Structure and Function of Ion Channels Regulating Sperm Motility—An Overview

Sperm motility is linked to the activation of signaling pathways that trigger movement. These pathways are mainly dependent on Ca²⁺, which acts as a secondary messenger. The maintenance of adequate Ca²⁺ concentrations is possible thanks to proper concentrations of other ions, such as K⁺ and Na⁺, among others, that modulate plasma membrane potential and the intracellular pH. Like in every cell, ion homeostasis in spermatozoa is ensured by a vast spectrum of ion channels supported by the work of ion pumps and transporters. To achieve success in fertilization, sperm ion channels have to be sensitive to various external and internal factors. This sensitivity is provided by specific channel structures. In addition, novel sperm-specific channels or isoforms have been found with compositions that increase the chance of fertilization. Notably, the most significant sperm ion channel is the cation channel of sperm (CatSper), which is a sperm-specific Ca²⁺ channel required for the hyperactivation of sperm motility. The role of other ion channels in the spermatozoa, such as voltage-gated Ca²⁺ channels (VGCCs), Ca²⁺-activated Cl-channels (CaCCs), SLO K⁺ channels or voltage-gated H⁺ channels (VGHCs), is to ensure the activation and modulation of CatSper. As the activation of sperm motility differs among metazoa, different ion channels may participate; however, knowledge regarding these channels is still scarce. In the present review, the roles and structures of the most important known ion channels are described in regard to regulation of sperm motility in animals.     

Computational Analyses of the AtTPC1 (Arabidopsis Two-Pore Channel 1) Permeation Pathway

Two Pore Channels (TPCs) are cation-selective voltage- and ligand-gated ion channels in membranes of intracellular organelles of eukaryotic cells. In plants, the TPC1 subtype forms the slowly activating vacuolar (SV) channel, the most dominant ion channel in the vacuolar membrane. Controversial reports about the permeability properties of plant SV channels fueled speculations about the physiological roles of this channel type. TPC1 is thought to have high Ca²⁺ permeability, a conclusion derived from relative permeability analyses using the Goldman–Hodgkin–Katz (GHK) equation. Here, we investigated in computational analyses the properties of the permeation pathway of TPC1 from Arabidopsis thaliana. Using the crystal structure of AtTPC1, protein modeling, molecular dynamics (MD) simulations, and free energy calculations, we identified a free energy minimum for Ca²⁺, but not for K⁺, at the luminal side next to the selectivity filter. Residues D269 and E637 coordinate in particular Ca²⁺ as demonstrated in in silico mutagenesis experiments. Such a Ca²⁺-specific coordination site in the pore explains contradicting data for the relative Ca²⁺/K⁺ permeability and strongly suggests that the Ca²⁺ permeability of SV channels is largely overestimated from relative permeability analyses. This conclusion was further supported by in silico electrophysiological studies showing a remarkable permeation of K⁺ but not Ca²⁺ through the open channel.     

The Important Role of Ion Transport System in Cervical Cancer

Cervical cancer is a significant gynecological cancer and causes cancer-related deaths worldwide. Human papillomavirus (HPV) is implicated in the etiology of cervical malignancy. However, much evidence indicates that HPV infection is a necessary but not sufficient cause in cervical carcinogenesis. Therefore, the cellular pathophysiology of cervical cancer is worthy of study. This review summarizes the recent findings concerning the ion transport processes involved in cell volume regulation and intracellular Ca²⁺ homeostasis of epithelial cells and how these transport systems are themselves regulated by the tumor microenvironment. For cell volume regulation, we focused on the volume-sensitive Cl⁻ channels and K⁺-Cl⁻ cotransporter (KCC) family, important regulators for ionic and osmotic homeostasis of epithelial cells. Regarding intracellular Ca²⁺ homeostasis, the Ca²⁺ store sensor STIM molecules and plasma membrane Ca²⁺ channel Orai proteins, the predominant Ca²⁺ entry mechanism in epithelial cells, are discussed. Furthermore, we evaluate the potential of these membrane ion transport systems as diagnostic biomarkers and pharmacological interventions and highlight the challenges.     

Augmented KCa2.3 Channel Feedback Regulation of Oxytocin Stimulated Uterine Strips from Nonpregnant Mice

Uterine contractions prior to 37 weeks gestation can result in preterm labor with significant risk to the infant. Current tocolytic therapies aimed at suppressing premature uterine contractions are largely ineffective and cause serious side effects. Calcium (Ca²⁺) dependent contractions of uterine smooth muscle are physiologically limited by the opening of membrane potassium (K⁺) channels. Exploiting such inherent negative feedback mechanisms may offer new strategies to delay labor and reduce risk. Positive modulation of small conductance Ca²⁺-activated K⁺ (K_Ca2.3) channels with cyclohexyl-[2-(3,5-dimethyl-pyrazol-1-yl)-6-methyl-pyrimidin-4-yl]-amine (CyPPA), effectively decreases uterine contractions. This study investigates whether the receptor agonist oxytocin might solicit K_Ca2.3 channel feedback that facilitates CyPPA suppression of uterine contractions. Using isometric force myography, we found that spontaneous phasic contractions of myometrial tissue from nonpregnant mice were suppressed by CyPPA and, in the presence of CyPPA, oxytocin failed to augment contractions. In tissues exposed to oxytocin, depletion of internal Ca²⁺ stores with cyclopiazonic acid (CPA) impaired CyPPA relaxation, whereas blockade of nonselective cation channels (NSCC) using gadolinium (Gd³⁺) had no significant effect. Immunofluorescence revealed close proximity of K_Ca2.3 channels and ER inositol trisphosphate receptors (IP₃Rs) within myometrial smooth muscle cells. The findings suggest internal Ca²⁺ stores play a role in K_Ca2.3-dependent feedback control of uterine contraction and offer new insights for tocolytic therapies.     

Na+/H+ Exchangers Involve in Regulating the pH-Sensitive Ion Channels in Mouse Sperm

Sperm-specific K⁺ ion channel (KSper) and Ca²⁺ ion channel (CatSper), whose elimination causes male infertility in mice, determine the membrane potential and Ca²⁺ influx, respectively. KSper and CatSper can be activated by cytosolic alkalization, which occurs during sperm going through the alkaline environment of the female reproductive tract. However, which intracellular pH (pH_i) regulator functionally couples to the activation of KSper/CatSper remains obscure. Although Na⁺/H⁺ exchangers (NHEs) have been implicated to mediate pH_i in sperm, there is a lack of direct evidence confirming the functional coupling between NHEs and KSper/CatSper. Here, 5-(N,N-dimethyl)-amiloride (DMA), an NHEs inhibitor that firstly proved not to affect KSper/CatSper directly, was chosen to examine NHEs function on KSper/CatSper in mouse sperm. The results of patch clamping recordings showed that, when extracellular pH was at the physiological level of 7.4, DMA application caused KSper inhibition and the depolarization of membrane potential when pipette solutions were not pH-buffered. In contrast, these effects were minimized when pipette solutions were pH-buffered, indicating that they solely resulted from pH_i acidification caused by NHEs inhibition. Similarly, DMA treatment reduced CatSper current and intracellular Ca²⁺, effects also dependent on the buffer capacity of pH in pipette solutions. The impairment of sperm motility was also observed after DMA incubation. These results manifested that NHEs activity is coupled to the activation of KSper/CatSper under physiological conditions.     

Mitochondrial Metal Ion Transport in Cell Metabolism and Disease

Mitochondria are vital to life and provide biological energy for other organelles and cell physiological processes. On the mitochondrial double layer membrane, there are a variety of channels and transporters to transport different metal ions, such as Ca²⁺, K⁺, Na⁺, Mg²⁺, Zn²⁺ and Fe²⁺/Fe³⁺. Emerging evidence in recent years has shown that the metal ion transport is essential for mitochondrial function and cellular metabolism, including oxidative phosphorylation (OXPHOS), ATP production, mitochondrial integrity, mitochondrial volume, enzyme activity, signal transduction, proliferation and apoptosis. The homeostasis of mitochondrial metal ions plays an important role in maintaining mitochondria and cell functions and regulating multiple diseases. In particular, channels and transporters for transporting mitochondrial metal ions are very critical, which can be used as potential targets to treat neurodegeneration, cardiovascular diseases, cancer, diabetes and other metabolic diseases. This review summarizes the current research on several types of mitochondrial metal ion channels/transporters and their functions in cell metabolism and diseases, providing strong evidence and therapeutic strategies for further insights into related diseases.     

Effect of Ion Concentration Changes in the Limited Extracellular Spaces on Sarcolemmal Ion Transport and Ca2+ Turnover in a Model of Human Ventricular Cardiomyocyte

We have developed a computer model of human cardiac ventricular myocyte (CVM), including t-tubular and cleft spaces with the aim of evaluating the impact of accumulation-depletion of ions in restricted extracellular spaces on transmembrane ion transport and ionic homeostasis in human CVM. The model was based on available data from human CVMs. Under steady state, the effect of ion concentration changes in extracellular spaces on [Ca²⁺]_i-transient was explored as a function of critical fractions of ion transporters in t-tubular membrane (not documented for human CVM). Depletion of Ca²⁺ and accumulation of K⁺ occurring in extracellular spaces slightly affected the transmembrane Ca²⁺ flux, but not the action potential duration (APD₉₀). The [Ca²⁺]_i-transient was reduced (by 2%–9%), depending on the stimulation frequency, the rate of ion exchange between t-tubules and clefts and fractions of ion-transfer proteins in the t-tubular membrane. Under non-steady state, the responses of the model to changes of stimulation frequency were analyzed. A sudden increase of frequency (1–2.5 Hz) caused a temporal decrease of [Ca²⁺] in both extracellular spaces, a reduction of [Ca²⁺]_i-transient (by 15%) and APD₉₀ (by 13 ms). The results reveal different effects of activity-related ion concentration changes in human cardiac t-tubules (steady-state effects) and intercellular clefts (transient effects) in the modulation of membrane ion transport and Ca²⁺ turnover.     

Gentamicin Blocks the ACh-Induced BK Current in Guinea Pig Type II Vestibular Hair Cells by Competing with Ca2+ at the l-Type Calcium Channel

Type II vestibular hair cells (VHCs II) contain big-conductance Ca²⁺-dependent K⁺ channels (BK) and l-type calcium channels. Our previous studies in guinea pig VHCs II indicated that acetylcholine (ACh) evoked the BK current by triggering the influx of Ca²⁺ ions through l-type Ca²⁺ channels, which was mediated by M2 muscarinic ACh receptor (mAChRs). Aminoglycoside antibiotics, such as gentamicin (GM), are known to have vestibulotoxicity, including damaging effects on the efferent nerve endings on VHCs II. This study used the whole-cell patch clamp technique to determine whether GM affects the vestibular efferent system at postsynaptic M2-mAChRs or the membrane ion channels. We found that GM could block the ACh-induced BK current and that inhibition was reversible, voltage-independent, and dose-dependent with an IC₅₀ value of 36.3 ± 7.8 μM. Increasing the ACh concentration had little influence on GM blocking effect, but increasing the extracellular Ca²⁺ concentration ([Ca²⁺]_o) could antagonize it. Moreover, 50 μM GM potently blocked Ca²⁺ currents activated by (−)-Bay-K8644, but did not block BK currents induced by NS1619. These observations indicate that GM most likely blocks the M2 mAChR-mediated response by competing with Ca²⁺ at the l-type calcium channel. These results provide insights into the vestibulotoxicity of aminoglycoside antibiotics on mammalian VHCs II.     

Calcium Ions Regulate K+ Uptake into Brain Mitochondria: The Evidence for a Novel Potassium Channel

The mitochondrial response to changes of cytosolic calcium concentration has a strong impact on neuronal cell metabolism and viability. We observed that Ca²⁺ additions to isolated rat brain mitochondria induced in potassium ion containing media a mitochondrial membrane potential depolarization and an accompanying increase of mitochondrial respiration. These Ca²⁺ effects can be blocked by iberiotoxin and charybdotoxin, well known inhibitors of large conductance potassium channel (BK_Ca channel). Furthermore, NS1619 – a BK_Ca channel opener – induced potassium ion–specific effects on brain mitochondria similar to those induced by Ca²⁺. These findings suggest the presence of a calcium-activated, large conductance potassium channel (sensitive to charybdotoxin and NS1619), which was confirmed by reconstitution of the mitochondrial inner membrane into planar lipid bilayers. The conductance of the reconstituted channel was 265 pS under gradient (50/450 mM KCl) conditions. Its reversal potential was equal to 50 mV, which proved that the examined channel was cation-selective. We also observed immunoreactivity of anti-β₄ subunit (of the BK_Ca channel) antibodies with ~26 kDa proteins of rat brain mitochondria. Immunohistochemical analysis confirmed the predominant occurrence of β₄ subunit in neuronal mitochondria. We hypothesize that the mitochondrial BK_Ca channel represents a calcium sensor, which can contribute to neuronal signal transduction and survival.     

KCa1.1 K+ Channel Inhibition Overcomes Resistance to Antiandrogens and Doxorubicin in a Human Prostate Cancer LNCaP Spheroid Model

Several types of K⁺ channels play crucial roles in tumorigenicity, stemness, invasiveness, and drug resistance in cancer. Spheroid formation of human prostate cancer (PC) LNCaP cells with ultra-low attachment surface cultureware induced the up-regulation of cancer stem cell markers, such as NANOG, and decreased the protein degradation of the Ca²⁺-activated K⁺ channel K_Ca1.1 by down-regulating the E3 ubiquitin ligase, FBXW7, compared with LNCaP monolayers. Accordingly, K_Ca1.1 activator-induced hyperpolarizing responses were larger in isolated cells from LNCaP spheroids. The pharmacological inhibition of K_Ca1.1 overcame the resistance of LNCaP spheroids to antiandrogens and doxorubicin (DOX). The protein expression of androgen receptors (AR) was significantly decreased by LNCaP spheroid formation and reversed by K_Ca1.1 inhibition. The pharmacological and genetic inhibition of MDM2, which may be related to AR protein degradation in PC stem cells, revealed that MDM2 was responsible for the acquisition of antiandrogen resistance in LNCaP spheroids, which was overcome by K_Ca1.1 inhibition. Furthermore, a member of the multidrug resistance-associated protein subfamily of ABC transporters, MRP5 was responsible for the acquisition of DOX resistance in LNCaP spheroids, which was also overcome by K_Ca1.1 inhibition. Collectively, the present results suggest the potential of K_Ca1.1 in LNCaP spheroids, which mimic PC stem cells, as a therapeutic target for overcoming antiandrogen- and DOX-resistance in PC cells.     

Cation Permeability of Voltage-Gated Hair Cell Ca2+ Channels of the Vertebrate Labyrinth

Some hearing, vestibular, and vision disorders are imputable to voltage-gated Ca²⁺ channels of the sensory cells. These channels convey a large Ca²⁺ influx despite extracellular Na⁺ being 70-fold more concentrated than Ca²⁺; such high selectivity is lost in low Ca²⁺, and Na⁺ can permeate. Since the permeation properties and molecular identity of sensory Ca²⁺ channels are debated, in this paper, we examine the Na⁺ current flowing through the L- and R-type Ca²⁺ channels of labyrinth hair cells. Ion currents and cytosolic free Ca²⁺ concentrations were simultaneously monitored in whole-cell recording synchronous to fast fluorescence imaging. L-type and R-type channels were present with different densities at selected sites. In 10 nM Ca²⁺, the activation and deactivation time constants of the L-type Na⁺ current were accelerated and its maximal amplitude increased by 6-fold compared to physiological Ca²⁺. The deactivation of the R-type Na⁺ current was not accelerated, and its current amplitude increased by 2.3-fold in low Ca²⁺; moreover, it was partially blocked by nifedipine in a voltage- and time-dependent manner. In conclusion, L channel gating is affected by the ion species permeating the channel, and its selectivity filter binds Ca²⁺ more strongly than that of R channel; furthermore, external Ca²⁺ prevents nifedipine from perturbing the R selectivity filter.     

Muscarinic Receptors and BK Channels Are Affected by Lipid Raft Disruption of Salivary Gland Cells

Activity-dependent fluid secretion is the most important physiological function of salivary glands and is regulated via muscarinic receptor signaling. Lipid rafts are important for G-protein coupled receptor (GPCR) signaling and ion channels in plasma membranes. However, it is not well understood whether lipid raft disruption affects all membrane events or only specific functions in muscarinic receptor-mediated water secretion in salivary gland cells. We investigated the effects of lipid raft disruption on the major membrane events of muscarinic transcellular water movement in human salivary gland (HSG) cells. We found that incubation with methyl-β-cyclodextrin (MβCD), which depletes lipid rafts, inhibited muscarinic receptor-mediated Ca²⁺ signaling in HSG cells and isolated mouse submandibular acinar cells. However, MβCD did not inhibit a Ca²⁺ increase induced by thapsigargin, which activates store-operated Ca²⁺ entry (SOCE). Interestingly, MβCD increased the activity of the large-conductance Ca²⁺-activated K⁺ channel (BK channel). Finally, we found that MβCD did not directly affect the translocation of aquaporin-5 (AQP5) into the plasma membrane. Our results suggest that lipid rafts maintain muscarinic Ca²⁺ signaling at the receptor level without directly affecting the activation of SOCE induced by intracellular Ca²⁺ pool depletion or the translocation of AQP5 into the plasma membrane.     

Electrophysiological Properties of Endogenous Single Ca2+ Activated Cl− Channels Induced by Local Ca2+ Entry in HEK293

Microdomains formed by proteins of endoplasmic reticulum and plasma membrane play a key role in store-operated Ca²⁺ entry (SOCE). Ca²⁺ release through inositol 1,4,5-trisphosphate receptor (IP₃R) and subsequent Ca²⁺ store depletion activate STIM (stromal interaction molecules) proteins, sensors of intraluminal Ca²⁺, which, in turn, open the Orai channels in plasma membrane. Downstream to this process could be activated TRPC (transient receptor potential-canonical) calcium permeable channels. Using single channel patch-clamp technique we found that a local Ca²⁺ entry through TRPC1 channels activated endogenous Ca²⁺-activated chloride channels (CaCCs) with properties similar to Anoctamin6 (TMEM16F). Our data suggest that their outward rectification is based on the dependence from membrane potential of both the channel conductance and the channel activity: (1) The conductance of active CaCCs highly depends on the transmembrane potential (from 3 pS at negative potentials till 60 pS at positive potentials); (2) their activity (NPo) is enhanced with increasing Ca²⁺ concentration and/or transmembrane potential, conversely lowering of intracellular Ca²⁺ concentration reduced the open state dwell time; (3) CaCC amplitude is only slightly increased by intracellular Ca²⁺ concentration. Experiments with Ca²⁺ buffering by EGTA or BAPTA suggest close local arrangement of functional CaCCs and TRPC1 channels. It is supposed that Ca²⁺-activated chloride channels are involved in Ca²⁺ entry microdomains.     

Pharmacological Dissection of the Crosstalk between NaV and CaV Channels in GH3b6 Cells

Thanks to the crosstalk between Na⁺ and Ca²⁺ channels, Na⁺ and Ca²⁺ homeostasis interplay in so-called excitable cells enables the generation of action potential in response to electrical stimulation. Here, we investigated the impact of persistent activation of voltage-gated Na⁺ (Na_V) channels by neurotoxins, such as veratridine (VTD), on intracellular Ca²⁺ concentration ([Ca²⁺]_i) in a model of excitable cells, the rat pituitary GH3b6 cells, in order to identify the molecular actors involved in Na⁺-Ca²⁺ homeostasis crosstalk. By combining RT-qPCR, immunoblotting, immunocytochemistry, and patch-clamp techniques, we showed that GH3b6 cells predominantly express the Na_V1.3 channel subtype, which likely endorses their voltage-activated Na⁺ currents. Notably, these Na⁺ currents were blocked by ICA-121431 and activated by the β-scorpion toxin Tf2, two selective Na_V1.3 channel ligands. Using Fura-2, we showed that VTD induced a [Ca²⁺]_i increase. This effect was suppressed by the selective Na_V channel blocker tetrodotoxin, as well by the selective L-type Ca_V channel (LTCC) blocker nifedipine. We also evidenced that crobenetine, a Na_V channel blocker, abolished VTD-induced [Ca²⁺]_i elevation, while it had no effects on LTCC. Altogether, our findings highlight a crosstalk between Na_V and LTCC in GH3b6 cells, providing a new insight into the mode of action of neurotoxins.     

Mechanisms of Pannexin 1 (PANX1) Channel Mechanosensitivity and Its Pathological Roles

Pannexins (PANX) were cloned based on their sequence homology to innexins (Inx), invertebrate gap junction proteins. Although there is no sequence homology between PANX and connexins (Cx), these proteins exhibit similar configurations. The PANX family has three members, PANX1, PANX2 and PANX3. Among them, PANX1 has been the most extensively studied. The PANX1 channels are activated by many factors, including high extracellular K⁺ ([K⁺]_e), high intracellular Ca²⁺ ([Ca²⁺]_i), Src family kinase (SFK)-mediated phosphorylation, caspase cleavage and mechanical stimuli. However, the mechanisms mediating this mechanosensitivity of PANX1 remain unknown. Both force-from-lipids and force-from-filaments models are proposed to explain the gating mechanisms of PANX1 channel mechanosensitivity. Finally, both the physiological and pathological roles of mechanosensitive PANX1 are discussed.     

A Calcium Guard in the Outer Membrane: Is VDAC a Regulated Gatekeeper of Mitochondrial Calcium Uptake?

Already in the early 1960s, researchers noted the potential of mitochondria to take up large amounts of Ca²⁺. However, the physiological role and the molecular identity of the mitochondrial Ca²⁺ uptake mechanisms remained elusive for a long time. The identification of the individual components of the mitochondrial calcium uniporter complex (MCUC) in the inner mitochondrial membrane in 2011 started a new era of research on mitochondrial Ca²⁺ uptake. Today, many studies investigate mitochondrial Ca²⁺ uptake with a strong focus on function, regulation, and localization of the MCUC. However, on its way into mitochondria Ca²⁺ has to pass two membranes, and the first barrier before even reaching the MCUC is the outer mitochondrial membrane (OMM). The common opinion is that the OMM is freely permeable to Ca²⁺. This idea is supported by the presence of a high density of voltage-dependent anion channels (VDACs) in the OMM, forming large Ca²⁺ permeable pores. However, several reports challenge this idea and describe VDAC as a regulated Ca²⁺ channel. In line with this idea is the notion that its Ca²⁺ selectivity depends on the open state of the channel, and its gating behavior can be modified by interaction with partner proteins, metabolites, or small synthetic molecules. Furthermore, mitochondrial Ca²⁺ uptake is controlled by the localization of VDAC through scaffolding proteins, which anchor VDAC to ER/SR calcium release channels. This review will discuss the possibility that VDAC serves as a physiological regulator of mitochondrial Ca²⁺ uptake in the OMM.     

Imaging of KCa3.1 Channels in Tumor Cells with PET and Small-Molecule Fluorescent Probes

The Ca²⁺ activated K⁺ channel K_Ca3.1 is overexpressed in several human tumor cell lines, e. g. clear cell renal carcinoma, prostate cancer, non-small cell lung cancer. Highly aggressive cancer cells use this ion channel for key processes of the metastatic cascade such as migration, extravasation and invasion. Therefore, small molecules, which are able to image this K_Ca3.1 channel in vitro and in vivo represent valuable diagnostic and prognostic tool compounds. The [¹⁸F]fluoroethyltriazolyl substituted senicapoc was used as positron emission tomography (PET) tracer and showed promising properties for imaging of K_Ca3.1 channels in lung adenocarcinoma cells in mice. The novel senicapoc BODIPY conjugates with two F-atoms ( 9 a ) and with a F-atom and a methoxy moiety ( 9 b ) at the B-atom led to the characteristic punctate staining pattern resulting from labeling of single K_Ca3.1 channels in A549-3R cells. This punctate pattern was completely removed by preincubation with an excess of senicapoc confirming the high specificity of K_Ca3.1 labeling. Due to the methoxy moiety at the B-atom and the additional oxyethylene unit in the spacer, 9 b exhibits higher polarity, which improves solubility and handling without reduction of fluorescence quantum yield. Docking studies using a cryo-electron microscopy (EM) structure of the K_Ca3.1 channel confirmed the interaction of 9 a and 9 b with a binding pocket in the channel pore.     

TRPM2 Oxidation Activates Two Distinct Potassium Channels in Melanoma Cells through Intracellular Calcium Increase

Tumor microenvironments are often characterized by an increase in oxidative stress levels. We studied the response to oxidative stimulation in human primary (IGR39) or metastatic (IGR37) cell lines obtained from the same patient, performing patch-clamp recordings, intracellular calcium ([Ca²⁺]_i) imaging, and RT-qPCR gene expression analysis. In IGR39 cells, chloramine-T (Chl-T) activated large K⁺ currents (KROS) that were partially sensitive to tetraethylammonium (TEA). A large fraction of KROS was inhibited by paxilline—a specific inhibitor of large-conductance Ca²⁺-activated BK channels. The TEA-insensitive component was inhibited by senicapoc—a specific inhibitor of the Ca²⁺-activated KCa3.1 channel. Both BK and KCa3.1 activation were mediated by an increase in [Ca²⁺]_i induced by Chl-T. Both KROS and [Ca²⁺]_i increase were inhibited by ACA and clotrimazole—two different inhibitors of the calcium-permeable TRPM2 channel. Surprisingly, IGR37 cells did not exhibit current increase upon the application of Chl-T. Expression analysis confirmed that the genes encoding BK, KCa3.1, and TRPM2 are much more expressed in IGR39 than in IGR37. The potassium currents and [Ca²⁺]_i increase observed in response to the oxidizing agent strongly suggest that these three molecular entities play a major role in the progression of melanoma. Pharmacological targeting of either of these ion channels could be a new strategy to reduce the metastatic potential of melanoma cells, and could complement classical radio- or chemotherapeutic treatments.