Ca2+-activated Cl- Channels Research Articles

Inhibition of Ca2+-activated Cl− channel ANO1/TMEM16A expression suppresses tumor growth and invasiveness in human prostate carcinoma

The etiology of prostatic adenocarcinoma remains unclear. Prostate cancer cells of varying metastatic potential and apoptotic resistance show altered expression of plasma membrane ion channels and unbalanced Ca2+ homeostasis. Ca2+-activated Cl− channels (CaCCs) are robustly expressed in epithelial cells and function to regulate epithelial secretion and cell volume for maintenance of ion and tissue homeostasis in proliferation, differentiation and apoptosis. ANO1/TMEM16A was recently identified as a CaCC, and it is of interest to determine whether ANO1 plays a role in development and metastasis of prostate carcinoma. Here we show that ANO1 mRNA and protein are highly expressed in human metastatic prostate cancer LNCaP and PC-3 cells by quantitative analysis of real-time PCR and Western blot. These findings were confirmed by whole-cell patch clamp recording of LNCaP and PC-3 cells with increased current density of ANO1 channels. Immunohistochemistry staining further revealed overexpression of ANO1 in human prostate cancer tissues, which correlated with the clinical TNM stage and Gleason score. Experiments with small hairpin RNAs (shRNAs) targeting human ANO1 resulted in a significant reduction of proliferation, metastasis and invasion of PC-3 cells using WST-8, colony formation, wound-healing and transwell assays. Moreover, intratumoral injection of ANO1 shRNA completely inhibited established tumor growth and survival in orthotopic nude mice implanted with PC-3 cells. Our findings provide compelling evidence that upregulation of CaCC ANO1 is involved in the proliferation, progression and pathogenesis of metastatic prostate cancer. Membrane ANO1 protein may therefore serve as a biomarker, and inhibition of overexpressed ANO1 has potential for use in prostate cancer therapy.

Anoctamin 1 (Tmem16A) Ca 2 + -activated chloride channel stoichiometrically interacts with an ezrin–radixin–moesin network

The newly discovered Ca(2+)-activated Cl(-) channel (CaCC), Anoctamin 1 (Ano1 or TMEM16A), has been implicated in vital physiological functions including epithelial fluid secretion, gut motility, and smooth muscle tone. Overexpression of Ano1 in HEK cells or Xenopus oocytes is sufficient to generate Ca(2+)-activated Cl(-) currents, but the details of channel composition and the regulatory factors that control channel biology are incompletely understood. We used a highly sensitive quantitative SILAC proteomics approach to obtain insights into stoichiometric protein networks associated with the Ano1 channel. These studies provide a comprehensive footprint of putative Ano1 regulatory networks. We find that Ano1 associates with the signaling/scaffolding proteins ezrin, radixin, moesin, and RhoA, which link the plasma membrane to the cytoskeleton with very high stoichiometry. Ano1, ezrin, and moesin/radixin colocalize apically in salivary gland epithelial cells, and overexpression of moesin and Ano1 in HEK cells alters the subcellular localization of both proteins. Moreover, interfering RNA for moesin modifies Ano1 current without affecting its surface expression level. Another network associated with Ano1 includes the SNARE and SM proteins VAMP3, syntaxins 2 and -4, and syntaxin-binding proteins munc18b and munc18c, which are integral to translocation of vesicles to the plasma membrane. A number of other regulatory proteins, including GTPases, Ca(2+)-binding proteins, kinases, and lipid-interacting proteins are enriched in the Ano1 complex. These data provide stoichiometrically prioritized information about mechanisms regulating Ano1 function and trafficking to polarized domains of the plasma membrane.

δ-Opioid receptor agonist SNC80 induces central antinociception mediated by Ca2+-activated Cl- channels

The aim of this study was to determine whether Ca(2+)-activated Cl(-) channels (CaCCs) are involved in central antinociception induced by the activation of µ-, δ- and κ-opioid receptors. The nociceptive threshold for thermal stimulation was measured using the tail-flick test in Swiss mice. The drugs were administered via the intracerebroventricular route. Probabilities values of P < 0.05 were considered to be statistically significant (analysis of variance/Bonferroni test). The results demonstrate that exposure to the CaCC blocker niflumic acid (2, 4 and 8 µg) partially reverses the central antinociception induced by the δ-opioid receptor agonist SNC80 ((+)-4-[(αR)-α-((2S,5R)-4-allyl-2,5-dimethyl-1-piperazinyl)-3-methoxybenzyl]-N,N-diethylbenzamide; 4 µg). In contrast, niflumic acid did not modify the antinociceptive effect of the µ-opioid receptor agonist [D-Ala(2), N-Me-Phe(4), Gly(5)-ol]-enkephalin (0.5 µg) or κ-opioid receptor agonist bremazocine (4 µg). These data provide evidence for the involvement of CaCCs in δ-opioid receptor-induced central antinociception resulting from receptor activation by the agonist SNC80. CaCC activation does not appear to be involved when µ- and κ-opioid receptors are activated.

750 Phosphodiesterase Inhibitors Stimulate Intestinal CA Activated Cl Channels (CaCCS) by an Epac1-Mediated Mechanism: an Example of cAMP-CA+2 Cross-Talk in the Intestine

750 Phosphodiesterase Inhibitors Stimulate Intestinal CA Activated Cl Channels (CaCCS) by an Epac1-Mediated Mechanism: an Example of cAMP-CA+2 Cross-Talk in the Intestine

Prostaglandin E2 Activates Basal EP4 Receptors to Stimulate Chloride Secretion Via Both cAMP and Calcium Mediated Pathways

The renal collecting duct (CD) maintains salt (NaCl) balance by fine‐tuning NaCl excretion in response to dietary salt intake. Under conditions of high dietary salt intake (HDS), prostaglandin‐E2 (PGE‐2) production is enhanced and promotes NaCl excretion. The molecular mechanisms by which PGE‐2 works are not well defined. Under conditions of HDS, Na+ reabsorption is suppressed, and the regulation of Cl− secretion may predominate. The initial segment of the inner medullary CD (IMCD) is the only segment known to secrete Cl− under HDS. We used the mIMCD‐K2 cell line, a mouse cell line derived from the initial IMCD, to study the effects of PGE‐2 on Cl− transport in the IMCD.In mIMCD‐K2 cells, PGE‐2 activates basolateral EP‐4 receptors (EP‐4R) to increase Cl− secretion via both the Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) and Ca2+ activated Cl− channels (CACC). EP‐4R activation enhances cAMP production which activates CFTR; unexpectedly, we found that EP4R activation also increases intracellular Ca2+ to stimulate CACC mediated Cl− secretion. We also determined that PGE‐2 stimulated secretion involves crosstalk between cAMP and Ca2+ signaling pathways in which intracellular Ca2+ influences CFTR activity and cAMP influences CACC activity. These two pathways, and the crosstalk between them, may represent a concerted mechanism by which PGE‐2 acts to enhance NaCl excretion under conditions of HDS.

Expression and functional significance of Ca2+‐activated Cl‐channels in dendritic cells

The functions of dendritic cells (DCs), professional antigen presenting cells linking innate and adaptive immunity, are under regulation of Ca2+. Ca2+ entry is sensitive to the membrane potential and may depend on Cl− channel activity. The present study employed whole‐cell patch clamp experiments on mouse bone marrow‐derived DCs to identify expression and regulation of Cl− channels. Outwardly rectifying Cl− currents were activated by elevation of cytosolic Ca2+ triggered either by ionomycin in the presence of extracellular Ca2+ or by inositol‐triphosphate‐induced Ca2+ release from intracellular stores. Most importantly, Ca2+‐activated Cl− channels (CaCCs) were activated by the chemokine CCL21, which induces DC migration to the lymph nodes. The currents were inhibited by Cl− channel and CaCC blockers: tannic, digallic and niflumic acids and AO1. According to Fura2 fluorescence, the CaCC blockers blunted the store operated Ca2+ entry following depletion of intracellular Ca2+ stores with thapsigargin or upon stimulation with CCL21. The CaCC blockers further inhibited spontaneous, and chemokine‐ (CXCL12, CCL21)‐induced migration and secretion of interleukin‐2 (IL‐2) and IL‐6. According to RT‐PCR and Western blot data, Anoctamin 6 is expressed and can contribute to CaCCs in DCs. Our data identify Ca2+‐activated Cl− channels in mouse DCs as major players in Ca2+ homeostasis and Ca2+‐regulated DC functions.

Purinergic regulation of ion transport in Capan‐1 cell monolayers

The aim of the present study was to develop a human pancreatic duct model and elucidate the purinergic signaling regulation of ion transport in pancreatic ducts. Human pancreatic duct cells, capan‐1 were grown on permeable supports. Transepithelial potential (Vte) was measured in open‐circuit Ussing chamber in physiological buffers. Capan‐1 cells formed monolayers with resting transepithelial resistance 404±25 Ω·cm2 (n=35). Serosal ATP (100 μM) transiently increased Vte from −1.4±0.2 to −4.7±0.4 mV and short‐circuit current (Isc) by 14± 0.8 μA/cm2, while mucosal ATP increased Vte by −3.6±0.3 mV and Isc by 13±1 μA/cm2 (n=28). The Ca2+‐activated Cl− channel (CaCC) blocker niflumic acid (NA, mucosa, 100 μM, n=5) inhibited 44% of mucosal but not serosal ATP effect. The CFTR Cl− channel blocker CFTRinh172 blocked both mucosal and serosal ATP effects (mucosa,10 μM, n=5). The intermediate conductance K+ (IK) channel opener DCEBIO (mucosa, 100 μM, n=5) increased mucosal ATP effect, and DCEBIO stimulation was completely inhibited by NA and CFTRinh172 (n=8). The IK channel blocker, clotrimazole, abolished mucosal and serosal ATP effects (30 μM, n=6). In conclusion, mucosal ATP opens both CFTR and CaCC and may stimulate pancrea secretion, while serosal ATP regulates secretion partly through CFTR. In addition, ATP stimulation also regulates IK channels that provide the driving force for Cl− channel dependent secretions.

Upregulation of TMEM16A Underlies Chronic Hypoxia‐induced Increase of Ca2+‐activated Cl− Current in Pulmonary Arterial Smooth Muscle

Ca2+‐activated Cl− channels (CaCCs), encoded by TMEM16A, play an important role in the regulation of vascular tone. We have previously observed an increase in CaCC current (ICl.Ca) and TMEM16A expression in pulmonary arterial smooth muscle cells (PASMCs) and tissue of rats with chronic hypoxic pulmonary hypertension (CPHP, 10 ±0.5% O2 for 3–4 weeks). This study aimed to determine whether chronic hypoxia (CH) alters the Ca2+‐or voltage‐dependence of CaCCs in PASMCs, thereby contributes to the enhanced ICl.Ca. Perforated and conventional whole‐cell patch clamp techniques were used to record the caffeine‐induced ICl.Ca and Ca2+ transients simultaneously on fluo 3/AM‐loaded PASMCs, and ICl.Ca evoked by depolarization at constant high Ca2+, respectively. The activation rates of caffeine‐induced ICl.Ca and Ca2+ release were not different between control and hypoxic PASMCs, and their strong correlation was unaltered by CH at both 0 and −80 mV. At 750 nM of [Ca2+]I, the current‐voltage relation exhibited a strong outward rectification in both control and CH cells with similar rectification index (I+60/I−60=8.7±0.5 vs 8.2±0.8, P&gt;0.05). The steady‐state activation curves from both groups were almost overlapped. The time‐dependent activation of ICl.Ca during depolarization, reflecting the voltage‐dependent Ca2+ binding to the channel, were unchanged in hypoxic PASMCs. These results suggest Ca2+‐ or voltage‐dependent properties of CaCC were unaffected by CH. Thus, the enhanced ICl.Ca is mainly attributable to increased channel expression.Supported by grants from NIH and AHA.

Ca2+-activated Cl- channels at a glance.

Cl− channels are membrane proteins that are responsible for the passive flow of Cl− into and out of the cell. The Ca2+-activated Cl− channels (CaCCs) are attracting a lot of attention lately. The molecular identity of these channels remained elusive (for a review, see [Hartzell et al., 2005][1

The voltage dependence of the TMEM16B/anoctamin2 calcium-activated chloride channel is modified by mutations in the first putative intracellular loop

Ca2+-activated Cl− channels (CaCCs) are involved in several physiological processes. Recently, TMEM16A/anoctamin1 and TMEM16B/anoctamin2 have been shown to function as CaCCs, but very little information is available on the structure–function relations of these channels. TMEM16B is expressed in the cilia of olfactory sensory neurons, in microvilli of vomeronasal sensory neurons, and in the synaptic terminals of retinal photoreceptors. Here, we have performed the first site-directed mutagenesis study on TMEM16B to understand the molecular mechanisms of voltage and Ca2+ dependence. We have mutated amino acids in the first putative intracellular loop and measured the properties of the wild-type and mutant TMEM16B channels expressed in HEK 293T cells using the whole cell voltage-clamp technique in the presence of various intracellular Ca2+ concentrations. We mutated E367 into glutamine or deleted the five consecutive glutamates 386EEEEE390 and 399EYE401. The EYE deletion did not significantly modify the apparent Ca2+ dependence nor the voltage dependence of channel activation. E367Q and deletion of the five glutamates did not greatly affect the apparent Ca2+ affinity but modified the voltage dependence, shifting the conductance–voltage relations toward more positive voltages. These findings indicate that glutamates E367 and 386EEEEE390 in the first intracellular putative loop play an important role in the voltage dependence of TMEM16B, thus providing an initial structure–function study for this channel.

Peripheral antinociception induced by δ-opioid receptors activation, but not μ- or κ-, is mediated by Ca2 +-activated Cl− channels

Studies have demonstrated that the L-arginine/NO/cGMP pathway and the potassium and calcium channels are involved in the mechanisms underlying opioid receptor activation. As additional pathways may participate in the observed antinociceptive effects following opioid exposure, the aim of our study was to determine whether Ca2+-activated Cl− channels (CaCCs) are involved in peripheral antinociception induced by μ-, δ- and κ-opioid receptor activation. Hyperalgesia was induced by intraplantar injection of prostaglandin E2 (PGE2, 2μg). Nociceptive thresholds to pressure (grams) were measured using an algesimetric apparatus 3h following injection. The μ-opioid receptor agonist morphine (200μg), δ-opioid receptor agonist (+)-4-[(alphaR)-alpha-((2S,5R)-4-Allyl-2,5-dimethyl-1-piperazinyl)-3-methoxybenzyl]-N,N-diethylbenzamide (SNC80, 80μg), κ-opioid receptor agonist bremazocine (50μg), CaCCs blocker niflumic acid (8–64μg), CaCCs blocker 5-Nitro-2-(3-phenylpropylamino) benzoic acid (NPPB, 32–128μg), nitric oxide donor sodium nitroprusside (SNP, 500μg) and cGMP exogenous analogs dibutyryl cGMP (db-cGMP, 100μg) were also administered into the paw. The CaCCs blocker niflumic acid and NPPB partially reversed the peripheral antinociception induced by exposure to the SNC80 in a dose-dependent manner. In contrast, niflumic acid did not modify the antinociceptive effect observed following exposure to morphine or bremazocine. Additionally, the peripheral antinociception induced by the NO donor SNP or by db-cGMP was not inhibited by niflumic acid. These results provide evidence for the involvement of CaCCs in the peripheral antinociception induced by SNC80. CaCCs activation does not appear to be involved when μ- and κ-opioid receptors are activated. In addition, we did not observe a link between CaCCs and the L-arginine/NO/GMPc pathway.

ANOs 3–7 in the anoctamin/Tmem16 Cl− channel family are intracellular proteins

Ca(2+)-activated Cl(-) channels (CaCCs) participate in numerous physiological functions such as neuronal excitability, sensory transduction, and transepithelial fluid transport. Recently, it was shown that heterologously expressed anoctamins ANO1 and ANO2 generate currents that resemble native CaCCs. The anoctamin family (also called Tmem16) consists of 10 members, but it is not known whether all members of the family are CaCCs. Expression of ANOs 3-7 in HEK293 cells did not generate Cl(-) currents activated by intracellular Ca(2+), as determined by whole cell patch clamp electrophysiology. With the use of confocal imaging, only ANO1 and ANO2 traffic to the plasma membrane when expressed heterologously. Furthermore, endogenously expressed ANO7 in the human prostate is predominantly intracellular. We took a chimeric approach to identify regions critical for channel trafficking and function. However, none of the chimeras of ANO1 and ANO5/7 that we made trafficked to the plasma membrane. Our results suggest that intracellular anoctamins may be endoplasmic reticulum proteins, although it remains unknown whether these family members are CaCCs. Determining the role of anoctamin family members in ion transport will be critical to understanding their functions in physiology and disease.

Flash Photolysis of Caged Compounds in the Cilia of Olfactory Sensory Neurons

Photolysis of caged compounds allows the production of rapid and localized increases in the concentration of various physiologically active compounds. Caged compounds are molecules made physiologically inactive by a chemical cage that can be broken by a flash of ultraviolet light. Here, we show how to obtain patch-clamp recordings combined with photolysis of caged compounds for the study of olfactory transduction in dissociated mouse olfactory sensory neurons. The process of olfactory transduction (Figure 1) takes place in the cilia of olfactory sensory neurons, where odorant binding to receptors leads to the increase of cAMP that opens cyclic nucleotide-gated (CNG) channels. Ca entry through CNG channels activates Ca-activated Cl channels. We show how to dissociate neurons from the mouse olfactory epithelium and how to activate CNG channels or Ca-activated Cl channels by photolysis of caged cAMP or caged Ca. We use a flash lamp to apply ultraviolet flashes to the ciliary region to uncage cAMP or Ca while patch-clamp recordings are taken to measure the current in the whole-cell voltage-clamp configuration.

Activation of P2Y1 and P2Y2 receptors induces chloride secretion via calcium-activated chloride channels in kidney inner medullary collecting duct cells

Dysregulation of urinary sodium chloride (NaCl) excretion can result in extracellular fluid (ECF) volume expansion and hypertension. Recent studies demonstrated that urinary nucleotide excretion increases in mice ingesting a high-salt diet and that these increases in extracellular nucleotides can signal through P2Y(2) receptors in the kidney collecting duct to inhibit epithelial Na(+) channels (ENaC). However, under conditions of ECF volume expansion brought about by high-dietary salt intake, ENaC activity should already be suppressed. We hypothesized that alternative pathways exist by which extracellular nucleotides control renal NaCl excretion. We used an inner medullary collecting duct (mIMCD-K2) cell line in an Ussing chamber system as a model to study additional ion transport pathways that are regulated by extracellular nucleotides. When ENaC was inhibited, the addition of adenosine triphosphate (ATP) to the basal side of cell sheets activated both P2Y(1) and P2Y(2) receptors, inducing a transient increase in short-circuit current (I(sc)); addition of ATP to the apical side activated only P2Y(2) receptors, inducing first a transient and then a sustained increase in I(sc). The ATP-induced increases in I(sc) were blocked by pretreatment with a phospholipase C (PLC) inhibitor, a calcium (Ca(2+)) chelator, or Ca(2+)-activated Cl(-) channel (CACC) inhibitors, suggesting that ATP signals through both PLC and intracellular Ca(2+) to activate CACC. We propose that P2Y(1) and P2Y(2) receptors operate in tandem in IMCD cells to provide an adaptive mechanism for enhancing urinary NaCl excretion in the setting of high-dietary NaCl intake.

Voltage- and calcium-dependent gating of TMEM16A/Ano1 chloride channels are physically coupled by the first intracellular loop

Ca(2+)-activated Cl(-) channels (CaCCs) are exceptionally well adapted to subserve diverse physiological roles, from epithelial fluid transport to sensory transduction, because their gating is cooperatively controlled by the interplay between ionotropic and metabotropic signals. A molecular understanding of the dual regulation of CaCCs by voltage and Ca(2+) has recently become possible with the discovery that Ano1 (TMEM16a) is an essential subunit of CaCCs. Ano1 can be gated by Ca(2+) or by voltage in the absence of Ca(2+), but Ca(2+)- and voltage-dependent gating are very closely coupled. Here we identify a region in the first intracellular loop that is crucial for both Ca(2+) and voltage sensing. Deleting (448)EAVK in the first intracellular loop dramatically decreases apparent Ca(2+) affinity. In contrast, mutating the adjacent amino acids (444)EEEE abolishes intrinsic voltage dependence without altering the apparent Ca(2+)affinity. Voltage-dependent gating of Ano1 measured in the presence of intracellular Ca(2+) was facilitated by anions with high permeability or by an increase in [Cl(-)](e). Our data show that the transition between closed and open states is governed by Ca(2+) in a voltage-dependent manner and suggest that anions allosterically modulate Ca(2+)-binding affinity. This mechanism provides a unified explanation of CaCC channel gating by voltage and ligand that has long been enigmatic.

Enhanced Ca2+‐activated Cl− conductance and TMEM16A expression in pulmonary arterial smooth muscle in a rat model of chronic hypoxic pulmonary hypertension

Ca2+‐activated Cl− Channel (CaCC) encoded by TMEM16A plays an important role in the regulation of vascular tone. Here we sought to determine whether TMEM16A expression and CaCC currents (ICl.Ca) in pulmonary arterial smooth muscle cells (PASMCs) are altered in a rat model of chronic hypoxic pulmonary hypertension (CHPH). TMEM16A mRNA was quantified by qRT‐PCR and protein by immunoblotting. ICl.Ca was recorded using whole‐cell or perforated patch clamp technique under K+ free conditions. The TMEM16A mRNA was more abundant in pulmonary arteries (PA) compared to cerebral, tail, mesenteric and renal arteries in normoxic rats. In Fluo‐3/AM‐loaded PASMCs, caffeine‐induced Ca2+ release evoked robust outward currents at 0 mV (19.9±2.4 pA/pF) and inward current at −80 mV (−21.7±3.1 pA/pF). The current was inhibited by niflumic acid, and reversed at the predicted ECl following the changes of [Cl−] gradient, as consistent with ICl.Ca. Chronic hypoxia (10% O2, 4 weeks) caused an increase in ICl.Ca density evoked by caffeine (30.0 ± 6.8 pA/pF at 0 mV) or by depolarization with [Ca2+]i clamped at 750 nM (124.2 ± 24.1 vs. 24.4 ± 3.3 pA/pF at +120 mV) in PASMCs, accompanied by ~ 2‐fold increase in TMEM16A mRNA and 5‐fold increase in protein levels in PA. Our results show that TMEM16A and functional CaCC are highly expressed in rat PA. Their upregulation by hypoxia may contribute to the enhanced pulmonary vasoconstriction observed in CHPH.

Basolateral membrane Cl conductance in guinea pig distal colon activates during electrogenic K secretion and Cl secretion

Epinephrine (epi) activates transient Cl secretion (sec) and sustained K sec across isolated distal colonic mucosa of guinea pig. The Ca-activated Cl channel inhibitor CaCCinh-A01 [30μM] reduced electrogenic K sec, detected as short-circuit current (Isc), consistent with basolateral Cl channels providing an exit pathway for Cl entering via Na/K/2Cl-cotransporters. CaCCinh-A01 inhibited both Isc and transepithelial conductance in a concentration dependent manner, IC50 = 6.3μM. GlyH-101, another Cl channel inhibitor, also reduced K secretory Isc (IC50 = 9.4μM). Epi activated whole-cell Cl current in isolated intact colonic crypts. This epi-activated Cl current also was inhibited by CaCCinh-A01 or GlyH-101. In contrast to K sec, CaCCinh-A01 augmented epi-activated Cl secretory Isc as well as PGE2-activated Cl sec. Synergistic Cl sec activated by cholinergic/PGE2 stimulation was insensitive to CaCCinh-A01. Colonic expression of Tmem16A, a Ca-activated Cl channel, was supported by RT-PCR of Tmem16A-mRNA, immuno-blot with Tmem16A-antibodies, and immuno-reactivity in lateral membranes of epithelial cells. Alternative splices of Tmem16A were detected for exons involved in channel activation. Inhibition of K sec and augmentation of Cl sec by CaCCinh-A01 supports a common colonic cell model for these two ion secretory processes with basolateral membrane Cl channels that contribute to production of electrogenic K sec and limiting Cl sec. Maximal Cl sec occurs only for synergistic activation mechanisms that inactivate these basolateral membrane Cl channels. [NIH DK65845]

Tmem16a Ca 2+ Activated Chloride Channels in Sensory Neurons can Discriminate between Different Intracellular Ca 2+ Sources

Proteins of Tmem16 (anoctamin) family are the candidate subunits for Ca2+-activated Cl- channels (CaCC). Both Tmem16a expression and CaCC currents were reported in small (predominantly nociceptive) sensory neurons from dorsal root ganglia (DRG). In contrast to CNS neurons, nociceptors accumulate high intracellular Cl- concentration and thus activation of CaCC in these neurons was shown to be excitatory and to contribute to the inflammatory nociceptive responses. We show that CaCC in nociceptors is activated by the release of Ca2+ from the IP3-sensitive intracellular stores in response to bradykinin (BK; 44/47 responsive neurons) or proteases (through PAR-2 receptors; 8/12 responsive neurons). This current was attenuated by 48% by siRNA knock-down of the Tmem16a (n=7). Interestingly, while in the majority of small DRG neurons CACC was induced by Ca2+ release from the stores, only in 12/58 neurons CaCC was activated by the Ca2+ influx through the voltage-gated Ca2+ channels triggered by the voltage pulse. Chelating intracellular Ca2+ with the slow Ca2+ buffer EGTA did not affect CaCC activation by PAR2-AP (5/7 responsive neurons) while the fast buffer BAPTA abolished such activation (1/9 responsive neurons) suggesting a close proximity of the Ca2+ release sites and CaCC. Acute treatment of DRG neurons with lipid raft disrupting agent methyl-beta-cyclodextrin (MBCD) partially restored coupling of CaCC to VGCC but disrupted coupling with the intracellular stores. Thus, after the MBCD treatment activation of VGCC by voltage pulse induced −106.5±51.4 pA CaCC currents in 17/25 neurons while BK and PAR2-AP failed to induce measurable CaCC in 4 and 8 tested neurons respectively (PAR2-AP induced −100±20 pA CaCC in 9/13 MBCD-untreated neurons). Or data suggest that Tmem16a/CaCC channels in small DRG neurons are functionally coupled to the IP3-sensitive Ca2+ stores.

Interactive Calcium and Voltage Gating of TMEM16A/Anoctamin1 Chloride Channels

Ca2+-activated Cl− channels (CaCCs) play important physiological roles, including epithelial fluid transport and secretion, sensory transduction and adaptation, regulation of smooth muscle contraction, control of neuronal and cardiac excitability, and nociception. Recently, Ano1 and Ano2 have been identified as CaCCs, but the mechanisms of gating by Ca2+ and voltage remain unknown. Here we use excised macropatch and single channel recording of Ano1 transiently expressed in HEK cells to explore its gating mechanisms. The studies show that Ano1 is essentially a Ca2+ ligand-gated channel whose voltage dependence is conferred not by a voltage sensor composed of charged amino acids but by permeant ions stabilizing channel open state. Increasing internal [Ca2+] in excised inside-out patches dramatically shifts the G-V curve to the left, consistent with Ca2+ causing an increase in po. Allosteric interaction between Ca2+ binding and anion permeation is shown by the observation that the position of the G-V curve on the voltage axis is dependent on the permeant anion, with a leftward shift when Cl- is replaced by SCN- or NO3-. Single channel analysis confirms that channel open probability increases with [Ca2+] and depolarization, but, in addition, single channel conductance is several-fold larger at positive potentials than at negative potentials. This supports the idea that occupancy of the pore by permeant anion stabilizes a conductive state and that occupancy is higher at positive potentials than at negative potentials. Gating of Ano1 is controlled by at least two Ca2+ binding sites. Acidic amino acids (E444-448, D452, E457, E464, E470, and E475) in the first intracellular loop are important for both voltage and Ca2+ gating of Ano1. These data provide new insights into the mechanisms of CaCC channel gating by voltage and ligand that have long been enigmatic.

Scan: A Novel Small-Conductance Ca2+-Activated Non-Selective Cation Channel Encoded by TMEM16F

Raising internal calcium level causes channel opening to regulate a wide range of cellular functions including neuronal signalling, muscle contraction and excitation-secretion coupling. Named after their ion selectivity, Ca2+-activated K+ channels such as the BK and SK channels with big and small single channel conductance respectively, Ca2+-activated Cl- channels (CaCC), and Ca2+-activated non-selective cation channels (CAN) respond to Ca2+ entry through ligand-gated or voltage-gated ion channels, or Ca2+ release from internal stores. Recently, we and others identified two of the ten members of the mammalian TMEM16 family, TMEM16A and TMEM16B, as CaCC. Here we show that, surprisingly, expression of another family member, TMEM16F, in Axolotloocytes yielded Ca2+-activated non-selective cation channels with novel properties. In contrast to the CAN channels that permeate mainly monovalent cations and show relatively little voltage sensitivity, the TMEM16F-induced channels permeate divalent cations as well and exhibit synergistic gating by Ca2+ and voltage - the higher the internal Ca2+ level, the less depolarization necessary for channel activation. Moreover, the sub picosiemens single channel conductance of the Ca2+-permeable TMEM16F channel is much smaller than the single channel conductance of the CAN channels whose discovery was ushered in by the advent of single channel recording three decades ago. This surprise finding of the novel SCAN channel (small-conductance Ca2+-activated non-selective cation channel) generated by TMEM16F, which is broadly expressed in many different cell types, will enable genetic studies of its physiological functions as exemplified by recent studies of Tmem16a mutant mice, and facilitate development of specific channel modulators of potential therapeutic applications.