCCTGAAGGA	57	155/1574	TRPM2	H/MK
hTRPM2-1r	GAGCAGTTTCTGGGCTCTCTCTTC	59		TRPM2	H/MK
hTRPM3-1	GAAGTGTTTGCGGACCAGATAGACC	59	105/37825	TRPM3	H/MK
hTRPM3-1r	CACGATCCAAGCTCCTGTCTTGC	59		TRPM3	H/MK
hTRPM4-1	CTGGATGAGCTGCGTTTGGCTG	59		TRPM4	M/H/MK
hTRPM4-1r	GAACTCAGGCCGGTCATTCAGCA	59	141/1257	TRPM4	H/MK
TRPM5-1	CAACTGGAACAAGTGTGACATGGTG	58		TRPM5	M/H/MK
hTRPM5-1r	CAGCGTGAACACCATGAAGTCCATG	59	127/494	TRPM5	H/MK
TRPM6-1	GAATCAACATGCAGGTCCATATGTGAC	58	174/3159	TRPM6	M/H/MK
hTRPM6-1r	TGGCTCAAATACAATATCTCGAGCTAG	57		TRPM6	H/MK
hTRPM7-1	GGTATGTGCGTTTGCTAGATTTTCTAGC	58	180/5749	TRPM7	H/MK
hTRPM7-1r	GAGTCCAAGATGGTGCTTCATGAG	57		TRPM7	H/MK
TRPM8-1	AAGCTTCTGCTGGAGTGGAACCAG	59	126/4818	TRPM8	M/H/MK
TRPM8-1r	CTTGGGTCTGTCCTTTATGAGAGCC	59		TRPM8	M/H/MK
TRPV1-2	GAGAGCAAGAACATCTGGAAGCTGC	59	204/1502	TRPV1	M/H/MK
TRPV1-2r	GATGATGCCCACGTTGGTGTTCC	59		TRPV1	M/H/MK
hTRPV2-1	GATCTGGCTGGACTTCCAGAGTAC	59	110/2460	TRPV2	H/MK
TRPV2-1r	TTCAGCACAGCCTTCATCAGGCAC	59		TRPV2	M/H/MK
hTRPV3-1	GCTGAAGAAGCGCATCTTTGCAGC	59	135/910	TRPV3	H/Mk
TRPV3-1r	GTCAGCTTGTGCATGAGGAAGTCAG	59		TRPV3	M/H/MK
TRPV4-1	GTGCTGGAGATCCTGGTGTACAAC	59	136/2369	TRPV4	M/H/MK
TRPV4-1r	TAGGAGACCACGTTGATGTAGAAGG	58		TRPV4	M/H/MK
hTRPV5-1	CTGCACATCGCTGTTGTGAACCAG	59	159/466	TRPV5	H/MK
TRPV5-1r	CACACAGGCAGCAAAGGACAAAG	57		TRPV5	M/H/MK
TRPV6-1	TGGTGCTGGTGACCATGGTGATG	59	189/524	TRPV6	M/H/MK
TRPV6-1r	CCATCAGCCAGCAGAATCGCATC	59		TRPV6	M/H/MK
hB2M-1	CTCTTTCTGGCCTGGAGGCTATC	59		B2M	H/MK
hB2M-1r	GAAGTTGACTTACTGAAGAATGGAGAGCTCTCCATTCTTCAGTAAGTCAACTTC	57	152	B2M	H/MK

Reference cDNA sequences for each gene in the NCBI were used to design oligonucleotides.

TRP, transient receptor potential; M, mouse; H, human; MK, monkey.

Statistical analysis.

Data are reported as means ± SE; n is the number of cells tested. Statistical significance was evaluated by Student's t-test. P values <0.05 were considered significant.

RESULTS

Basally activated NSCC in human and monkey colonic SMC.

Whole cell voltage-clamp experiments were performed on single colonic SMC isolated from human and monkey colon smooth muscle under physiological extracellular (CaPSS) and intracellular K⁺-rich solution (E_Cl = 0 mV) (see materials and methods). We recorded basally activated inward currents that were composed of two types of inward currents. One was a net inward “holding current” (HC, see red dotted line, Fig. 1, A and Aa), which was the measured current between 0 pA and the red-dotted line as an average over 60 s of recording. The other type of inward currents was spontaneous transient inward currents (STICs, green arrow and asterisk, Fig. 1, A and Aa). Figure 1Aa illustrates how STICs were measured by taking an average of peak currents (green asterisk) over a period of 60 s from the red-dotted line (which represents 0 mV for measuring STIC currents). At a holding potential of −80 mV, the HC recorded from human colonic SMC was −46 ± 3 pA (n = 18, Fig. 1A). The amplitude of STICs ranged from −8 to −60 pA, with an average of −21 ± 2 pA (Fig. 1A). Similar STIC activity was recorded in monkey colonic SMC, where the average amplitude was −12 ± 2 pA (n = 19, Fig. 1B). The average HC was −30 ± 4 pA (Fig. 1B).

Fig. 1.

Spontaneous transient inward currents (STICs) recorded in colonic smooth muscle cell (SMC). A and B: under voltage clamp at a holding potential of −80 mV, ongoing inward currents were recorded in human and monkey colonic SMC. This activity was divided into two components: holding current (HC, red-dotted line, A and B) and STICs (green-dotted arrow and asterisk, A and B). Aa: HC that was the measured current (red arrow) between 0 pA and the red-dotted line as an average over a 60-s recording. STICs were measured by taking an average of peak currents (green asterisk, Aa) over a period of 60 s from the red-dotted line (which represents 0 mV for measuring STIC currents). HC and STICs were collectively termed basally activated nonselective cation currents or bI_NSCC. r denotes single-ramp depolarization from −80 to +80 mV. B: bI_NSCC were also recorded in monkey colonic SMC. Ca and Da: representative traces illustrating differences in HC and STIC activity at different potentials when E_Cl was set to −40 mV. Cb–Cc and Db–Dc: current-voltage (I-V) relationships showing average HC and STIC activity at different potentials and this activity reversed at ∼0 mV.

STICs normally refer to activation of Ca²⁺-activated Cl⁻ channels (4, 10, 20). Therefore, in the next experiments, internal Cl⁻ concentration was decreased to 30 mM in a Cs⁺-rich solution (see materials and methods), setting E_Cl to −40 mV. Cells were held from −80 mV to +80 mV for ∼2 min at tested potentials. In human colonic SMC, HC and STICs reversed at ∼0 mV (n = 5, Fig. 1, Ca–Cc). HC and STICs also reversed at ∼0 mV in monkey colonic SMC (n = 4, Fig. 1, Da–Dc). These data indicate that a Cl⁻ conductance does not contribute to basally activated inward currents in human and monkey colonic SMC. Therefore, we termed these basally activated inward currents as “basally activated nonselective cation currents” or bI_NSCC.

Na⁺ contributed to bI_NSCC and membrane potential.

Na⁺ is a major charge carrier in NSCC (9). In these experiments, Cs⁺-rich internal solutions were used to exclude contamination by K⁺ conductances (see materials and methods). External Na⁺ (135 mM) was replaced with equimolar TEA to test the effect of Na⁺ removal. HC was significantly reduced from −44 ± 5 to −16 ± 2 pA (n = 5, P < 0.001, Fig. 2, A and C), and STIC activity was also decreased from −22 ± 0.1 to −9 ± 0.2 pA at −80 mV (n = 5, P < 0.001, Fig. 2, A and D) in human colonic SMC. In monkey colonic SMC, HC was significantly reduced from −28 ± 8 to −11 ± 3 pA (n = 6, P < 0.05, Fig. 2, B and C), and STIC activity was decreased from −11 ± 0.1 to −4 ± 0.04 pA at −80 mV (n = 5, P < 0.001, Fig. 2, B and D). We also tested the effect of Na⁺ replacement with NMDG under voltage-clamp conditions. In human colonic SMC, HC was significantly reduced from −29 ± 3 to −14 ± 1 pA (n = 4, P < 0.05, Fig. 2, E and G), and STIC activity was also decreased from −13 ± 3 to −5 ± 1 pA at −80 mV (n = 4, P < 0.05, Fig. 2, E and H). In monkey colonic SMC, HC was also significantly reduced from −55 ± 8 to −25 ± 2 pA (n = 4, P < 0.05, Fig. 2, F and G), and STIC activity was decreased from −13 ± 2 pA to −5 ± 1 pA at −80 mV (n = 4, P < 0.05, Fig. 2, F and H).

Fig. 2.

Na⁺ contributed to bI_NSCC. A and B: under voltage clamp, replacement of external Na⁺ (135 mM) with equimolar tetraethylammonium (TEA) significantly reduced HC and STIC activity in human and monkey colonic SMC. C: summary of the effect of TEA on HC in colonic SMC (human, n = 5; monkey, n = 6). D: summary of the effect of TEA on STICs in colonic SMC (human, n = 5; monkey, n = 5). E and F: similar effect on HC and STIC was seen with replacement of external Na⁺ (135 mM) with equimolar N-methyl-d-glucamine (NMDG) in human and monkey colonic SMC. G: summary of the effect of NMDG on HC in colonic SMC (human, n = 4; monkey, n = 4). H: summary of the effect of NMDG on STICs in colonic SMC (human, n = 4; monkey, n = 4). *P < 0.05 and ***P < 0.001.

Current-clamp recordings (I = 0) were performed to explore the significance of bI_NSCC in regulating the RMP in colonic SMC. The pipette solution contained 140 mM K⁺ in a conventional dialyzed whole cell configuration (see materials and methods). Reduction in external Na⁺ from 135 mM to 5 mM (replaced with 130 mM NMDG) induced hyperpolarization in human SMC from −31 ± 6 mV to −49 ± 4 mV (n = 4, P < 0.05, Fig. 3A) and in monkey SMC from −34 ± 5 mV to −47 ± 5 mV (n = 4, P < 0.01, Fig. 3C). Transient hyperpolarization was recorded under control conditions in human and monkey SMC. To test the involvement of Ca²⁺-activated K⁺ conductance for transient hyperpolarization, we performed NMDG experiments in the presence of TEA (1 mM) (to block large-conductance Ca²⁺-activated K⁺ channels) and apamin (300 nM) (a small-conductance Ca²⁺-activated K⁺ channel blocker). TEA and apamin treatment gradually reduced the spontaneous transient hyperpolarization (Fig. 3B). In the continued presence of TEA and apamin, application of NMDG (130 mM) still induced significant hyperpolarization from −22 ± 4 mV to −44 mV ± 5 mV (n = 4, P < 0.001, Fig. 3B). Monkey colonic SMC also showed transient hyperpolarization, which was also gradually inhibited by pretreatment of TEA and apamin (Fig. 3D). Furthermore, replacement with NMDG induced significant hyperpolarization from −17 ± 3 mV to −33 ± 3 mV (n = 4, P < 0.001, Fig. 3D). These data suggest that a Na⁺-permeable conductance (bI_NSCC) contributes to RMP in colonic SMC.

Fig. 3.

Na⁺ contributed to membrane potential. A: in current clamp (I = 0), a reduction in external Na⁺ from 135 mM to 5 mM (130 mM NMDG) induced hyperpolarization in human colonic SMC (n = 4). B: pretreatment with TEA (1 mM) and apamin (300 nM) gradually decreased spontaneous transient hyperpolarization. Addition of NMDG in the presence of TEA and apamin also caused hyperpolarization. C and D: similar effect of Na⁺ replacement with NMDG was recorded in monkey colonic SMC in the absence (C) and presence (D) of TEA and apamin (n = 4).

The effects of NSCC blockers on bI_NSCC and membrane potential.

The trivalent cations, Gd³⁺ and La³⁺, are widely used as NSCC/TRP channel blockers (30). Therefore, we tested their effects on bI_NSCC. Under voltage-clamp conditions, La³⁺ (10 μM) significantly decreased the HC from −45 ± 7 pA to −20 ± 2 pA (n = 5, P < 0.001, Fig. 4, A and E) and STIC activity from −17 ± 0.2 pA to −6 ± 0.1 pA (n = 5, P < 0.001, Fig. 4, A and F) in human colonic SMC. Application of La³⁺ (10 μM) significantly inhibited bI_NSCC in monkey colonic SMC (HC: n = 4, P < 0.05, and STICs: n = 4, P < 0.001, Fig. 4, B, E, and F). Gd³⁺ (10 μM) also significantly decreased the HC from −44 ± 7 pA to 16 ± 5 pA (n = 4, P < 0.05, Fig. 4, C and E) and STICs from −24 ± 0.2 pA to −10 ± 0.2 pA (n = 4, P < 0.001, Fig. 4, C and F) in human colonic SMC. Similarly, Gd³⁺ inhibited both components of bI_NSCC in monkey colonic SMC (HC: n = 7, P < 0.05, and STICs: n = 6, P < 0.005, Fig. 4, D, E, and F). These inhibitory effects were not reversible after 20-min washout, as previously reported for NSCC in other cells (17, 24).

Fig. 4.

NSCC blockers inhibited bI_NSCC and influenced membrane potential. A and B: addition of the NSCC blocker, La³⁺ (10 μM), significantly decreased HC and STICs in human and monkey colonic SMC. C and D: Gd³⁺ also significantly decreased HC and STIC in human and monkey colonic SMC. E: summary of the effects of La³⁺ and Gd³⁺ on HC in colonic SMC (La³⁺ human, n = 5; La³⁺ monkey, n = 4; Gd³⁺ human, n = 4; Gd³⁺ monkey, n = 7). F: summary of the effect of La³⁺ and Gd³⁺ on STICs in colonic SMC (La³⁺ human, n = 5; La³⁺ monkey, n = 4; Gd³⁺ human, n = 4; Gd³⁺ monkey, n = 6). *P < 0.05 and ***P < 0.001. G: in current clamp (I = 0), application of the NSCC blocker, La³⁺ (10 μM), induced hyperpolarization in human colonic SMC (n = 4, P < 0.05). H: in monkey colonic SMC, Gd³⁺ (10 μM) induced membrane hyperpolarization (n = 4, P < 0.01).

From these data, we hypothesized that application of these NSCC blockers would significantly alter membrane potential as a result of inhibition of bI_NSCC. Under current-clamp conditions (I = 0), La³⁺ (1–10 μM) induced hyperpolarization in human SMC from −37 ± 6 mV to −48 ± 9 mV (n = 4, P < 0.05, Fig. 4G). In monkey colonic SMC, Gd³⁺ resulted in significant hyperpolarization from −21 ± 2 mV to −34 ± 5 mV (n = 4, P < 0.01, Fig. 4H). These data demonstrate that bI_NSCC are an important depolarizing influence in colonic SMC.

The effects of divalent cations and TRPM4 blocker on bI_NSCC.

Replacement of external Ca²⁺ (2 mM) with equimolar Mn²⁺ significantly decreased both components of bI_NSCC in human and monkey colonic SMC (Fig. 5, A–D). We also tested the effects of Mn²⁺ in the presence of Ca²⁺ (2 mM). Bath perfusion of Mn²⁺ (0.5–2 mM) inhibited STICs and HC (data not shown), suggesting that Mn²⁺ is a blocker of bI_NSCC. When external Ca²⁺ (2 mM) was replaced with equimolar Ba²⁺ (BaPSS, see materials and methods), STICs and HC in monkey colonic SMC were not affected (n = 4, Fig. 5E). We also tested whether Ca²⁺ influx via voltage-dependent Ca²⁺ channels affects bI_NSCC. Blockade of low-threshold voltage-activated Ca²⁺ channels with Ni²⁺ (50 μM) had no significant effect on HC or STICs in human SMC (data not shown). The L-type Ca²⁺ channel blocker, nicardipine, also had no effect on bI_NSCC in monkey colonic SMC (data not shown). These data suggest that neither T-type nor L-type Ca²⁺ channels are sources of Ca²⁺ that contribute to bI_NSCC activity.

Fig. 5.

The effects of divalent cations and transient receptor potential (TRP)M4 blocker on bI_NSCC. A and B: under voltage clamp, replacement of external Ca physiological salt solution (CaPSS) with MnPSS significantly reduced HC and STIC activity in human and monkey colonic SMC. C: summary of the effect of MnPSS on HC in human and monkey SMC (human, n = 4; monkey, n = 7). D: summary of the effect of MnPSS on STICs in human and monkey SMC (human, n = 4; monkey, n = 4). E: replacement of CaPSS with BaPSS had no effect on bI_NSCC in monkey SMC. F: representative trace illustrating that the TRPM4 blocker, 9-phenanthrol (100 μM), significantly reduced bI_NSCC in monkey colonic SMC (n = 4). *P < 0.05 and ***P < 0.001.

It is now well established that TRP channel proteins underlie many NSCC (29). One of the unitary conductances in human and monkey colonic SMC is ∼25 pS (see Figs. 6 and 7). This unitary conductance is similar to TRPM4 channels (23), which are also expressed in human and monkey colonic SMC (see Fig. 8). Thus we tested the effects of the hydroxytricyclic compound, 9-phenanthrol, a selective blocker of TRPM4 currents (15, 16). Application of 9-phenanthrol (100 μM) significantly reduced HC (from −49 ± 9 mV to −29 ± 9 mV, n = 4, P < 0.05) and STICs (from −15 ± 2 pA to −6 ± 1 pA, n = 4, P < 0.05) in monkey colonic SMC (Fig. 5F). These data suggest that TRPM4 currents may be important contributors to bI_NSCC and setting membrane potential in colonic SMC.

Fig. 6.

Single-channel conductances recorded in human colonic SMC. A–C: using the inside-out configuration with symmetrical Cs⁺ (140 mM, E_Cl = −40 mV), 3 conductances were recorded in human colonic SMC. A: at a holding potential of −20 mV, single-channel openings were recorded that were −0.8 pA in amplitude (see all-points amplitude histogram, middle). A conductance of 27 pS was calculated by plotting peak channel amplitude at different voltages and fit with linear regression (top, right) (n = 5 of 26 patches). B: at a holding potential of −60 mV, single-channel openings were recorded that had an amplitude of −1.4 pA (middle). The calculated single conductance was 7.5 pS (right) (n = 6 of 26 patches). C: small-conductance single-channel activity revealed noisy-like openings at −60 mV but no channel openings at 0 mV (right) (n = 25 of 26 patches). HP, holding potential.

Fig. 7.

Single-channel conductances recorded in monkey colonic SMC. A–C: using the inside-out configuration with symmetrical Cs⁺ (140 mM), 3 conductances were recorded in monkey colonic SMC. A: at a holding potential of −60 mV, single-channel openings were recorded that were −1.5 pA in amplitude (see all-points amplitude histogram, middle). A conductance of 25 pS was calculated by plotting peak channel amplitude at different voltages and fit with linear regression (top, right) (n = 12 of 68 patches). B: at a holding potential of −80 mV, single-channel openings were recorded that had an amplitude of −0.6 pA (middle). The calculated single conductance was 5 pS (right) (n = 19 of 68 patches). C: small-conductance single-channel activity revealed noisy-like openings. Bottom: expanded time scale from box in top (n = 65 of 68 patches).

Fig. 8.

TRP channels expressed in human and monkey colonic tissue and SMC. A and B: RT-PCR analysis was performed on human and monkey colonic tissue and SMC using TRP isoform specific primers for members of the TRPC, TRPM, and TRPV subfamilies. β-2-Macroglobulin (B2M) was used as a control. A and B, top: representative agarose gels illustrating TRP transcripts present in human and monkey colonic tissue, respectively (human tissue, n = 4; monkey tissue, n = 4). A and B, bottom: representative agarose gels of TRP transcripts expressed in human and monkey colonic SMC, respectively. In human colonic tissue TRPC1, -C3, -C4, -C6, -C7, -M2, -M3, -M4, -M6, -M7, -V1, -V2, -V3, -V4, and -V5 transcripts were detected, whereas in human colonic SMC, TRPC6, -M3, -V3, -V4, and -V5 were not expressed. An identical pattern of TRP transcript expression was found in monkey colonic SMC (human colonic SMC samples, n = 4; monkey colonic SMC samples, n = 4). (Note: other bands on gels are primer dimers.)

Single-channel conductances recorded in human and monkey colonic SMC.

Inside-out single-channel recordings were performed to identify single-channel conductances that may be responsible for bI_NSCC recorded at the whole cell level. Symmetrical Cs⁺ (140/140 mM, E_Cl = −40 mV) was used to amplify NSCC (because they are highly permeable to Cs⁺) and block contamination from K⁺ conductances. Figure 6A shows single-channel openings recorded at −20 mV in a human colonic SMC. The current-voltage (I-V) relationship was constructed using the all-points amplitude histogram (Fig. 6A, middle and right). The calculated single-channel conductance by linear regression was 27 ± 0.9 pS and reversed at ∼0 mV (n = 5 of 26 patches, Fig. 6A, right). In addition, a 7.5 ± 0.5 pS channel (n = 6 of 26 patches) was recorded at −60 mV (Fig. 6B) as well as noisy small-conductance NSCC channels (n = 25 of 26 patches, Fig. 6C). Because the small-conductance NSCC did not exhibit clear channel openings at −60 mV, an amplitude histogram could not be constructed. No channel openings were found at 0 mV (Fig. 6C, right).

Single channels with conductances similar to those in human SMC were recorded in monkey colonic SMC. Figure 7A demonstrates single-channel openings recorded at −60 mV (−1.5 ± 0.2 pA at −60 mV), which had a slope conductance of 25 ± 0.8 pS (n = 12 of 68 patches). We also recorded 5 ± 0.3 pS channels (n = 19 of 68 patches, Fig. 7B) and small-conductance NSCC channels with indiscrete channel openings (n = 65 of 68 patches, Fig. 7C).

TRP channels expressed in human and monkey colonic tissue and SMC.

We investigated transcriptional expression of TRPC, M and V subfamily members in human and monkey colonic tissue. In Fig. 8, A and B, representative gels show TRP-subfamily member transcripts present from homogenates of muscularis tissues (human samples, n = 4; monkey samples, n = 4). TRP family members found at the tissue level in preliminary experiments (data not shown) were probed and included in representative gels in Fig. 8, A and B (top). Because transcripts at the tissue level could include genes expressed in a variety of cell types (e.g., neurons, ICC, etc), we also examined transcript expression in SMC collected by micropipette (∼200 cells) following enzyme dispersion (see materials and methods). Kit (ICC marker) and PGP9.5 (neuron marker) transcripts were not detected in these cells. In isolated human colonic SMC, TRPC1, C3, C4, C7, V1, V2, M2, M4, M6, and M7 were resolved (bottom, n = 4, Fig. 8A), and the same compliment of TRP channels was expressed in monkey SMC (bottom, n = 4, Fig. 8B). These data indicate that expression of specific TRPC, TRPV, and TRPM subfamily members in SMC is a subset of the total TRP channel expression in the whole muscularis.

DISCUSSION

RMP plays a critical role in setting the basal excitability of GI smooth muscles. The RMP of colonic muscles in humans and nonhuman primates is regulated to be within the range of membrane potentials in which the open probability of voltage-dependent Ca²⁺ is regulated dynamically (32). Thus small changes in membrane potential can alter Ca²⁺ entry and affect SMC contraction dramatically. Responses to all other regulatory mechanisms (e.g., ICC pacemaker activity and SMC responses to inflammatory mediators) are superimposed upon this basic, intrinsic property of GI SMC; thus understanding regulation of RMP and the open probability of basally active NSCC is fundamental to understanding colonic motility and the changes in motility that might occur in disease, pregnancy, and aging. In the present study we have shown that NSCC, possibly encoded by specific TRP channel genes, are expressed by human and nonhuman primates.

Studies of vascular smooth muscles and a few visceral muscles have suggested that the relatively positive potentials of SMCs may be related to the balance between basal activation of K⁺ channels and inward conductances (2, 26). Previous reports have suggested that basally activated NSCC are involved in regulating the RMP in some SMC (1, 2, 26, 27). In the present study, basally activated inward currents reversed at 0 mV in human and monkey SMC (E_Cl- = −40 mV), suggesting that these currents were nonselective cation conductances (bI_NSCC). bI_NSCC were composed of two major types of current: 1) HC, which was a tonic inward current observed at negative holding potentials; 2) STICs, which were transient in nature, but summation of STICs, occurring independently in myriad SMCs within a physiological muscle, could yield a tonic depolarizing influence on the tissue. In voltage-clamp experiments, replacement of external Na⁺ with equimolar TEA or NMDG significantly reduced STICs and HC, suggesting that Na⁺ is a major charge carrier for bI_NSCC. Thus bI_NSCC raises the net permeability of colonic SMC to Na⁺ ions, resulting in ongoing Na⁺ entry. In current-clamp (I = 0), decreasing the influx of Na⁺ through bI_NSCC caused significant hyperpolarization of the RMP. Taken together, bI_NSCC contributes significantly to the regulation of RMP in human and monkey SMC. In addition to the depolarizing influence of constant Na⁺ entry, this ionic flux would necessitate ongoing Na⁺/K⁺ ATPase activity and energy expenditure to continuously maintain the Na⁺ gradient that powers NSCC. Na⁺/K⁺ ATPase has been shown to be independently electrogenic in a variety of smooth muscles, (6, 19); thus this is a secondary regulatory mechanism of RMP in GI SMC that might depend upon bI_NSCC.

Replacement of Ca²⁺ with equimolar Mn²⁺ inhibited bI_NSCC, suggesting that Ca²⁺ could be a charge carrier contributing to bI_NSCC. However, additional studies showed that Mn²⁺ added in the presence of CaPSS also inhibited bI_NSCC, suggesting that Mn²⁺ blocks bI_NSCC. Further experiments demonstrated that Ni²⁺ (low-voltage-activated Ca²⁺ channel blocker), nicardipine (high-voltage-activated Ca²⁺ channel blocker), and Ba²⁺ replacement did not affect the magnitude of bI_NSCC. These data, together with the effects of Na⁺ replacement, which totally blocked bI_NSCC, suggest that Ca²⁺ is only a minor charge carrier in bI_NSCC. Furthermore, we found that Gd³⁺ and La³⁺, which are used to block a variety of NSCC in SMC (24, 26), decreased HC and STICs and induced hyperpolarization. These data support the view that bI_NSCC play an important role in setting RMP to levels significantly less negative than E_K.

TRP channel proteins are widely accepted as molecular candidates for NSCC (31). Several TRP channels have been proposed to play important roles in the regulation of RMP in certain cell types (11, 12). In the present study, analysis of human and monkey colonic SMC revealed TRPC1, C3, C4, C7, M2, M4, M6, M7, V1, and V2 transcript expression. Single-channel experiments revealed three different unitary conductances in human and monkey colonic SMC. Molecular analysis found that SMC from human and monkey expressed identical TRP channels (from the TRPC, M and V subfamilies). An important characteristic that the TRP candidates for these three unitary conductances must exhibit is constitutive activity. Among the TRP channels expressed in human and monkey SMC, TRPC3, C7, M4, M6, M7, and V2 (2, 7, 14, 22, 28) have been reported to exhibit basal activity.

In human colonic SMC, 27-pS, 7.5-pS, and “small” channels were recorded, and, interestingly, near identical conductances were recorded in monkey colonic SMC (25 pS, 5 pS, and small). The reported unitary conductances for TRPM2, C3, M6, M7, and V2 all exceed 40 pS (9). Therefore, on the basis of the two important parameters, single-channel conductance (≤27 pS) and constitutive activity, only TRPM4 and TRPC7 channels satisfy these two criteria. In the present study we found that 1) replacement of Na⁺ from the external solution inhibited bI_NSCC and caused hyperpolarization, 2) Ca²⁺ replacement with Ba²⁺ did not affect bI_NSCC activity, 3) 9-phenanthrol blocked HC and STICs in bI_NSCC of monkey colonic SMC, and 4) one of the single-channel conductances from both species was ∼25 pS. These properties are very similar to those exhibited by TRPM4 including Na⁺ permeability and divalent impermeability (15, 21), 9-phenanthrol sensitivity (15, 16), and unitary conductance (21). Thus these data suggest that TRPM4 is a major component of bI_NSCC.

Molecular identification of the two smaller conductances (7.5 pS and small in human; 5 pS and small in monkey) is harder to determine. If the TRP channels expressed in colonic SMC are homomultimers, the smallest conductance expected to be recorded is ∼16 pS through TRPC1 (9). However, specific TRP subunits can form heteromeric channels (8) and can have smaller conductances than their homomeric counterparts (i.e., ≤7.5 pS). Unfortunately few studies have determined the conductances of different combinations of TRP heteromultimers. Future studies will need to involve coimmunoprecipitation analysis of the specific TRP subunits present in colonic SMC.

In summary, our findings indicate that bI_NSCC play an important role in the maintenance of the RMP in human and monkey SMC. We have also identified that human and monkey SMC express identical TRPC, M and V channels. In particular, TRPM4 and specific TRPC heteromultimer combination(s) could be molecular candidates for bI_NSCC activity in colonic SMC.

GRANTS

This work was supported by DK 41315 NIH/NIDDK.

DISCLOSURES

No conflicts of interest, financial or otherwise, are declared by the authors.