Inhibition of Kv2.1 potassium channels by the antidepressant drug sertraline.

Sertraline is a commonly used antidepressant of the selective serotonin reuptake inhibitors (SSRIs) class. In this study, we have used the patch-clamp technique to assess the effects of sertraline on Kv2.1 channels heterologously expressed in HEK-293 cells and on the voltage-gated potassium currents (IKv) of Neuro 2a cells, which are predominantly mediated by Kv2.1 channels. Our results reveal that sertraline inhibits Kv2.1 channels in a concentration-dependent manner. The sertraline-induced inhibition was not voltage-dependent and did not require the channels to be open. The kinetics of activation and deactivation were accelerated and decelerated, respectively, by sertraline. Moreover, the inhibition by this drug was use-dependent. Notably, sertraline significantly modified the inactivation mechanism of Kv2.1 channels; the steady-state inactivation was shifted to hyperpolarized potentials, the closed-state inactivation was enhanced and accelerated, and the recovery from inactivation was slowed, suggesting that this is the main mechanism by which sertraline inhibits Kv2.1 channels. Overall, this study provides novel insights into the pharmacological actions of sertraline on Kv2.1 channels, shedding light on the intricate interaction between SSRIs and ion channel function.

Crosstalk between cholesterol and PIP2 in the regulation of Kv7.2/Kv7.3 channels.

The activity of neuronal Kv7.2/Kv7.3 channels is critically dependent on PIP2 and finely modulated by cholesterol. Here, we report the crosstalk between cholesterol and PIP2 in the regulation of Kv7.2/Kv7.3 channels. Our results show that currents passing through Kv7.2/Kv7.3 channels in cholesterol-depleted cells, by acute application of methyl-ß-cyclodextrin (MßCD), were less sensitive to PIP2 dephosphorylation strategies than those of control cells, suggesting that cholesterol depletion enhances the Kv7.2/Kv7.3-PIP2 interaction. In contrast, the sensitivity of Kv7.2/Kv7.3 channels to acute membrane cholesterol depletion by MßCD was not altered in mutant channels with different apparent affinities for PIP2.

Phosphatidylinositol 4,5-Bisphosphate and Cholesterol Regulators of the Calcium-Activated Chloride Channels TMEM16A and TMEM16B.

Chloride fluxes through homo-dimeric calcium-activated channels TMEM16A and TMEM16B are critical to blood pressure, gastrointestinal motility, hormone, fluid and electrolyte secretion, pain sensation, sensory transduction, and neuronal and muscle excitability. Their gating depends on the voltage-dependent binding of two intracellular calcium ions to a high-affinity site formed by acidic residues from α-helices 6-8 in each monomer. Phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2), a low-abundant lipid of the inner leaflet, supports TMEM16A function; it allows TMEM16A to evade the down-regulation induced by calcium, poly-L-lysine, or PI(4,5)P2 5-phosphatase. In stark contrast, adding or removing PI(4,5)P2 diminishes or increases TMEM16B function, respectively. PI(4,5)P2-binding sites on TMEM16A, and presumably on TMEM16B, are on the cytosolic side of α-helices 3-5, opposite the calcium-binding sites. This modular structure suggested that PI(4,5)P2 and calcium cooperate to maintain the conductive state in TMEM16A. Cholesterol, the second-largest constituent of the plasma membrane, also regulates TMEM16A though the mechanism, functional outcomes, binding site(s), and effects on TMEM16A and TMEM16B remain unknown.

Determination of the size of lipid rafts studied through single-molecule FRET simulations.

Fluorescence resonance energy transfer (FRET) is a high-resolution technique that allows the characterization of spatial and temporal properties of biological structures and mechanisms. In this work, we developed an in silico single-molecule FRET methodology to study the dynamics of fluorophores inside lipid rafts. We monitored the fluorescence of a single acceptor molecule in the presence of several donor molecules. By looking at the average fluorescence, we selected events with single acceptor and donor molecules, and we used them to determine the raft size in the range of 5-16 nm. We conclude that our method is robust and insensitive to variations in the diffusion coefficient, donor density, or selected fluorescence threshold.

Kir4.1/Kir5.1 channels possess strong intrinsic inward rectification determined by a voltage-dependent K+-flux gating mechanism.

Inwardly rectifying potassium (Kir) channels are broadly expressed in both excitable and nonexcitable tissues, where they contribute to a wide variety of cellular functions. Numerous studies have established that rectification of Kir channels is not an inherent property of the channel protein itself, but rather reflects strong voltage dependence of channel block by intracellular cations, such as polyamines and Mg2+. Here, we identify a previously unknown mechanism of inward rectification in Kir4.1/Kir5.1 channels in the absence of these endogenous blockers. This novel intrinsic rectification originates from the voltage-dependent behavior of Kir4.1/Kir5.1, which is generated by the flux of potassium ions through the channel pore; the inward K+-flux induces the opening of the gate, whereas the outward flux is unable to maintain the gate open. This gating mechanism powered by the K+-flux is convergent with the gating of PIP2 because, at a saturating concentration, PIP2 greatly reduces the inward rectification. Our findings provide evidence of the coexistence of two rectification mechanisms in Kir4.1/Kir5.1 channels: the classical inward rectification induced by blocking cations and an intrinsic voltage-dependent mechanism generated by the K+-flux gating.

Riluzole inhibits Kv4.2 channels acting on the closed and closed inactivated states.

Riluzole is an anticonvulsant drug also used to treat the amyotrophic lateral sclerosis and major depressive disorder. This compound has antiglutamatergic activity and is an important multichannel blocker. However, little is known about its actions on the Kv4.2 channels, the molecular correlate of the A-type K+ current (IA) and the fast transient outward current (Itof). Here, we investigated the effects of riluzole on Kv4.2 channels transiently expressed in HEK-293 cells. Riluzole inhibited Kv4.2 channels with an IC50 of 190 ± 14 µM and the effect was voltage- and frequency-independent. The activation rate of the current (at +50 mV) was not affected by the drug, nor the voltage dependence of channel activation, but the inactivation rate was accelerated by 100 and 300 µM riluzole. When Kv4.2 channels were maintained at the closed state, riluzole incubation induced a tonic current inhibition. In addition, riluzole significantly shifted the voltage dependence of inactivation to hyperpolarized potentials without affecting the recovery from inactivation. In the presence of the drug, the closed-state inactivation was significantly accelerated, and the percentage of inactivated channels was increased. Altogether, our findings indicate that riluzole inhibits Kv4.2 channels mainly affecting the closed and closed-inactivated states.

In vitro and in silico characterization of the inhibition of Kir4.1 channels by aminoglycoside antibiotics.

BACKGROUND AND PURPOSE: Aminoglycoside antibiotics are positively charged molecules that are known to inhibit several ion channels. In this study, we have shown that aminoglycosides also inhibit the activity of Kir4.1 channels. Aminoglycosides inhibit Kir4.1 channels by a pore-blocking mechanism, plugging the central vestibule of the channel. EXPERIMENTAL APPROACH: Patch-clamp recordings were made in HEK-293 cells transiently expressing Kir4.1 channels to analyse the effects of gentamicin, neomycin and kanamycin. In silico modelling followed by mutagenesis were realized to identify the residues critical for aminoglycosides binding to Kir4.1. KEY RESULTS: Aminoglycoside antibiotics block Kir4.1 channels in a concentration- and voltage-dependent manner, getting access to the protein from the intracellular side of the plasma membrane. Aminoglycosides block Ki4.1 with a rank order of potency as follows: gentamicin ˃ neomycin ˃ kanamycin. The residues T128 and principally E158, facing the central cavity of Kir4.1, are important structural determinants for aminoglycosides binding to the channel, as determined by our in silico modelling and confirmed by mutagenesis experiments. CONCLUSION AND IMPLICATIONS: Kir4.1 channels are also target of aminoglycoside antibiotics, which could affect potassium transport in several tissues.

Voltage-induced structural modifications on M2 muscarinic receptor and their functional implications when interacting with the superagonist iperoxo.

It has been reported that muscarinic type-2 receptors (M2R) are voltage sensitive in an agonist-specific manner. In this work, we studied the effects of membrane potential on the interaction of M2R with the superagonist iperoxo (IXO), both functionally (using the activation of the ACh-gated K+ current (IKACh) in cardiomyocytes) and by molecular dynamics (MD) simulations. We found that IXO activated IKACh with remarkable high potency and clear voltage dependence, displaying a larger effect at the hyperpolarized potential. This result is consistent with a greater affinity, as validated by a slower (τ = 14.8 ± 2.3 s) deactivation kinetics of the IXO-evoked IKACh than that at the positive voltage (τ = 6.7 ± 1.2 s). The voltage-dependent M2R-IXO interaction induced IKACh to exhibit voltage-dependent features of this current, such as the 'relaxation gating' and the modulation of rectification. MD simulations revealed that membrane potential evoked specific conformational changes both at the external access and orthosteric site of M2R that underlie the agonist affinity change provoked by voltage on M2R. Moreover, our experimental data suggest that the 'tyrosine lid' (Y104, Y403, and Y426) is not the previously proposed voltage sensor of M2R. These findings provide an insight into the structural and functional framework of the biased signaling induced by voltage on GPCRs.

Functional marriage in plasma membrane: Critical cholesterol level-optimal protein activity.

In physiology, homeostasis refers to the condition where a system exhibits an optimum functional level. In contrast, any variation from this optimum is considered as a dysfunctional or pathological state. In this review, we address the proposal that a critical cholesterol level in the plasma membrane is required for the proper functioning of transmembrane proteins. Thus, membrane cholesterol depletion or enrichment produces a loss or gain of direct cholesterol-protein interaction and/or changes in the physical properties of the plasma membrane, which affect the basal or optimum activity of transmembrane proteins. Whether or not this functional switching is a generalized mechanism exhibited for all transmembrane proteins, or if it works just for an exclusive group of them, is an open question and an attractive subject to explore at a basic, pharmacological and clinical level.

Chloroquine inhibits tumor-related Kv10.1 channel and decreases migration of MDA-MB-231 breast cancer cells in vitro.

Chloroquine (CQ) is an old antimalarial drug currently being investigated for its anti-tumor properties. As chloroquine has been shown to inhibits several potassium channels, we decided to study its effect on the tumor-related Kv10.1 channel by using patch-clamp electrophysiology and cell migration assays. We found that chloroquine inhibited Kv10.1 channels transiently expressed in HEK-293 cells in a concentration- and voltage-dependent manner acting from the cytoplasmic side of the plasma membrane. Chloroquine also inhibited the outward potassium currents from MDA-MB-231 cells, which are mainly carried through Kv10.1 channels as was confirmed using astemizole. Additionally, chloroquine decreased MDA-MB-231 cell migration in the in vitro scratch wound healing assay. In conclusion, our data suggest that chloroquine decreases MDA-MB-231 cell migration by inhibiting Kv10.1 channels. The inhibition of Kv10.1 channels could represent another mechanism of the antitumoral action of chloroquine, besides autophagy inhibition and tumor vessel normalization.

Cytoskeleton disruption affects Kv2.1 channel function and its modulation by PIP2.

Voltage-gated potassium channels are expressed in a wide variety of excitable and non-excitable cells and regulate numerous cellular functions. The activity of ion channels can be modulated by direct interaction or/and functional coupling with other proteins including auxiliary subunits, scaffold proteins and the cytoskeleton. Here, we evaluated the influence of the actin-based cytoskeleton on the Kv2.1 channel using pharmacological and electrophysiological methods. We found that disruption of the actin-based cytoskeleton by latrunculin B resulted in the regulation of the Kv2.1 inactivation mechanism; it shifted the voltage of half-maximal inactivation toward negative potentials by approximately 15 mV, accelerated the rate of closed-state inactivation, and delayed the recovery rate from inactivation. The actin cytoskeleton stabilizing agent phalloidin prevented the hyperpolarizing shift in the half-maximal inactivation potential when co-applied with latrunculin B. Additionally, PIP2 depletion (a strategy that regulates Kv2.1 inactivation) after cytoskeleton disruption does not regulate further the inactivation of Kv2.1, which suggests that both factors could be regulating the Kv2.1 channel by a common mechanism. In summary, our results suggest a role for the actin-based cytoskeleton in regulating Kv2.1 channels.

Dual regulation of hEAG1 channels by phosphatidylinositol 4,5-bisphosphate.

The ether-à-go-go1 (EAG1, Kv10.1) K+ channel is a member of the voltage-gated K+ channel family mainly expressed in the central nervous system and cancer cells. Membrane lipids regulate several voltage-gated K+ channels but their influence on EAG1 channels has been poorly explored. Here we have studied the regulation of hEAG1 channels by phosphatidylinositol 4,5-bisfofate (PIP2) by using different strategies to manipulate the levels of this lipid, and the patch clamp technique. We found that depletion of endogenous PIP2 by activation of the voltage-sensing phosphatase from Danio rerio (Dr-VSP) or the human muscarinic type-1 receptor (hM1R) inhibits hEAG1 currents; however, the application of exogenous PIP2 to increase the level of this lipid on the plasma membrane, also induced an inhibition of hEAG1. In summary, our results indicate that PIP2 have dual effects on hEAG1 channels and its action as activator or inhibitor depends on its initial level on the plasma membrane.

Regulation of Kv7.2/Kv7.3 channels by cholesterol: Relevance of an optimum plasma membrane cholesterol content.

Kv7.2/Kv7.3 channels are the molecular correlate of the M-current, which stabilizes the membrane potential and controls neuronal excitability. Previous studies have shown the relevance of plasma membrane lipids on both M-currents and Kv7.2/Kv7.3 channels. Here, we report the sensitive modulation of Kv7.2/Kv7.3 channels by membrane cholesterol level. Kv7.2/Kv7.3 channels transiently expressed in HEK-293 cells were significantly inhibited by decreasing the cholesterol level in the plasma membrane by three different pharmacological strategies: methyl-ß-cyclodextrin (MßCD), Filipin III, and cholesterol oxidase treatment. Surprisingly, Kv7.2/Kv7.3 channels were also inhibited by membrane cholesterol loading with the MßCD/cholesterol complex. Depletion or enrichment of plasma membrane cholesterol differentially affected the biophysical parameters of the macroscopic Kv7.2/Kv7.3 currents. These results indicate a complex mechanism of Kv7.2/Kv7.3 channels modulation by membrane cholesterol. We propose that inhibition of Kv7.2/Kv7.3 channels by membrane cholesterol depletion involves a loss of a direct cholesterol-channel interaction. However, the inhibition of Kv7.2/Kv7.3 channels by membrane cholesterol enrichment could include an additional direct cholesterol-channel interaction, or changes in the physical properties of the plasma membrane. In summary, our results indicate that an optimum cholesterol level in the plasma membrane is required for the proper functioning of Kv7.2/Kv7.3 channels.

Regulation of Kv2.1 channel inactivation by phosphatidylinositol 4,5-bisphosphate.

Phosphatidylinositol 4,5-bisphosphate (PIP2) is a membrane phospholipid that regulates the function of multiple ion channels, including some members of the voltage-gated potassium (Kv) channel superfamily. The PIP2 sensitivity of Kv channels is well established for all five members of the Kv7 family and for Kv1.2 channels; however, regulation of other Kv channels by PIP2 remains unclear. Here, we investigate the effects of PIP2 on Kv2.1 channels by applying exogenous PIP2 to the cytoplasmic face of excised membrane patches, activating muscarinic receptors (M1R), or depleting endogenous PIP2 using a rapamycin-translocated 5-phosphatase (FKBP-Inp54p). Exogenous PIP2 rescued Kv2.1 channels from rundown and partially prevented the shift in the voltage-dependence of inactivation observed in inside-out patch recordings. Native PIP2 depletion by the recruitment of FKBP-Insp54P or M1R activation in whole-cell experiments, induced a shift in the voltage-dependence of inactivation, an acceleration of the closed-state inactivation, and a delayed recovery of channels from inactivation. No significant effects were observed on the activation mechanism by any of these treatments. Our data can be modeled by a 13-state allosteric model that takes into account that PIP2 depletion facilitates inactivation of Kv2.1. We propose that PIP2 regulates Kv2.1 channels by interfering with the inactivation mechanism.

Phytochemicals genistein and capsaicin modulate Kv2.1 channel gating.

BACKGROUND: Phytochemicals are a large group of plant-derived compounds that have a broad range of pharmacological effects. Some of these effects are derived from their action on transport proteins, including ion channels. The present study investigates the effects of the phytochemicals genistein and capsaicin on voltage-gated potassium Kv2.1 channels. METHODS: The whole-cell patch clamp technique was used to explore the regulation of Kv2.1 channels expressed in HEK293 cells by genistein and capsaicin. RESULTS: Both phytochemicals had a profound effect on the gating properties of Kv2.1 channels; the voltage dependence of activation and inactivation was shifted to hyperpolarized potentials, the closed-state inactivation was accelerated, and the recovery from inactivation was delayed. Moreover, genistein and capsaicin inhibited Kv2.1 currents in a concentration dependent manner. CONCLUSION: This study effectively demonstrated the inhibitory effects of genistein and capsaicin on Kv2.1 channels. As Kv2.1 channels play a prominent role in glucose-stimulated insulin secretion, our findings contribute to our understanding of the putative mechanism by which these phytochemicals exert their reported hypoglycemic effects.

High-potency block of Kir4.1 channels by pentamidine: Molecular basis.

Inward rectifier potassium (Kir) channels are expressed in almost all mammalian tissues and contribute to a wide range of physiological processes. Kir4.1 channel expression is found in the brain, inner ear, eye, and kidney. Loss-of-function mutations in the pore-forming Kir4.1 subunit cause an autosomal recessive disorder characterized by epilepsy, ataxia, sensorineural deafness and tubulopathy (SeSAME/EST syndrome). Despite its importance in physiological and pathological conditions, pharmacological research of Kir4.1 is limited. Here, we characterized the effect of pentamidine on Kir4.1 channels using electrophysiology, mutagenesis and computational methods. Pentamidine potently inhibited Kir4.1 channels when applied to the cytoplasmic side under inside-out patch clamp configuration (IC50 = 97nM). The block was voltage dependent. Molecular modeling predicted the binding of pentamidine to the transmembrane pore region of Kir4.1 at aminoacids T127, T128 and E158. Mutation of each of these residues reduced the potency of pentamidine to block Kir4.1 channels. A pentamidine analog (PA-6) inhibited Kir4.1 with similar potency (IC50 = 132nM). Overall, this study shows that pentamidine blocks Kir4.1 channels interacting with threonine and glutamate residues in the transmembrane pore region. These results can be useful to design novel compounds with major potency and specificity over Kir4.1 channels.

Inhibition of Kir4.1 potassium channels by quinacrine.

Inwardly rectifying potassium (Kir) channels are expressed in many cell types and contribute to a wide range of physiological processes. Particularly, Kir4.1 channels are involved in the astroglial spatial potassium buffering. In this work, we examined the effects of the cationic amphiphilic drug quinacrine on Kir4.1 channels heterologously expressed in HEK293 cells, employing the patch clamp technique. Quinacrine inhibited the currents of Kir4.1 channels in a concentration and voltage dependent manner. In inside-out patches, quinacrine inhibited Kir4.1 channels with an IC50 value of 1.8±0.3µM and with extremely slow blocking and unblocking kinetics. Molecular modeling combined with mutagenesis studies suggested that quinacrine blocks Kir4.1 by plugging the central cavity of the channels, stabilized by the residues E158 and T128. Overall, this study shows that quinacrine blocks Kir4.1 channels, which would be expected to impact the potassium transport in several tissues.

Chloroquine blocks the Kir4.1 channels by an open-pore blocking mechanism.

Kir4.1 channels have been implicated in various physiological processes, mainly in the K+ homeostasis of the central nervous system and in the control of glial function and neuronal excitability. Even though, pharmacological research of these channels is very limited. Chloroquine (CQ) is an amino quinolone derivative known to inhibit Kir2.1 and Kir6.2 channels with different action mechanism and binding site. Here, we employed patch-clamp methods, mutagenesis analysis, and molecular modeling to characterize the molecular pharmacology of Kir4.1 inhibition by CQ. We found that this drug inhibits Kir4.1 channels heterologously expressed in HEK-293 cells. CQ produced a fast-onset voltage-dependent pore-blocking effect on these channels. In inside-out patches, CQ showed notable higher potency (IC50 ≈0.5µM at +50mV) and faster onset of block when compared to whole-cell configuration (IC50 ≈7µM at +60mV). Also, CQ showed a voltage-dependent unblock with repolarization. These results suggest that the drug directly blocks Kir4.1 channels by a pore-plugging mechanism. Moreover, we found that two residues (Thr128 and Glu158), facing the central cavity and located within the transmembrane pore, are particularly important structural determinants of CQ block. This evidence was similar to what was previously reported with Kir6.2, but distinct from the interaction site (cytoplasmic pore) CQ-Kir2.1. Thus, our findings highlight the diversity of interaction sites and mechanisms that underlie amino quinolone inhibition of Kir channels.

Extracellular protons enable activation of the calcium-dependent chloride channel TMEM16A.

KEY POINTS: The calcium-activated chloride channel TMEM16A provides a pathway for chloride ion movements that are key in preventing polyspermy, allowing fluid secretion, controlling blood pressure, and enabling gastrointestinal activity. TMEM16A is opened by voltage-dependent calcium binding and regulated by permeant anions and intracellular protons. Here we show that a low proton concentration reduces TMEM16A activity while maximum activation is obtained when the external proton concentration is high. In addition, protonation conditions determine the open probability of TMEM16A without changing its calcium sensitivity. External glutamic acid 623 (E623) is key for TMEM16A's ability to respond to external protons. At physiological pH, E623 is un-protonated and TMEM16A is activated when intracellular calcium increases; however, under acidic conditions E623 is partially protonated and works synergistically with intracellular calcium to activate the channel. These findings are critical for understanding physiological and pathological processes that involve changes in pH and chloride flux via TMEM16A. ABSTRACT: Transmembrane protein 16A (TMEM16A), also known as ANO1, the pore-forming subunit of a Ca2+ -dependent Cl- channel (CaCC), is activated by direct, voltage-dependent, binding of intracellular Ca2+ . Endogenous CaCCs are regulated by extracellular protons; however, the molecular basis of such regulation remains unidentified. Here, we evaluated the effects of different extracellular proton concentrations ([H+ ]o ) on mouse TMEM16A expressed in HEK-293 cells using whole-cell and inside-out patch-clamp recordings. We found that increasing the [H+ ]o from 10-10 to 10-5.5  m caused a progressive increase in the chloride current (ICl ) that is described by titration of a protonatable site with pK = 7.3. Protons regulate TMEM16A in a voltage-independent manner, regardless of channel state (open or closed), and without altering its apparent Ca2+ sensitivity. Noise analysis showed that protons regulate TMEM16A by tuning its open probability without modifying the single channel current. We found a robust reduction of the proton effect at high [Ca2+ ]i . To identify protonation targets we mutated all extracellular glutamate and histidine residues and 4 of 11 aspartates. Most mutants were sensitive to protons. However, mutation that substituted glutamic acid (E) for glutamine (Q) at amino acid position 623 (E623Q) displayed a titration curve shifted to the left relative to wild type channels and the ICl was nearly insensitive to proton concentrations between 10-5.5 and 10-9.0  m. Additionally, ICl of the mutant containing an aspartic acid (D) to asparagine (N) substitution at position 405 (D405N) mutant was partially inhibited by a proton concentration of 10-5.5  m, but 10-9.0  m produced the same effect as in wild type. Based on our findings we propose that external protons titrate glutamic acid 623, which enables voltage activation of TMEM16A at non-saturating [Ca2+ ]i .

Revealing the activation pathway for TMEM16A chloride channels from macroscopic currents and kinetic models.

TMEM16A (ANO1), the pore-forming subunit of calcium-activated chloride channels, regulates several physiological and pathophysiological processes such as smooth muscle contraction, cardiac and neuronal excitability, salivary secretion, tumour growth and cancer progression. Gating of TMEM16A is complex because it involves the interplay between increases in intracellular calcium concentration ([Ca(2+)]i), membrane depolarization, extracellular Cl(-) or permeant anions and intracellular protons. Our goal here was to understand how these variables regulate TMEM16A gating and to explain four observations. (a) TMEM16A is activated by voltage in the absence of intracellular Ca(2+). (b) The Cl(-) conductance is decreased after reducing extracellular Cl(-) concentration ([Cl(-)]o). (c) ICl is regulated by physiological concentrations of [Cl(-)]o. (d) In cells dialyzed with 0.2 µM [Ca(2+)]i, Cl(-) has a bimodal effect: at [Cl(-)]o <30 mM TMEM16A current activates with a monoexponential time course, but above 30 mM, [Cl(-)]o ICl activation displays fast and slow kinetics. To explain the contribution of Vm, Ca(2+) and Cl(-) to gating, we developed a 12-state Markov chain model. This model explains TMEM16A activation as a sequential, direct, and Vm-dependent binding of two Ca(2+) ions coupled to a Vm-dependent binding of an external Cl(-) ion, with Vm-dependent transitions between states. Our model predicts that extracellular Cl(-) does not alter the apparent Ca(2+) affinity of TMEM16A, which we corroborated experimentally. Rather, extracellular Cl(-) acts by stabilizing the open configuration induced by Ca(2+) and by contributing to the Vm dependence of activation.