Interactions of nanomaterials with ion channels and related mechanisms

Abstract

The pharmacological potential of nanotechnology, especially in drug delivery and bioengineering, has developed rapidly in recent decades. Ion channels, which are easily targeted by external agents, such as nanomaterials (NMs) and synthetic drugs, due to their unique structures, have attracted increasing attention in the fields of nanotechnology and pharmacology for the treatment of ion channel‐related diseases. NMs have significant effects on ion channels, and these effects are manifested in many ways, including changes in ion currents, kinetic characteristics and channel distribution. Subsequently, intracellular ion homeostasis, signalling pathways, and intracellular ion stores are affected, leading to the initiation of a range of biological processes. However, the effect of the interactions of NMs with ion channels is an interesting topic that remains obscure. In this review, we have summarized the recent research progress on the direct and indirect interactions between NMs and ion channels and discussed the related molecular mechanisms, which are crucial to the further development of ion channel‐related nanotechnological applications.

Abbreviations

[Ca²⁺]_i

intracellular calcium concentration

AP

action potential

Au NPs

gold nanoparticles

BK channel

big‐conductance K⁺ channel

c‐MWCNTs

carboxylated multi‐walled carbon nanotubes

ER

endoplasmic reticulum

G

conductivity

G_max

maximal conductance

I_K

delayed rectifier K⁺ current

I_K1

inward rectifier K⁺ current

IP₃

inositol 1,4,5‐triphosphate

I_to

transient outward K⁺ current

k

slope factor

MCU

mitochondrial calcium uniporter

MWCNTs

multi‐walled carbon nanotubes

NMs

nanomaterials

QDs

quantum dots

RP

resting potential

RyRs

ryanodine receptors

SF

selectivity filter

SiO₂ NPs

silicon dioxide nanoparticles

SR

sarcoplasmic reticulum

STIM1

stromal interaction molecule 1

SWCNTs

single‐walled carbon nanotubes

V_1/2

half‐maximum activation membrane potential

V_m

testing membrane potential

VSD

voltage sensor domain

1. INTRODUCTION

With the rapid development of nanotechnology, the biological effects of nanomaterials (NMs), ranging in size from 1–100 nm, size range have attracted substantial attention (Ou et al., 2016; L. Wang, Hu, & Shao, 2017). Because of their high surface energy, volume effects, and quantum size effects, NMs have prospective applications in biomedical fields, including biomedical imaging (Rosado‐de‐Castro, Morales, Pimentel‐Coelho, Mendez‐Otero, & Herranz, 2018), medical diagnosis (Sharifi et al., 2019), sterilization (L. Wang, Hu, & Shao, 2017), and drug delivery (Y. Wang, Zhang, Wang, & Liang, 2018). However, NMs have some disadvantages, and the potential hazards of NMs are an important issue that warrants investigation. Some studies have reported that NMs can penetrate various defence barriers in vivo (Beloqui, des Rieux, & Preat, 2016; Furtado et al., 2018; Hawkins et al., 2018; L. Wu, Shan, Zhang, & Huang, 2018) and induce cellular and molecular alterations and physiological changes in tissues and organs, which could subsequently induce a variety of pathological changes, including pneumoconiosis, metal fume fever (Monse et al., 2018) and neurological disorders. During these interactions, abnormalities in the electrophysiological properties regulated by ion channels are important factors (Hille, 1986; Zaydman, Silva, & Cui, 2012).

Ion channels embedded in the plasma membrane and organelle membranes are essential for regulating cellular physiological functions and maintaining cellular homeostasis. Because of their distinctive molecular architecture and accessible location, ion channels are potential targets for extracellular stimulants, including synthetic drugs and some potentially hazardous materials, such as NMs. In recent years, many studies have reported that NMs can interact with ion channels (Kang et al., 2016; Lin et al., 2017; Park, Chhowalla, Iqbal, & Sesti, 2003) and alter their currents, channel kinetics, subcellular localization, and even the expression levels of ion channel‐related RNA and proteins. However, the detailed mechanisms remain unclear, and more comprehensive information is needed. In this review, we summarize the structural and functional changes in ion channels after treatment with NMs and discuss the detailed mechanisms underlying the direct and indirect interactions of NMs with ion channels (Figure 1). We have also summarized induced ion homeostasis alterations and biological effects (Figures 3 and 4). We believe that our review provides insights regarding the effects of the interactions between NMs and ion channels and provides new insights into NMs and their role in preventing and treating ion channel‐related cellular dysfunctions and diseases.

Figure 1.

Diagram of direct and indirect interactions of nanomaterials (NMs) with ion channels. (a) Many NMs such as C₆₀ form stable contacts with the domains (Chhowalla et al., 2005; Gonzalez‐Durruthy et al., 2017; Park et al., 2003) of ion channels in many directions by (A) directly blocking the channel pore from extracellular sites, (B) binding to hydrophobic residues in extracellular and intracellular sites or (C) penetrating into the lipid bilayer of the cell membrane, and (D) causing the rotation of functional areas, such as voltage sensor domains (VSDs). The structure and function of ion channels are affected by these interactions (Gu et al., 2018), leading to partial or complete block of current (Calvaresi, Furini, Domene, Bottoni, & Zerbetto, 2015; Kraszewski, Tarek, Treptow, & Ramseyer, 2010). Copyright 2015 American Chemical Society. (b) The size, charge, and surface modifications of NMs affect their resultant binding to ion channels. (A) Small‐diameter nanotubes and C₆₀ showed more efficient blockage because they had an appropriate size to fit into channel pores (Calvaresi et al., 2015). NMs with (B) specific charges (Q. Wang, Sun, Zhang, & Duan, 2015), (C) hydrophobic groups, and (D) surface modifications (Hilder & Chung, 2013) showed affinity for specific types of ion channels. (c) NMs led to abnormal changes in cell membrane structure and function, which could indirectly affect embedded ion channels. (A) ZnO nanoparticles (NPs) interacted with the hERG channel gating process by changing the lipid composition (Piscopo & Brown, 2018). (B) Carbon nanotubes penetrated the platelet plasma membrane, caused injury to the calcium store membrane, and subsequently activated store‐operated calcium entry (SOCE), causing a Ca²⁺ influx (Lacerda et al., 2011). (C) Graphene (Perreault, de Faria, & Elimelech, 2015) could directly puncture the cell membrane, leading to the extraction of phospholipids from the lipid bilayer, and graphene sheets adhering to the cell surface affect ion channels located on the cell membrane. (D) Some hydrophobic NMs, such as C₆₀ (Russ et al., 2016), can diffuse into membranes and cause membrane deformation, potentially impeding normal conformational changes in ion channels. (d) Metal cations released from NMs could disrupt the function of ion channels. (A) The neuronal Ca_V2.2 channel was inhibited by yttrium ions (Y³⁺) released from carbon nanotubes (Jakubek et al., 2009). (B) Ion‐shedding NMs, such as ZnO NPs (Jia et al., 2017), entered an acidic environment, such as the lysosome, to release a large number of ions into the lysosomes and cytoplasm, and then modulated the functions of various ion channels located in the lysosomal and cell membranes (Y. X. Liu & Wang, 2013; Peralta & Huidobro‐Toro, 2016). (C) Metal ions could bind to the channel surface or to the pore, thereby affecting the VSDs or changing the gating (Elinder & Arhem, 2003)

Figure 3.

The effects of nanomaterials (NMs) on K⁺/Na⁺ channels and subsequent dysfunction of ion homeostasis. NMs, such as MoS₂ nanoparticles (NPs; Gu et al., 2018), multi‐walled carbon nanotubes (Xu et al., 2009), ZnO NPs (Piscopo & Brown, 2018), Ag NPs (Lin et al., 2017), and Au NPs (Chin, 2014), could influence various types of K⁺ channels located on the cell membrane, including voltage‐gated K⁺ channels, calcium‐activated K⁺ channels, and inward rectifier K⁺ channels. NMs exposure altered several K⁺ currents, such as I _to, I _K, and I _K1. Crucially, the effects of NMs on K⁺ channels could lead to a decreased intracellular K⁺ concentration ([K⁺]_i) and the following subsequent biological effects: autophagy, inflammation, and apoptosis. For example, decreased [K⁺]_i could cause phospholipid phosphorylation, thereby activating the AMPK pathway and initiating autophagy. A decrease in [K⁺]_i could activate an inflammatory process by accelerating inflammasome formation. NMs‐induced mitochondrial Ca²⁺ overload could be decreased after the influx of K⁺ via the activation of mitochondrial K_Ca channels, leading to a lower redox state of the NAD system; thus, intracellular oxidative stress was down‐regulated (Kulawiak, Kudin, Szewczyk, & Kunz, 2008). In addition, an NMs‐induced decrease in [K⁺]_i could increase cytochrome c release and caspase activation and then initiate apoptosis. Conversely, an NMs‐induced increase in [K⁺]_i could inhibit apoptosis via the same pathway. Additionally, nano red elemental selenium (Yuan, Lin, & Lan, 2006) and Ag NPs (Z. Liu, Ren, Zhang, & Yang, 2009) have been reported to affect Na⁺ channels and their corresponding currents, but studies on the subsequent biological effects induced via Na⁺ homeostasis dysfunction are limited. I _K, delayed rectifier K⁺ current; I _K1, inward rectifier K⁺ current; I _to, transient outward K⁺ current; K_Ca channel, calcium‐activated potassium channel

Figure 4.

The effects of nanomaterials (NMs) on Ca²⁺ channels located on the cell membrane and Ca²⁺ stores and subsequent dysfunction of ion homeostasis. A [Ca²⁺]_i increase was one of the crucial effects of NMs exposure. NMs, such as Au NMs (Fusi, Sgaragli, & Valoti, 2018) and CdSeZnS QDs (Gosso et al., 2011), prompted the opening of Ca²⁺ channels or decreased the activity of Ca²⁺‐ATPase (Cao et al., 2013), allowing increased Ca²⁺ influx and decreased Ca²⁺ flux. In addition, NMs (Tang, Wang, et al., 2008) could be internalized into cells and damage the Ca²⁺ store (ER and mitochondria), allowing Ca²⁺ to flow into the cytoplasm. Furthermore [Ca²⁺]_i, which is a key second messenger, regulates many cellular processes, including oxidative stress, inflammation, apoptosis, and autophagy. For example, an increased [Ca²⁺]_i could affect the tricarboxylic acid cycle and electron transport chain (ETC) of the mitochondria, leading to oxidative stress initiation. An increase in [Ca²⁺]_i could also activate an inflammatory process by accelerating inflammasome formation. Ca²⁺ overload could result in apoptosis through the activation of mitochondria‐mediated intrinsic apoptotic pathways or the calpain–caspase pathway. Calpain also participates in the Ca²⁺‐induced autophagy process. An increased [Ca²⁺]_i could also directly activate the calcium/calmodulin‐dependent protein kinase II (CAMKII)–AMPK pathway to induce autophagy. The up‐regulation of PKC phosphorylation under an increased [Ca²⁺]_i status also led to increased LC3‐I conversion to LC3‐II, suggesting autophagosome formation. However, [Ca²⁺]_i could decrease autophagic activity in some situations by mTOR activation. ER, endoplasmic reticulum; IP₃R, inositol 1,4,5‐trisphosphate receptor; MCU, mitochondrial calcium uniporter; NCX, Na⁺/Ca²⁺ exchanger; PEG, polyethylene glycol; PMCA, plasma membrane Ca²⁺ ATPase; RyR, ryanodine receptor; STIM1, stromal interaction molecule 1; TRPC, transient receptor potential canonical; TRPM, transient receptor potential mucolipin; TRPV, transient receptor potential vanilloid

2. CLASSIFICATIONS, STRUCTURES, AND BIOLOGICAL ROLES OF ION CHANNELS

According to their ion selectivity, ion channels can be divided into several families, including potassium (K⁺), sodium (Na⁺), calcium (Ca²⁺), and chloride (Cl⁻) channels. Ion channels can also be classified based on different gating mechanisms that respond to chemical or electrical signals, temperature, or mechanical forces, and the categories include voltage‐gated, ligand‐gated, and other gated channels (Figure 2a).

Figure 2.

Ion channel classification, detection, components, and kinetics. (a) Ion channels classified on the basis of different gating mechanisms, such as (A) voltage‐gated channels, (B) ligand‐gated channels, and (C) other gated ion channels, including mechanical‐, light‐, and temperature‐gated channels. (b) A diagram of the whole‐cell patch clamp experiment. (c) In addition to the cavity through which ions flow, other main functional domains form and functionalize ion channels, mainly including the selectivity filter (SF; PDB 1BL8), voltage sensor domain (VSD; PDB 5EK0), and ligand‐binding domain (LBD; PDB 1LNQ). (d) Various types of curves were fitted to describe the ion channel kinetics (e.g., a study conducted by Lin et al., 2017). (A) Ag nanoparticles (NPs) shifted the steady‐state activation curve to the right and decreased half‐maximum activation membrane potential (V _1/2) and the slope value k, suggesting that I _K1 currents were decreased and the activation of I _K1 channels was delayed. (B) Ag NPs shifted the inactivation curve of I _Na to the left and increased V _1/2 and the slope value k, suggesting that the inactivation of Na⁺ channels occurred at relatively negative membrane potentials and that inactivation was accelerated. (C) Ag NPs shifted the recovery curve of I _Na to the right and increased the recovery time constant (τ) value, suggesting a delayed recovery of Na⁺ channels from the inactivation state. I, current value; I _max, maximal current value; V _m, testing membrane potential. Copyright 2015 Taylor & Francis Group

An ion channel is a hydrophilic channel surrounded by pore‐forming and non‐pore‐forming subunits on the plasma membrane or organelle membrane. These ordered arrayed proteins form a tunnel that allows ions to undergo transmembrane migration. In addition to the tunnel, ion channels also consist of several functional domains, such as selectivity filters (SFs), voltage sensor domains (VSDs), and ligand‐binding domains (Figure 2c). These domains participate in regulating ion channels in response to a broad range of stimuli. SFs are formed by several specific sequences of amino acids and are present in nearly all ion channels. The coordination between amino acids and ions within SFs gives the ion channels unique ion selectivity. VSDs are widely present in voltage‐gated ion channels and possess several transmembrane segments with positive or negative charges to respond to membrane potential changes near the channel. Thus, changes in the membrane potential can alter the conformational states and regulate the opening, closing, and inactivation of the channels. Similarly, ligands and ions can selectively bind to the ligand‐binding domains of ligand‐gated ion channels from the intracellular or extracellular side of cells, regulating the opening and closing of the channels. Therefore, these functional areas are of crucial importance, and their interactions with NMs can lead to ion channel dysfunction.

Ion channels play a key role in regulating the excitability of excitable cells and mainly function to establish the cellular resting potential (RP) and shape the action potentials (APs) that regulate cell excitability and conductance. Additionally, by mediating the concentration of intracellular secondary messengers, such as Ca²⁺, ion channels can trigger the following series of physiological effects: muscle contraction, neuronal synaptic transmissions, and normal cell volume regulation.

Currently, investigations of ion channels often involve biophysical, electrophysiological, and pharmacological approaches using patch clamp, immunohistochemistry, X‐ray crystallography, fluoroscopy, and RT‐PCR techniques. The patch clamp technique is mainly used to detect the electrical activity of ion channels (Figure 2b). This technique can be used to analyse current–voltage relationships and the kinetics of activation, inactivation, recovery, and other functions. The detection of changes in the opened–closed and activation–inactivation statuses of ion channels is valuable for revealing functional changes in the characteristics of ion channels. For example, activation, inactivation, and de‐inactivation curves (also named recovery curves) are used to describe the kinetics of ion channels. Activation curves indicate the speed and difficulty of channel opening, inactivation curves indicate the degree of voltage‐gated ion channel inactivation, and de‐inactivation curves indicate the duration of recovery after ion channel inactivation. According to the impulse voltage values (V _m) and measured current values (I), the conductivity (G) under various V _m and the maximal conductance (G _max) can be obtained. These indicators are used to establish steady‐state activation and inactivation curves, and recovery intervals (t) are plotted on the horizontal axis to establish de‐inactivation curves. These curves are mainly fitted by the following equations:

• For activation curves: G/G _max = 1 − {1 + exp[(V _m − V _1/2)/k]}⁻¹;

• for inactivation curves: I/I _max = {1 + exp[(V _m − V _1/2)/k]}⁻¹; and

• for de‐inactivation curves: I/I _max = 1 − exp(−t/τ).

On the basis of the fitted curves, the half‐maximum activation membrane potential (V _1/2), slope factor (k), and time constant (τ) are obtained. These values can be used to describe kinetic changes in ion channels after exposure to NMs. For example, the k in activation curves indicates the activation speed of ion channels, and higher k values indicate a slower activation speed. The curve shifts and the V _1/2 are also useful for evaluating the effects of NMs exposure on ion channels. More detailed analytical procedures for the above‐mentioned curves are shown in Figure 2d.

3. THE EFFECTS OF INTERACTIONS BETWEEN NMs AND ION CHANNELS

The cell membrane, which contains a broad distribution of ion channels, is a cellular structure targeted by NMs. Thus, direct and/or indirect interactions between NMs and ion channels are inevitable. When NMs contact ion channels, they block the ion channels or alter ion channel kinetics, thereby affecting intracellular ion homeostasis and regulating cellular physiological activity.

3.1. NM‐induced effects on K⁺ and Na⁺ channels

K⁺ and Na⁺ channels are widely found in all living cells and are especially important in excitable cells, such as cardiomyocytes and neuronal cells. In these cells, K⁺ and Na⁺ channels coordinately control the flow of K⁺ and Na⁺, which are the basis of action potential (AP) depolarization, repolarization, and RP resetting. For example, AP initiation is caused by a voltage‐dependent inward Na⁺ current, while AP termination depends on the activation of voltage‐dependent K⁺ currents. Studies focused on NM‐induced biological effects on K⁺ and Na⁺ channels are summarized in Table 1.

Table 1.

Interaction of K⁺, Na⁺, and Ca²⁺ channels and NMs

Cell model	NMs	Size	Dose and duration of exposure	Ion channel	Changes to ion channel	Reference
Mouse chromaffin cells	MWCNTsa	Diameter: 67 ± 2 nm Length: 1,120 ± 500 nm	3 × 104 μg·ml−1 for 24 hr	BK channels	Channels inactivation prolonged	Gavello et al. (2012)
Rat thoracic aorta smooth muscle cells	Colloidal Au NPs	<5 nm	1–300 μM for 1–2 min	BK channels	Channels opening activated	Soloviev et al. (2015)
RAW264.7 cells	c‐MWCNTsa	Diameter: 300–1,500 nm Length: 926–945 nm	50 μg·ml−1 for 24 hr	KCa3.1	Channels assembled in a polarized distribution manner	H. Li et al. (2017)
Calu‐3 cells	Negatively charged polystyrene NPs	20 nm	10 μg·ml−1, immediately	cAMP‐dependent K+ channels	K+ currents activated in a cAMP‐dependent manner	McCarthy, Gong, Nahirney, Duszyk, and Radomski (2011)
CHO cell	SWCNTsa	Diameter: 0.9–1.3 nm Length: 1,000 nm	100 μg·ml−1, immediately	EXP‐2, KVS‐1, human KCNQ1 and Kv4.2, and Kv11.1	Channels' activation and deactivation accelerated	Park et al. (2003)
Undifferentiated PC12 cells	c‐MWCNTsa	Diameter: 40–50 nm Length: 300–800 nm	5 μg·ml−1 for 6, 12, and 24 hr	VGKCs	I to, I K, and I K1 current densities suppressed	Xu et al. (2009)
HUVECs	SiO2 NPs	56.8 ± 14.1 nm	40 and 20 μg·ml−1 for 24 hr	Kv1.3	I K current increased, steady‐state activation curves of I K moved towards left, and the fluorescence intensity of the Kv1.3 protein increased	L. Yang et al. (2013)
HEK 239 cells	ZnO NPs	45 ± 30 nm	10 μg·ml−1 for 20 min	Kv11.1	The amplitude of the steady‐state current increased, and the rate of the current inactivation during steady‐state depolarization decreased	Piscopo and Brown (2018)
Hippocampal CA3 neurons	MWCNTa	Diameter: 20–30 nm Length: 300–1,500 nm	3 × 10−3 μM for 5 min	Kv4.2/4.3	The K+ current decreased	X. Q. Tan et al. (2014)
Mice native ventricular myocytes	Citrate‐coated Ag NPs	75 nm	0.01 μg·ml−1 for 5 min	VGKCs	I K decreased, and the activation of VGKCs delayed	Lin et al. (2017)
BEL‐7402/LO2 cells	PEG‐modified NPs	<100 nm	—	VGKCs	Outwardly rectifying K+ current increased	Q. Wang et al. (2015)
661W cells	ZnO NPs	30 nm	0, 31.25, 62.5, and 125.0 μM for 5 min	VGKCs	I K current decreased	C. Chen, Bu, et al. (2017)
Mice left ventricular tissue	Fe3O4 NPs	30 nm	0.58 mg·kg−1 every other day for 14 days	KCNQ1 channels	mRNA and protein expression enhanced	L. Liu et al. (2010)
HEK 293 cells	Triphenylphosphine‐stabilized Au NPs	1.4 nm	3.1, 6.5, and 16.25 μM for 10 min	Kv11.1	The tail current decreased	Leifert et al. (2013)
HL‐1 cells	Negatively charged Au NPs	0.8 nm	50 nM for 15 min	Inward rectifier K+ channel	The amplitude of the K+ current decreased	Chin (2014)
Jurkat E6‐1 cells	Dimercaptosuccinic acid‐coated γ‐Fe2O3 NPs	10 nm	20 μg·ml−1 for 1, 3, and 6 hr	Kv1.3	The current density decreased, and the channels inactivation and recovery delayed	Yan et al. (2015)
Hippocampal neurons	CdSe QDs	10 nm	1, 10, and 20 × 10−3 μM for 5 min	VGSCs	Ca2+ flux increased	Tang, Wang, et al. (2008)
Hippocampal neurons	CdSe QDs	2.38 nm	1, 10, and 20 × 10−3 μM for 5 min	VGSCs	Properties of activation and inactivation altered, recovery slowed, and fraction reduced	Tang, Xing, et al. (2008)
Juvenile Epinephelus coioides	Cu NPs	85 ± 29 nm	0.1 μg·ml−1 for 25 days	Na+/K+‐ATPase	Na+/K+‐ATPase activity decreased in the liver, gills, and muscle but increased in brain	T. Wang, Long, Cheng, Liu, and Yan (2014)
661W cells	ZnO NPs	30 nm	0, 31.25, 62.5, and 125.0 μM for 2 and 6 hr	Na+/K+‐ATPase	mRNA and protein levels reduced	C. Chen, Bu, et al. (2017)
Fish (Labeo rohita)	SiO2 NPs	80–100 nm	1, 5, and 25 μg·ml−1 for 96 hr	Na+/K+‐ATPase	Na+/K+‐ATPase activity increased	Krishna, Ramesh, Saravanan, and Ponpandian (2015)
Tail artery myocytes	Polyvinylpyrrolidone‐coated Au NMs	5 nm	500 μM, immediately	CaV1.2 and CaV3.1	Ca2+ current increased	Fusi et al. (2018)
Hippocampal neurons	PbS NPs	36.8 nm	15/30 mg·kg−1 once every 7 days for 3 months	Ca2+‐ATPase	Ca2+‐ATPase activities increased	Cao et al. (2013)
Hippocampal neurons	PbS NPs	36.8 nm	15/30 mg·kg−1 once every 7 days for 3 months	L‐type calcium channel	The expression of channel subunits increased	Cao et al. (2013)
Rat retinal ganglion cells	ZnO NPs	30 nm	2.5, 5, and 10 μg·ml−1 for 2 and 5 hr	Ca2+‐ATPase	Activity of Ca2+‐ATPase reduced	Guo, Bi, Wang, and Wu (2013)
Chromaffin cells	Carboxyl QDs with CdSe core and ZnS shell	9.3 nm	5, 8, 16, or 36 × 10−3 μM for 24 hr	L‐, N‐, P/Q‐, and R‐type Ca2+ channels	Ca2+ currents reduced	Gosso et al. (2011)
Cerebellum granule cells	Citrate‐coated Ag NPs	23.3 nm	10, 20, 50, and 100 μg·ml−1 for 24 hr	Cav2.1	The expression of channel protein (CACNA1A) decreased	N. Yin et al. (2015)
Hippocampal neurons	CdSe QDs	2.38 nm	10 × 10−3 μM for 5 min		[Ca2+]i increased via extracellular Ca2+ flux and internal Ca2+ release	Tang, Xing, et al. (2008)
GT1‐7 cell line	SiO2 NPs	50 nm	20 μg·ml−1, immediately	TRPV4	Ca2+ flux	Gilardino et al. (2015)
ChaGo‐K1 cell line	TiO2 NPs	75 nm	750 and 1,000 μg·ml−1, immediately	L‐type Ca2+ channels	Ca2+ flux	E. Y. Chen, Garnica, Wang, Chen, and Chin (2011)

Abbreviations: BK channels, big‐conductance K⁺ channels; CdSe, cadmium selenium; c‐MWCNTs, carboxylated multi‐walled carbon nanotubes; I _K, delayed rectifier K⁺ current; I _K1, inward rectifier K⁺ current; I _Na, voltage‐gated sodium current; I _to, transient outward K⁺ current; MWCNTs, multi‐walled carbon nanotubes; NMs, nanomaterials; NPs, nanoparticles; QDs, quantum dots; SWCNTs, single‐walled carbon nanotubes; TRPV4, transient receptor potential channel 4; VGKCs, voltage‐gated potassium channels; VGSCs, voltage‐gated sodium channels.

^aCarbon nanotubes' size was described with a diameter and length.

3.1.1. K⁺ channels

K⁺ channels contribute to signal conduction across chemical synapses and form K⁺ currents in different stages of the AP in neurons. They are predominantly responsible for cardiac, skeletal, and smooth muscle contractions (Hille, 1986; Zaydman et al., 2012). Generally, the cellular K⁺ current consists of the following three main components: transient outward K⁺ current (I _to), delayed rectifier K⁺ current (I _K), and inward rectifier K⁺ current (I _K1). I _to is the main contributing current during repolarization and is caused by the flux of K⁺. I _K, which does not have deactivation kinetic characteristics, plays a key role in repolarization, and I _K1 helps to stabilize the resting potential (RP).

Generally, most studies have focused on the K⁺ current in cells and the identification of K⁺ currents, including I _to (Gu et al., 2018), I _K (C. Chen, Bu, et al., 2017; Xu et al., 2009), and I _K1 (Gu et al., 2018; Lin et al., 2017), which are inhibited after exposure to NMs. In detail, a study conducted by Gu et al. (2018) showed that MoS₂ nanoflakes block I _to and I _K1 in a concentration‐dependent manner in vitro. Similar results were found in PC12 cells treated with carboxylated multi‐walled carbon nanotubes (c‐MWCNTs; Xu et al., 2009). In addition, zinc oxide (ZnO) nanoparticles (NPs) decreased I _K in murine photoreceptor cells (C. Chen, Bu, et al., 2017), and I _K1 inhibition was observed in ventricular myocytes after exposure to citrate‐coated silver (Ag) NPs (Lin et al., 2017) and in HL‐1 cells after exposure to gold (Au) NPs (Chin, 2014). However, some NMs have been reported to activate K⁺ currents. Colloidal Au NPs can activate Ca²⁺‐activated K⁺ currents (I _KCa) in vascular smooth muscle cells by increasing the opening of Ca²⁺‐activated K⁺ channels (Soloviev et al., 2015), and ZnO NPs can increase I _to and I _K in rat hippocampal neurons (Zhao, Xu, Zhang, Ren, & Yang, 2009).

The effect of NMs on K⁺ channels is indicated not only by changes in the K⁺ current but also by corresponding alterations in kinetics. When the current density of I _K was decreased by treatment with MWCNTs, the kinetics of voltage‐gated K⁺ channels were also altered as follows: the steady‐state activation curves moved towards the right, and V _1/2 was shifted towards a higher membrane potential, while the k values significantly increased (Xu et al., 2009). These phenomena suggested that the K⁺ channel sensitivity was decreased, and activation became more difficult after exposure to MWCNTs. However, certain NMs have also been reported to increase the K⁺ current. After treatment with silicon dioxide (SiO₂) NPs, the amplitudes of I _K in human vascular endothelial cells increased, the steady‐state activation curves of I _K shifted left, and the V _1/2 values of I _K and the k values decreased (L. Yang et al., 2013).

Moreover, NMs can change the distribution of K⁺ channels on the cell membrane. A study conducted by H. Li et al. (2017) showed that after treatment with c‐MWCNTs, K_Ca3.1 channels assembled uncharacteristically in the corners of RAW264.7 cells. Similar phenomena were also observed in SiO₂ NPs‐treated human umbilical vascular endothelial cells (L. Yang et al., 2013).

In summary, K⁺ channels are definitely affected by NMs exposure, resulting in changes in the K⁺ current, K⁺ channel open probability, and channel distribution. However, related information remains limited. For example, many studies have focused on the transient interaction between NMs and K⁺ channels, but the potential effects of prolonged NMs treatment on ion channels should also be considered. Moreover, K⁺ channel dysfunction could disturb the equilibrium of ionic homeostasis and other physiological and pathological conditions, which require further investigation.

3.1.2. Na⁺ channels

Similar to K⁺ channels, Na⁺ channels and Na⁺ currents (I _Na) modulate the excitability and activity of excitable cells. I _Na determines many neuronal properties, including AP generation, AP propagation to synapse terminals, and the local depolarization of neurons. It also participates in the transport of amino acid neurotransmitters and monoamines. Studies have revealed that after exposure to NMs, cells exhibit varying degrees of changes in I _Na (Schultz et al., 2012; Yuan et al., 2006). In most cases, NMs inhibit I _Na, although a few potentiating effects have been reported (Zhao et al., 2009).

In neurons, some NMs decrease the amplitude of I _Na (Busse et al., 2010; Z. Liu et al., 2009; Yuan et al., 2006), resulting in a hyperpolarized shift in the activation curve, altered inactivation properties, and delayed recovery of I _Na after inactivation. While Ag NPs showed no observable effects on the inactivation curve of I _Na in hippocampal neurons (Z. Liu et al., 2009), ZnO NPs showed some positive effects, such as increasing the peak amplitudes of I _Na by increasing the open number of Na⁺ channels and enhancing the excitability of hippocampal neurons (Zhao et al., 2009). However, studies have shown differences in the effects of the same NMs (e.g., Ag NPs) on neurons and cardiomyocytes. For example, Na⁺ and K⁺ channels in cardiomyocytes are more sensitive than those in the hippocampal neurons (Z. Liu et al., 2009). For cardiomyocytes, the outcomes were somewhat similar, for example, treatment with Ag NPs inhibited I _Na by accelerating the activation and inactivation processes and by delaying the recovery from inactivation, thereby decreasing the excitability of myocytes (Lin et al., 2017).

In general, similar to the functions and kinetic characteristics of K⁺ channels, the functions and kinetic characteristics of Na⁺ channels are also affected by NMs. These observed phenomena indicate that NMs may alter the voltage sensitivity of Na⁺ channels in neurons and cardiomyocytes, affecting the transition from the closed state to the open state of the channel. However, in addition to the kinetic characteristics, increased attention should be focused on correlations between the Na⁺ and K⁺ channels and subsequent effects on ion homeostasis.

3.1.3. Na⁺‐ and K⁺‐stimulated ATPase

Na⁺/K⁺‐stimulated ATPase (Na⁺/K⁺‐ATPase) is an ATP‐consuming transporter of Na⁺ and K⁺ that helps to maintain the membrane potential, regulate the osmotic pressure of cells, and provide energy for nutrient absorption. Moreover, it is essential for maintaining the normal impulse conduction of neurons and muscle cells. However, the activity of Na⁺/K⁺‐ATPase has been shown to be significantly inhibited after Ag NPs exposure (Osborne et al., 2015; Schultz et al., 2012). In contrast, other NMs, such as SiO₂ NPs (Krishna et al., 2015) and Au NPs (Petrovic, Vodnik, Stanojevic, Rakocevic, & Vasic, 2012), have been shown to increase Na⁺/K⁺‐ATPase activity.

K⁺ and Na⁺ channels synergistically play a crucial role in the biological function of cardiomyocytes and neurons, and metabolic disturbances could lead to serious myocardial and neurological diseases such as arrhythmia and epilepsy. We have summarized, in Figure 3, the targeted effects of NMs on K⁺ and Na⁺ channels, including Na⁺/K⁺‐ATPase, which are manifested as changes in the current, channel kinetics, channel construction, and channel distribution. These effects provide additional prospective applications of NMs for myocardial and neurological disease treatments, such as the use of Na⁺ channel blockers for pain relief (Emery, Luiz, & Wood, 2016).

3.2. Calcium channels

Ca²⁺ channels regulate the intracellular calcium concentration ([Ca²⁺]_i), which plays a crucial role in muscle contraction, cardiac conduction, self‐regulatory AP generation, neurotransmitter release, and cell proliferation and growth. The composition of Ca²⁺ channels is varied and complex and includes voltage‐gated calcium channels, ligand‐gated calcium channels, store‐operated channels, plasma membrane Ca²⁺ ATPase, and Na⁺/Ca²⁺exchangers (NCXs). Intracellular calcium stores, such as the endoplasmic reticulum (ER) and mitochondria, also help to regulate intracellular calcium homeostasis (Figure 4).

Exposure to NMs may affect the current and the [Ca²⁺]_i, and the distribution of Ca²⁺ channels on the cell membrane can also be altered, similar to the alterations of K⁺ channel distribution mentioned above. After treatment with c‐MWCNTs, the stromal interaction molecule 1 (STIM1, a type of Ca²⁺ channel located on the ER membrane) was translocated to the plasma membrane in RAW264.7 cells, activating Orai1 Ca²⁺ channels on the plasma membrane and leading to an influx of Ca²⁺ into the ER (H. Li et al., 2017).

3.2.1. Current and subunit changes in ion channels

Whole‐cell patch clamp studies showed that Ca²⁺ currents were altered after exposure to NMs (Fusi et al., 2018; Gosso et al., 2011), while the kinetics and gating of the channels seemed to be unaffected. Au NPs potentiated the Ca²⁺ current of the L‐type Ca_V1.2 and Ca_V1.3 channels in a concentration‐ and voltage‐dependent manner. However, neither the inactivation nor the activation kinetics were affected (Fusi et al., 2018). High concentrations of CdSeZnS quantum dots (QDs) inhibited Ca²⁺ currents in a voltage‐independent manner but did not affect channel gating (Gosso et al., 2011). Generally, changes in the current are accompanied by changes in the channel kinetics. The above‐mentioned experimental results are very contradictory but are likely to be attributed to increased channel protein expression.

After exposure to PbS NPs, the α1 and β subunits of the L‐type Ca²⁺ channels were overexpressed, which increased the opening of Ca²⁺ channels in hippocampal neurons, resulting in increased Ca²⁺ influx and [Ca²⁺]_i (Cao et al., 2013). Si NPs inhibited the calcium signalling pathway via the down‐regulation of the expression of related genes, such as ATPase‐related genes (ATP2A1L, ATP1B2B, and ATP1A3B), Ca²⁺ channel‐related genes (CACNA1AB and CACNA1DA), and the regulatory gene TNNC1A for cardiac troponin C in zebrafish embryos (Duan et al., 2016). Ag NPs also decreased the Ca²⁺ channel protein and mRNA expression levels of CACNA1A in the cerebellum and in primary cultured cerebellum granule cells (N. Yin et al., 2015).

3.2.2. [Ca²⁺]_i alterations

Many studies have shown that exposure to NMs increase [Ca²⁺]_i (Cao et al., 2013; E. Y. Chen et al., 2011; Dubes et al., 2017; Huang, Aronstam, Chen, & Huang, 2010; Onodera et al., 2017; Tang, Wang, et al., 2008; Xiang et al., 2016). For example, magnetic NPs triggered Ca²⁺ influx via N‐type Ca²⁺ channels in rat cortical neurons (Tay & Di Carlo, 2017), while other studies have shown that [Ca²⁺]_i does not significantly change after treatment with NMs, such as MWCNTs (Xu et al., 2009). Nanotoxicology studies revealed that the influx of extracellular Ca²⁺ and the subsequent [Ca²⁺]_i increase play important roles in NM‐induced cytotoxicity (Huang et al., 2010). When cells are stimulated to release Ca²⁺ from intracellular stores and/or when Ca²⁺ enters cells through ion channels located on the plasma membrane, [Ca²⁺]_i undergoes a spatiotemporal fluctuation. Ca²⁺ signals are then triggered and participate in many physiological processes, and they function as initiators to induce different reactions, which are described below.

3.2.3. The influence of NMs on calcium stores

Calcium stores, such as the ER (including the sarcoplasmic reticulum [SR]) and mitochondria, are cell organelles with the ability to store intracellular Ca²⁺. After entering cells, NMs directly or indirectly influence calcium stores and induce ER stress or mitochondrial dysfunction (Figure 4).

When the ER, which is the main organelle for Ca²⁺ storage, becomes a target cell organelle, it can be activated and produce an ER stress response (Christen, Camenzind, & Fent, 2014; Hou et al., 2013; Q. Yang, Wang, et al., 2017), which can further disrupt intracellular Ca²⁺ homeostasis. The ER stress‐induced disorder of Ca²⁺ regulation after exposure to NMs, such as ZnO NPs (R. Chen et al., 2014), has been shown to initiate toxicological effects, including unfolded protein responses and apoptosis (Pirot, Eizirik, & Cardozo, 2006). Furthermore, NMs could also impair mitochondrial functions (Tsai et al., 2018) by decreasing the mitochondrial membrane potential (Gurunathan & Kim, 2018; Peng et al., 2018) and/or increasing the mitochondrial membrane permeability (Y. Yang, Xiao, et al., 2017), leading to an imbalance in mitochondria‐mediated Ca²⁺ homeostasis (Tang, Wang, et al., 2008). Intriguingly, ER–mitochondria membrane contact sites, which are important components that provide a conduit to transport Ca²⁺ from the ER lumen to the mitochondrial matrix to increase the mitochondrial Ca²⁺ uptake efficiency (H. X. Wu, Carvalho, & Voeltz, 2018), could also be affected by NMs exposure. For example, the length of ER–mitochondria membrane contact sites increased after exposure to Ag NPs (L. Li et al., 2018), leading to an increase in the mitochondrial Ca²⁺ concentration ([Ca²⁺]_mito).

ER and mitochondria both play key roles in maintaining a normal cellular physiological state and responding to pathological conditions. Their dysfunction and the above‐mentioned disturbance in Ca²⁺ homeostasis, which is regulated by ion channels located in the ER and mitochondria, could trigger further biological effects and even lead to cell death. We describe more related information in the next section, which discusses NMs‐induced biological effects involving metabolic disorders of cellular ions.

In summary, there is experimental evidence that NMs can regulate Ca²⁺ homeostasis through Ca²⁺ channels, [Ca²⁺]_i and calcium stores. However, the detailed interaction(s) between NMs and Ca²⁺ channel composition has not been reported. Most previous studies have mainly focused on how NMs interact with Ca²⁺ channels on the cell membrane, and more information regarding how the interaction begins and the effects of the interaction is needed. In addition, more studies should be performed on other important Ca²⁺ channels, such as plasma membrane Ca²⁺ ATPases and store‐operated channels (two types of Ca²⁺ channels located on the plasma membrane). When the detailed mechanism of the interaction between NMs and ion channels is clearly revealed, appropriate types of NMs could be applied to target these ion channels, thereby regulating Ca²⁺ signals and influencing subsequent biological activities. The process of [Ca²⁺]_i changes is another topic that warrants investigation. Some studies have reported that [Ca²⁺]_i can be monitored by new techniques that record Ca²⁺ signal changes over time (Camire & Topolnik, 2018; Ellefsen & Parker, 2018). The detection of the structures of purified and reconstituted ion channels should also be more consistent with the development of nanoscale detection technologies (Q. Chen, She, et al., 2017). Currently, studies on the interaction between Ca²⁺ channels and NMs have become increasingly feasible and necessary, and clarifying the specific targets of NMs for Ca²⁺ channels could help reveal more detailed mechanisms. In the future, the application of NMs to regulate intracellular Ca²⁺ signals could be explored as a therapeutic method.

3.3. TRP channels

The TRP channels are transmembrane ion channels that form a non‐selective cationic passage through the cell membrane. TRP channels are divided into the following six subfamilies: TRPA, TRPC, TRPML TRPM, TRPP (PKD) and TRPV ion channels. These channels are responsible for various sensory reactions and play important roles in regulating many physiological processes, including BP, intestinal peristalsis, mineral absorption, fluid balance, and cell survival and death. Therefore, studies focused on TRP channels and the interactions with NMs were reviewed.

A study conducted by Dryn et al. (2018) showed that C₆₀ fullerene aqueous colloid solution irreversibly inhibited the TRPC4/TRPC6 channel, and Si NPs inhibited the activation of TRPV4 channels but enhanced the activation of TRPV1 channels in cultured airway epithelial cells (Sanchez et al., 2017). In addition, fullerol reversed the TNF‐α‐induced change in TRPV1 expression and attenuated the [Ca²⁺]_i increase in dorsal root ganglion neurons (Xiao et al., 2018). Other studies have found that some types of TRPV channels (TRPV1, Dubes et al., 2017; Veronesi, de Haar, Roy, & Oortgiesen, 2002; TRPV4, Dubes et al., 2017; Gilardino et al., 2015; and TRPM2, P. Yu et al., 2015) participate in NMs‐induced Ca²⁺ influx and increasing [Ca²⁺]_i. Moreover, in vivo studies found that treatment with hydrated C₆₀ fullerene increased the TRPM2 gene expression level in mouse brains (Etem et al., 2014). In summary, TRP channels have a close relationship with [Ca²⁺]_i, and exposure to NMs not only change the expression level of TRP channels but also regulate [Ca²⁺]_i by interacting with TRP channels. cationic

3.4. Influence of ion channels on organelles

When NMs enter cells by endocytosis, they inevitably exert effects on certain organelles, namely, the ER, mitochondria, and Golgi body, which are the main intracellular ion stores and contain diverse organelle ion channels. However, limited research has shown that NMs interact with organelle ion channels, affecting their open and closed state, thereby causing organelle dysfunction and disturbing intracellular ion homeostasis.

3.4.1. Ion channels on the ER

The inositol 1,4,5‐trisphosphate receptor (IP₃R) is a membrane glycoprotein complex that acts as a Ca²⁺ channel on the ER and that binds with IP₃ to regulate [Ca²⁺]_i. The IP₃R is very sensitive to Ca²⁺ and progressively increases the open‐state probability of the channel after it binds with IP₃. At higher intracellular IP₃ concentrations, the susceptibility of IP₃R to Ca²⁺ is decreased, progressively inhibiting the opening of IP₃R (Foskett, White, Cheung, & Mak, 2007). Intriguingly, NMs could also affect Ca²⁺ channels on the ER, including the IP₃Rs. NMs such as graphene oxide and carbon nanotubes could promote the production of IP₃ by influencing the GPCR on cell membranes. As a consequence, IP₃ could bind to its receptor and induce a flux of Ca²⁺ from the ER (Lim et al., 2016; Matsumoto & Shimizu, 2013; H. Yin et al., 2017). In addition, after internalization, MWCNTs and Ag NPs activated PLC‐β, which is involved in the biosynthesis of IP₃, and promoted the flux of Ca²⁺ from the ER through IP₃Rs (H. Li et al., 2017; L. Li et al., 2018).

Ryanodine receptors (RyRs) also mediate and regulate Ca²⁺ release from the SR in muscle cells and from the ER in non‐muscle cells. RyR has major isoforms, including RyR1, RyR2, and RyR3, which develop in different tissues and participate in different signalling pathways to regulate Ca²⁺ release. RyR1 is activated by a molecular reconfiguration mediated by the dihydropyridine receptor domain of Ca_v1.1 plasma membrane channels in response to membrane depolarization. In contrast, membrane depolarization in cardiomyocytes activates Ca_v1.2 channels to stimulate Ca²⁺ entry, and these ions directly bind to RyR2 to induce RyR2 opening. The RyR open‐state probability is also influenced by several other factors, including Mg²⁺, cADP‐ribose, ATP, ryanodine, and the ER/SR luminal Ca²⁺ concentration (Endo, 2009; Lanner, Georgiou, Joshi, & Hamilton, 2010; Zalk, Lehnart, & Marks, 2007). CdSe QDs increased [Ca²⁺]_i and induced an increase in Ca²⁺ release from the ER through RyRs (Tang, Wang, et al., 2008).

Moreover, STIM1, which acts as an ER Ca²⁺ depletion sensor, could translocate to the contact sites between the ER and cell membrane, thus activating Orai1 calcium channels in the plasma membrane and leading to an influx of Ca²⁺ into the ER (Alsaleh, Persaud, & Brown, 2016; H. Li et al., 2017). Intriguingly, one study found that a type of upconversion NPs could modulate the structure of STIM1 to stimulate Orai1 calcium channels without ER Ca²⁺ depletion, ultimately enhancing dendritic cell maturation and antigen presentation (P. Tan, He, Han, & Zhou, 2017).

3.4.2. Ion channels on the mitochondria

Mitochondria are the “powerhouse” of cells because they continuously generate ATP to enable cellular physiological activities, including Ca²⁺ signalling, ROS generation, and cell death pathway regulation. As mentioned above, NMs can damage mitochondrial structures and disrupt mitochondrial function. The mitochondrial calcium uniporter (MCU) is one of the primary Ca²⁺ intake structures on the mitochondria. MCUs function with NCXs to regulate Ca²⁺ release from the mitochondria, playing a crucial role in mitochondrial Ca²⁺ metabolism. Some interesting data were also observed on ion channels located on mitochondria after NM exposure. CdSe QDs induced extracellular Na⁺ influx via voltage‐gated sodium channels (VGSCs), causing NCXs to release Ca²⁺ from the mitochondria (Tang, Wang, et al., 2008). After exposure to Au NPs, the function of MCUs was up‐regulated, leading to an increase in [Ca²⁺]_mito. However, the Au NP‐induced Ca²⁺ flux from the mitochondria could be buffered by MCU‐mediated Ca²⁺ influx (Arvizo et al., 2013).

3.4.3. Ion channels on the lysosomes

Other organelles containing ion channels are also affected by NMs. Lysosomes, which are membrane‐bound organelles, break down waste materials and cellular debris via lysosomal acid hydrolase enzymes and a specific acidic inner environment. Similar to the plasma membrane, the lysosomal membrane contains various ion channels that act together to regulate the lysosomal volume, maintain the pH, and coordinate with other organelles (e.g., K⁺‐permeable channels, such as Ca²⁺‐activated big K⁺ [BK] channels; TMEM175, Feng, Zhao, Li, & Tan, 2018; and the Ca²⁺‐permeable channel TRPML3; S. Zhang, Li, Zeng, Gao, & Yang, 2017). Most importantly, lysosomes are regarded as major targets for various types of NMs, which can cause lysosomal membrane permeability changes, acid hydrolase enzyme inactivation, and lysosomal alkalization (J. Liu et al., 2019; Ma et al., 2011; J. Wang, Yu, et al., 2017).

Many recent studies have designed and modified NMs to function as nanocarriers for the delivery of drugs or nucleic acids into target cells. During this process, these nanocarriers are internalized into the cytoplasm and translocated into lysosomes. Almost inevitably, a large portion of cargo is released in the lysosome before entering the nucleus to exert their function, suggesting that the lysosomal release process is a crucial step in nanocarrier modification. Our previous study (Jia et al., 2017) showed that soluble NMs, such as ZnO NPs, could be endocytosed into lysosomes followed by the release of Zn²⁺. However, ZnO NPs dissolution induces the consumption of lysosomal protons, leading to lysosomal alkalization and high lysosomal Zn²⁺‐induced cathepsin inactivation. Furthermore, lysosomal ion channels for Zn²⁺ transport, including zinc transporter 2, zinc transporter 4, and vesicle‐associated membrane protein 7, are all affected (J. Liu et al., 2019). Moreover, whether other lysosomal ion channels are affected by ZnO NPs exposure remains unclear, but Zn²⁺ has been reported to bind directly to channel proteins of several ion channels (Noh et al., 2015). In addition, ion channels on the lysosome membrane play substantial roles in modulating the pH of the lysosome and ensuring that autophagy proceeds normally. Related issues, including whether the autophagy process is affected by NM exposure to ion channels located on the lysosome membrane, should be considered mainly during the autophagic degradation process after autophagosomes fuse with lysosomes.

As discussed above, in addition to ion channels located on the cell membrane, ion channels located on organelle membranes should also be examined. The ER and mitochondria are the main ion stores that can be targeted by NMs, and ion channels located on the membrane are specific targets. However, information regarding how NMs affect ion channels is lacking, and the ensuing induced biological effects have not been addressed thus far. Therefore, more effort should be devoted to research on organelle ion channels, such as ER Ca²⁺‐ATPase. In addition, the relationship between NMs and ion channels on other organelle membranes, especially on the Golgi apparatus and ribosomes, is also of interest.

3.5. Other ion channels

Anion channels are also crucial for the regulation of cell physiological activity. Chloride (Cl⁻) channels conduct not only Cl⁻ but also many other anions (SCN⁻, Br⁻, NO³⁻, and I⁻). Chloride channels have many important physiological roles, including pH regulation, volume homeostasis, organic solute transport, cell migration, cell proliferation, and cell differentiation (Jentsch, Stein, Weinreich, & Zdebik, 2002). However, only a few studies have shown that exposure to NMs could affect the function of Cl⁻ channels (Ahmad et al., 2012; Gonzalez‐Durruthy et al., 2017). A molecular docking simulation suggested that three types of single‐walled carbon nanotubes (SWCNTs) can block the mitochondrial voltage‐dependent anion channel (Gonzalez‐Durruthy et al., 2017). Mustafa and Komatsu (2016) indicated that Al₂O₃ NPs affect voltage‐dependent anion channels by up‐regulating their protein expression. Other types of ion channels could also be affected by NMs treatment. For example, in the presence of anionic ZnO NPs, the conductance of the lysenin channel was substantially reduced. The decrease in the macroscopic conductance was asymmetrical with respect to electrostatic interactions between ZnO NPs and a potential binding site. Conversely, no inhibitory effects were observed for anionic SnO₂ NPs (Bryant et al., 2017).

4. RELATED CHANGES IN PHYSIOLOGICAL AND BIOLOGICAL EFFECTS

4.1. Nerve excitation

As summarized above, exposure to NMs can alter ion channel kinetics and intracellular ion homoeostasis, which may disturb the normal excitability of neurons. Carbon‐based NMs, such as MWCNTs, could affect cell excitability and reduce spontaneous firing in mouse adrenal chromaffin cells, suppressing the RP and decreasing the AP amplitude accompanied by an increased rate of firing (Gavello et al., 2012). In the CNS, the excitability and activity of neurons are regulated by ion channels, and long‐term disorders of ion channels may lead to neuronal dysfunction. Z. Liu et al. (2009) showed that Ag NPs potentially modulate the I _Na of hippocampal neurons, causing alterations in cellular activity and function. They found that the amplitude of the AP peak decreased, while the half‐width increased, and the pattern of repetitive firing did not change. These results suggested that a lower threshold value for AP generation was observed after Ag NPs exposure, and exposed neurons were more readily excited.

As mentioned above, Ca²⁺ also plays an important role in the excitatory functions of neurons. The hippocampal neuronal [Ca²⁺]_i level was significantly increased after PbS NPs treatment, indicating that PbS NPs influenced Ca²⁺ homeostasis and affected LTP, which are important for synaptic plasticity (Cao et al., 2013). CdSeZnS QDs decreased Ca²⁺ influx through voltage‐gated calcium channels, decreased the size of the readily releasable store of Ca²⁺, and decreased the probability of ion release, thus impairing the secretion ability of mouse chromaffin cells (Gosso et al., 2011).

4.2. Cardiac function

In fast‐responding cardiomyocytes (functioning cardiomyocytes and Purkinje cells in the conduction system), I _Na is responsible for AP depolarization, excitability, and conductivity. I _K1 plays a major role in maintaining the RP and participating in late‐phase AP repolarization, thus directly or indirectly contributing to regulating the myocardial excitation threshold, effective refractory period, excitability, and conductivity.

Changes in myocardial electrophysiological characteristics may lead to electrical remodelling of the heart, thereby increasing the possibility of arrhythmia. In nanotoxicology studies, Au NPs were reported to block K_v11.1 channels and prolong the QT interval (Leifert et al., 2013), which is a risk factor for ventricular tachycardia (Antzelevitch, 2007). Ag NPs have potential impacts on I _Na and I _K1, altering the electrophysiological activity of cardiomyocytes and resulting in a loss of excitability. Sinus bradycardia and completed atrioventricular conduction blocking were also observed after Ag NP treatment, ultimately resulting in cardiac asystole in mice (Lin et al., 2017). In addition, MWCNTs were reported to induce a prolonged action potential during, increase the vagal output, and cause myocardial inflammation (X. Q. Tan et al., 2014).

4.3. The role of ion homeostasis in following biological effects

As discussed above, NMs can interact with various ion channels embedded in the cell plasma membrane, inducing changes in the ion current and kinetics of ion channels. Furthermore, ion channels located on cellular organelles can also be affected by NMs. These interactions affect ion homeostasis, which likely induces various biological effects and could even initiate cell death pathways. Some studies have indicated that NMs‐induced ion homeostasis dysfunction is the key factor leading to oxidative stress (He et al., 2018), autophagy (Lim et al., 2016), apoptosis (R. Chen et al., 2014; He et al., 2018; Lim et al., 2016), and inflammation (Liang et al., 2018). However, although many other aspects remain to be addressed, the following discussion is mainly focused on the relationships between ion homeostasis and various important biological effects, with the goal of providing new insights into the development of intracellular fate after NMs‐induced ion channel changes.

4.3.1. Oxidative stress

Currently, it is clear that disordered ion homeostasis is involved in oxidative stress. For example, Ca²⁺ signalling is essential for ROS production (Hempel & Trebak, 2017). Specifically, Ca²⁺ can directly regulate the tricarboxylic acid cycle and electron transport chain enzymes, which are among the main sources of ROS. Ca²⁺ can lead to three‐dimensional conformational changes in respiratory chain complexes, which are key components in the electron transport chain, and then increase ROS generation (Brookes, Yoon, Robotham, Anders, & Sheu, 2004). Furthermore, Ca²⁺ could induce changes in the oxidative status via K⁺. Calcium‐activated potassium channels (K_Ca channels) are important for the transport of K⁺ across cell membranes and are regulated by [Ca²⁺]_i. K_Ca channels have been reported to decrease the production of ROS in some situations. For example, an increase in [Ca²⁺]_mito activates mitochondrial K_Ca channels and leads to a flux of K⁺ from the cytosol into mitochondria. The K⁺ flux decreases the mitochondrial membrane potential and redox state of the NAD system, diminishing the production of ROS (Kulawiak et al., 2008).

4.3.2. Autophagy

Changes in ion homeostasis can also regulate autophagy, which is a catabolic degradation event that is essential for metabolic equilibrium. Ca²⁺ is an important regulator of autophagy, and several classic pathways participate in [Ca²⁺]_i‐induced autophagy. An increased [Ca²⁺]_i directly activates Ca²⁺/calmodulin‐dependent protein kinase 2, which then activates the AMPK pathway to promote autophagy (Hoyer‐Hansen et al., 2007). Additionally, an increased [Ca²⁺]_i induces the phosphorylation of PKC, promoting the process of autophagy (evidenced by increased LC3‐I conversion to LC3‐II; Sakaki, Wu, & Kaufman, 2008). Calpain has also been shown to be instrumental in Ca²⁺‐activated autophagy (Demarchi et al., 2006). Conversely, an increased [Ca²⁺]_i can decrease autophagic activity in some situations. TRPM2 channel‐mediated Ca²⁺ flux has been shown to increase the level of [Ca²⁺]_i and then induce cell apoptosis, which then decreases the autophagy level (Q. Wang et al., 2016). Other studies proposed that an elevated [Ca²⁺]_i can activate mTOR, resulting in the inhibition of autophagy (Gulati et al., 2008).

K⁺ homeostasis mediated by ion channels is another factor that influences the autophagy process. The activation of K_v11.3 channels promotes autophagy by linking plasma membrane hyperpolarization‐mediated phospholipid phosphorylation with activated AMPK (Perez‐Neut et al., 2016). Moreover, ATP‐sensitive K⁺ channels (K_ATP), which mediate K⁺ flux, are also associated with autophagy. A study found that inhibition of mitochondrial K_ATP can suppress autophagy (K. Y. Yu et al., 2014).

4.3.3. Apoptosis

Apoptosis is a form of programmed cell death that occurs in multicellular organisms. An increased [Ca²⁺]_i can initiate intracellular apoptotic signalling, which may lead to cell death (Prudent & McBride, 2017). After NMs induce ER stress (R. Chen et al., 2014), Ca²⁺ flux from the ER and [Ca²⁺]_i increase. Intracellular Ca²⁺ can combine with and phosphorylate calpain, leading to the activation of caspase‐12‐ and caspase‐3‐mediated apoptosis (Zuo et al., 2018). Mitochondria‐mediated intrinsic apoptotic pathways are also initiated due to NMs‐induced mitochondrial dysfunction as follows: Pro‐apoptotic proteins, such as cytochrome c, are released into the cytosol, where they activate caspases, resulting in apoptotic cell death (Rasola & Bernardi, 2011). IP₃R‐mediated Ca²⁺ transfer from the ER to the mitochondria has also been reported to play an essential role in the apoptotic signalling pathway (Prudent & McBride, 2017). Suppression of IP₃R open probability prevents the transfer and inhibits apoptosis (M. Wang & Xu, 2019).

K⁺, which is a predominant intracellular ion, has also been reported to be involved with apoptosis. An increased intracellular K⁺ concentration ([K⁺]_i) has been shown to suppress apoptosis via the inhibition of cytochrome c release, while a diminished [K⁺]_i favours the activation of caspases, leading to apoptosis (Cain, Langlais, Sun, Brown, & Cohen, 2001). Several studies have demonstrated that [K⁺]_i depletion or the overexpression of plasma membrane K⁺ channels, such as K_ir1.1 and K_v2.1, induces apoptosis in various cell types (F. C. Liu et al., 2018; Nadeau, McKinney, Anderson, & Lester, 2000).

4.3.4. Inflammation

Changes in intracellular ionic homeostasis can induce inflammation. Ca²⁺ flux has been reported to strongly up‐regulate the expression of inflammation‐associated genes (Delgadillo‐Silva et al., 2019) and promote NLRP3 inflammasome assembly and IL‐1β secretion (Jo, Kim, Shin, & Sasakawa, 2016; Murakami et al., 2012). An increased [Ca²⁺]_i, which is mediated by many Ca²⁺ channels, including TRPC3, TRPM2, TRPC6, and L‐type Ca²⁺ channels, also participates in the development of inflammation (Jo et al., 2016; Ramirez et al., 2018; Roca‐Lapirot et al., 2018). In addition to Ca²⁺ flux, a decrease in [K⁺]_i also participates in the development of inflammation (Dostert et al., 2008). SiO₂ NPs increase the open probability of outward K⁺ channels, the expression of channel proteins, and K⁺ flux, inducing an inflammatory effect by a decrease in [K⁺]_i (L. Yang et al., 2013). Other reports have also suggested that a decrease in [K⁺]_i is essential for NLRP1 and NLRP3 inflammasome activation (Munoz‐Planillo, Franchi, Miller, & Nunez, 2009; Petrilli et al., 2007). This intracellular K⁺‐regulated inflammasome activation is necessary for IL‐1β maturation and secretion, leading to cellular inflammatory processes (Jo et al., 2016). Furthermore, voltage‐gated K⁺ channels, such as K_v1.3, are also required for pro‐inflammatory activation (Di Lucente, Nguyen, Wulff, Jin, & Maezawa, 2018).

5. THE MECHANISM UNDERLYING THE INTERACTION BETWEEN NMs AND ION CHANNELS

The previous sections have shown that NMs directly and indirectly alter the permeability and kinetics of ion channels, which affect the intracellular ion concentration and disrupt cellular homeostasis. NMs can be internalized into humans through various pathways, and some NMs first directly contact the subunits of ion channels located on the lateral margin of the cell membrane. These interactions largely depend on the physical and chemical properties of NMs. Internalization of NMs is typically accompanied by cell membrane destruction, oxidative stress, the release of ions from NMs, and disturbances of proteins that regulate ion channels, all of which can alter the function of ion channels (Figure 1). Although various types of NMs and ion channels exist, the detailed mechanism of the interaction between NMs and ion channels can be summarized as follows.

5.1. Direct effects

5.1.1. Physical blocking

Most NMs are foreign materials after introduction to the body and cells in vivo, and the cell membrane and ion channels located on the cell membrane are typically the first sites exposed to NMs. In addition to blocking the path of ions and obstructing the conformational movement of channels, NMs can block ion channels and/or change their kinetics. For instance, carbon‐based NMs, such as SWCNTs, exhibited a reversible blocking effect on K_v11.1 channels by fitting into the pores (Park et al., 2003). C₈₄ fullerene was also reported to bind and block VGSCs in bacteria (Hilder & Chung, 2013). Furthermore, ZnO NPs could block the ion permeation pathway of voltage‐gated K⁺ channels and influence the physiological exchange between intracellular Na⁺ and extracellular K⁺ (C. Chen, Bu, et al., 2017). Although all of these NMs have the ability to block ion channels, the effects of their blocking are related to their binding at different sites of ion channels (e.g., active structures of ion channels and/or subunits).

From the moment NMs arrive at the surface of the cell membrane, interactions between NMs and ion channels begin. The functions and structures of ion channels are inevitably disturbed by NMs throughout the process. Initially, investigators speculated that the process involved NMs being directly embedded into the pores of ion channels from the outside of the cell (Park et al., 2003). With the development of detection technologies (docking simulations, molecular dynamics simulations, and cryo‐electron microscopy), various combinations of NMs and other active sites, such as VSD, have been observed and considered. A study of C₆₀ showed several binding sites between C₆₀ and ion channels, including the VSDs and extracellular hydrophobic residues of ion channels but not SFs. The study also found that binding to intracellular hydrophobic residues was stronger than extracellular interactions and that binding can induce conformational changes in ion channels (Kraszewski et al., 2010). Similarly, Calvaresi et al. (2015) showed that the blockade of a channel in the open state via intracellular C₆₀ was stronger than that of extracellular C₆₀ diffusing into the cytoplasm. In addition, some NMs, such as fullerene, can partition into lipid membranes, leading to a small rotation of VSDs, which could also affect the function of K_v1.2 channels (Monticelli, Barnoud, Orlowski, & Vattulainen, 2012).

In addition to active sites, NMs have been reported to combine with other domains of ion channels due to specific NMs surface modifications. Chin (2014) synthesized an Au NPs–spermidine complex in which Au NPs could be led by spermidine to the ion pores of K⁺ channels after penetrating into the cell membrane, and the biological effects of the K⁺ channels could then be blocked due to gold–sulfur bonds between the Au NPs and the cysteine loop of the channels. Furthermore, increasing attention has been focused on the role of ion channel amino acid residues in the blocking process. Hilder and Chung (2013) designed a C₈₄ fullerene with six attached lysine derivatives and found that the [Lys]‐fullerene surface hydrophobically interacted with Met¹⁸¹ and Glu¹⁷⁷ residues in the VGSCs, hindering conformational changes of the channels. It was also observed that the binding energy was mainly attributed to three amino acids (Thr⁵⁹, Ile⁸⁴, and Ala⁸⁸) and C₆₀ (Calvaresi et al., 2015).

Essentially, direct blocking is the result of direct interactions between NMs and channel residues through hydrophobic interactions or chemical bonds. When this type of binding occurs near the pore, the ion channel is blocked. Moreover, the channel kinetics are affected when binding occurs near VSDs. However, most of the studies mentioned in this review did not consider the complexity of the in vivo environment. After internalization into humans, NMs are wrapped by proteins in the blood, including serum albumin, to form a protein corona on their surface (Shemetov, Nabiev, & Sukhanova, 2012). It has been reported that NMs with a protein corona are less likely to block ion channels (Piscopo & Brown, 2018). Proteins adsorbed on NMs should be incorporated into simulations to examine interference by the protein corona. Therefore, events that occur after NMs enter the body should be fully considered in future research rather than only focusing on NMs and ion channels isolated in vitro.

5.1.2. The physicochemical properties of NMs

NMs have unique physical and chemical properties, including the shape, size, surface to volume ratio, surface chemistry, surface charge, agglomeration state, and purity, which can cause unique effects on cells. These parameters can also affect interactions between NMs and ion channels (Johnston et al., 2010).

First, the blocking effect depends on the shape and dimensions of NMs. Regulated by shape complementarity, C₆₀ has been shown to position itself in the cavity of Mth K channels and block the passage (Calvaresi et al., 2015). In addition, small‐diameter SWCNTs and C₆₀ fullerene showed more efficient blocking effects on K⁺ channels than large‐diameter hyperfullerenes and MWCNTs (Park et al., 2003), and this type of block did not require any electrochemical interactions between NMs and ion channels. The second influencing factor is the surface modification of NMs. In addition to the two examples mentioned regarding the regulation of the design of surface‐modified NMs targeting specific sites of ion channels, other related studies have been performed. For example, compared with the original types of Si NPs, Si NPs with the same diameter and a carboxyl modification did not increase [Ca²⁺]_i and showed low toxicity in vitro (Onodera et al., 2017). The reason may be that the carboxyl modification increased the dispersion of the NMs in the medium and reduced the contact between the NMs and the ion channels. Intriguingly, unmodified MWCNTs did not block K⁺ channels (Park et al., 2003), while carboxy‐terminated SCWNTs (Chhowalla et al., 2005) and MWCNTs (Xu et al., 2009) blocked K⁺ channels. These results indicated that carboxylation may play an important role in carbon‐based NMs‐induced block. The differing results of carboxyl modification could be explained by the different structures of K⁺ channels and Ca²⁺ channels. In addition, Hilder and Chung (2013) designed a fullerene modified with μ‐conotoxin, which has a strong affinity to NavAb channels (a bacterial Na⁺ channel), and a polyethylene glycol modification also enhanced the targeted effects of NMs on ion channels (Q. Wang et al., 2015). However, when ligands around NMs were stripped over time in vivo, bare NPs were found to irreversibly block the ion channels (Leifert et al., 2013).

Thus, our review suggests that NMs can act as competitors and restrain the flow of ions in a subcellular functional system dependent on their size, shape, and surface modification. Their binding to different sites on ion channels causes various modulations of channel function. Current and future research should be focused on improving the biosafety and bioavailability of NMs by considering their physicochemical properties.

5.2. Indirect effects

5.2.1. NMs induce changes in the structural and elastic properties of the cell membrane

Ion channels are embedded in lipid bilayers, and a lipid bilayer with appropriate flexibility and fluidity is a prerequisite for the normal operation of ion channels. However, long needle‐like NMs (such as MWCNTs) and NMs with sharp edges (such as graphene) can destroy the integrity of the cell membrane and could lead to leakage of intracellular ions. Gavello et al. (2012) described the internalization of MWCNTs into chromaffin cells, resulting in damage to the cell membrane and a reduction in the number of spontaneously firing cells. Additionally, because of its hydrophobic characteristic, C₆₀ partitioned the membrane core and migrated laterally towards membrane proteins by diffusion (Kraszewski et al., 2010), which could also affect ion channels located on the cell membrane.

Changes in the physicochemical properties of cell membranes could also affect the function of ion channels. A study found that upon penetration, fullerene induced small distortions in the bilayer structure and modestly increased the membrane softness (Wong‐Ekkabut et al., 2008). When C₇₀ was embedded in the membrane, it changed the stiffness, strength, and toughness of the membrane, and a small rotation of the S3 and S4 helices in the VSD of the K⁺ channel was also observed (Monticelli et al., 2012). Metal oxide NMs also showed similar effects. For example, ZnO NPs may affect K⁺ channels in this manner (Piscopo & Brown, 2018). Furthermore, some studies have reported that functionalized NMs could be stably embedded in the cell membrane, serving as a functional substitute for ion channels in living cells (Amiri, Shepard, Nuckolls, & Sanchez, 2017; L. Liu, Xie, Li, & Wu, 2015; Y. L. Zhang, Tunuguntla, Choi, & Noy, 2017). Another study showed that a polyamidoamine dendrimer could be incorporated into the membrane through multiple polar interactions, disrupting the nearby membrane bilayer and forming a unique hydrophilic Na⁺‐permeable channel embedded in the cell membrane around the dendrimer (Nyitrai et al., 2013).

5.2.2. The antagonistic action via ion release from NMs

Because many types of metal cations, including Cu²⁺, Pb²⁺, Fe²⁺, Se²⁺, and Zn²⁺, can block ion channels, the interactions of ion‐shedding NMs and their dissolved ions and ion channels are interesting topics that warrant investigation. Metal cations bind to the channel surface, thereby changing the surface charge density, which affects VSDs and accelerates or slows the opening and closing of ion channels (Elinder & Arhem, 2003). For example, during the manufacture and purification of carbon nanotubes, Y³⁺ was dissolved and released from a nanotube growth catalyst and was shown to exert an inhibitory effect on the function of Ca²⁺ channels, which might be related to competition between Y³⁺ and Ca²⁺ for Ca²⁺ binding sites (Jakubek et al., 2009). Furthermore, when ZnO NPs entered an acidic environment, such as the lysosomal environment, their solubility increased, and a large amount of Zn²⁺ was released into the lysosomes and cytoplasm (Jia et al., 2017), leading to a cellular physiological response (promoting cell proliferation or inducing cytotoxicity). Zn²⁺ at the NPs surface or in the cytoplasm could then modulate various ion channel functions (Peralta & Huidobro‐Toro, 2016). Released Zn²⁺ in the cytoplasm could increase the amplitude of the steady‐state current and accelerate channel deactivation during resurgent tail currents on a K_v11.1 channel (Piscopo & Brown, 2018). However, Bryant et al. (2017) indicated that ion transport inhibition was not due to dissolved Zn²⁺ and the subsequent interactions of Zn²⁺ with ion channels. The differing results might be attributed to differences in the lysenin channel and the local accumulation of ZnO NPs.

5.2.3. Oxidative stress

Some of the above‐mentioned studies have shown that NMs can affect ion channels and thereby lead to increased ROS production and inflammatory responses. However, other studies also indicated that NMs‐induced oxidative stress could result in damage to ion channels. Notably, Fe₂O₃ NPs reduced the current density of the K_v1.3 channel in Jurkat cells by the oxidation of NADPH, delaying the inactivation and recovery kinetics of K_v1.3 channels. Specifically, Fe₂O₃ NPs inhibited the redox activity of K_vβ2 subunits and led to changes in the function of K_v1.3 channels (Yan et al., 2015). Moreover, ROS production may have affected the expression of Ca²⁺‐ATPase, thereby further disturbing [Ca²⁺]_i homeostasis and causing cell death (Guo et al., 2013).

6. SUMMARY

Nanomedicine is a rapidly developing field in which NMs are employed to serve as diagnostic tools and to deliver therapeutic agents to specific targeted sites. Nanotechnology offers many benefits in treating chronic human diseases by site‐specific and target‐oriented delivery of precise medicines. Furthermore, most data have indicated that NMs have the ability to penetrate biological barriers (especially by damage to the barriers), causing biological changes in tissues and organs and leading to organ dysfunction. The interaction between NMs and intracellular components, such as the mitochondria, cell nuclei, and ion channels, has attracted substantial attention in recent years. Ion channels, which are important regulators of ion flow and various biological signals, are indispensable in maintaining normal physiological activities. Considering the increasing opportunities for ion channels to be exposed to NMs, this review aims to summarize the interactions between NMs and ion channels and explore the underlying mechanisms.

As established in this review, NMs interact with ion channels in various manners. Generally, NMs directly and indirectly affect ion channels from the outside, inside, and even the centre of the cell membrane, causing a variety of excitatory or inhibitory effects on ion channels. Additionally, ion channels located on organelle membranes can also be affected. Abnormal gating and kinetic characteristics of ion channels have been detected after NMs exposure, leading to abnormal intracellular ion homeostasis. Interestingly, abnormal ion homeostasis is likely to induce subsequent nanotoxicological effects, such as oxidative stress, autophagy, apoptosis, and inflammation. In addition, we summarized the mechanisms involved in ion channel changes induced by NMs. The direct interaction between NMs and ion channels is likely to be the key factor, and these events depend on the physicochemical properties of NMs, such as the particle size, shape, surface charge, and modifications. Furthermore, the indirect effects of NMs, such as NMs‐induced cell membrane disruption, NMs‐derived metal ions, and contact between NMs and ion channel‐related proteins, are also involved.

In summary, NMs can induce a variety of changes in ion channels, leading to related alterations in biological function. However, further related studies are needed. For example, additional research on the structural changes of ion channels after NMs exposure, including the effects of the interactions of binding sites in ion channels with NMs with different properties, is required. Moreover, the above‐mentioned interactions between NMs and ion channels also provide new insights into NMs applications. Investigating the effects of NMs on ion channels can aid in understanding the roles of certain ion channels in different cell types and tissues and in exploring the presence of structural and functional abnormalities of ion channels in related diseases that contribute to NMs‐based drug loading and disease diagnosis.

6.1. Nomenclature of targets and ligands

Key protein targets and ligands in this article are hyperlinked to corresponding entries at http://www.guidetopharmacology.org, which is a common portal for data from the IUPHAR/BPS Guide to PHARMACOLOGY (Harding et al., 2018), and are permanently archived in the Concise Guide to PHARMACOLOGY 2017/18 (Alexander, Fabbro et al., 2017a,b; Alexander, Kelly et al., 2017a,b; Alexander, Peters et al., 2017; Alexander, Striessnig et al., 2017).

CONFLICT OF INTEREST

The authors declare no conflicts of interest.

Yin S, Liu J, Kang Y, Lin Y, Li D, Shao L. Interactions of nanomaterials with ion channels and related mechanisms. Br J Pharmacol. 2019;176:3754–3774. 10.1111/bph.14792