TMEM16 proteins: the long awaited calcium-activated chloride channels?

Currents mediated by calcium-activated chloride channels (CaCCs), observed for the first time in Xenopus oocytes, have been recorded in many cells and tissues ranging from different types of neurons to epithelial and muscle cells. CaCCs play a role in the regulation of excitability in neurons including sensory receptors. In addition, they are crucial mediators of chloride movements in epithelial cells where their activity regulates electrolyte and fluid transport. The roles of CaCCs, particularly in epithelia, are briefly reviewed with emphasis on their function in secretory epithelia. The recent identification by three independent groups, using different strategies, of TMEM16A as the molecular counterpart of the CaCC is discussed. TMEM16A is part of a family that has 10 other members in mice. The discovery of the potential TMEM16 anion channel activity opens the way for the molecular investigation of the role of these anion channels in specific cells and in organ physiology and pathophysiology. The identification of TMEM16A protein as a CaCC chloride channel molecule represents a great triumph of scientific perseverance and ingenuity. The varied approaches used by the three independent research groups also augur well for the solidity of the discovery.

TMEM16 proteins: the long awaited calcium-activated chloride channels?

Currents mediated by calcium-activated chloride channels (CaCCs), observed for the first time in Xenopus oocytes, have been recorded in many cells and tissues ranging from different types of neurons to epithelial and muscle cells. CaCCs play a role in the regulation of excitability in neurons including sensory receptors. In addition, they are crucial mediators of chloride movements in epithelial cells where their activity regulates electrolyte and fluid transport. The roles of CaCCs, particularly in epithelia, are briefly reviewed with emphasis on their function in secretory epithelia. The recent identification by three independent groups, using different strategies, of TMEM16A as the molecular counterpart of the CaCC is discussed. TMEM16A is part of a family that has 10 other members in mice. The discovery of the potential TMEM16 anion channel activity opens the way for the molecular investigation of the role of these anion channels in specific cells and in organ physiology and pathophysiology. The identification of TMEM16A protein as a CaCC chloride channel molecule represents a great triumph of scientific perseverance and ingenuity. The varied approaches used by the three independent research groups also augur well for the solidity of the discovery.

Gating of two-pore domain K+ channels by extracellular pH.

Potassium channels have a conserved selectivity filter that is important in determining which ions are conducted and at what rate. Although K+ channels of different conductance characteristics are known, they differ more widely in the way their opening and closing, the gating, is governed. TASK and TALK subfamily proteins are two-pore region KCNK K+ channels gated open by extracellular pH. We discuss the mechanism for this gating in terms of electrostatic effects on the pore changing the occupancy and open probability of the channels in a way reminiscent of C-type inactivation gating at the selectivity filter. Essential to this proposed mechanism is the replacement of two highly conserved aspartate residues at the pore mouth by asparagine or histidine residues in the TALK and TASK channels.

The role of the agonist binding site in Ca(2+) inhibition of the recombinant 5-HT(3A) receptor.

The mechanism and site of action of Ca(2+) at the recombinant murine 5-hydroxytryptamine (5-HT)(3A) receptor were investigated using whole-cell voltage clamp, radioligand binding and single-cell Ca(2+) imaging. Inhibition of the 5-HT (3 microM)-induced response by 10 mM Ca(2+) reached a plateau at 68.5% inhibition, with half-maximal effect at 2.6 mM. This was due to an increase in EC(50) from 2.35 to 3.87 microM and a 30% reduction in I(max). Ca(2+) also resulted in the inhibition of binding of both 5-HT(3) receptor agonist [3H]m-chlorophenylbiguanide and antagonist [3H]granisetron due to an increase in K(d), with no change in B(max). An increase in EC(50) from 2.6 (1 mM Ca(2+)) to 4.7 microM (10 mM Ca(2+)), with no change in maximal [Ca(2+)](i), was observed from Ca(2+) imaging studies. Largely similar effects were observed with Mg(2+). The combined data suggest that Ca(2+) acting at a site that directly or indirectly influences the agonist binding site plays a significant role in its inhibitory effect at the 5-HT(3) receptor.

Modulation of the two-pore domain acid-sensitive K+ channel TASK-2 (KCNK5) by changes in cell volume.

The molecular identity of K(+) channels involved in Ehrlich cell volume regulation is unknown. A background K(+) conductance is activated by cell swelling and is also modulated by extracellular pH. These characteristics are most similar to those of newly emerging TASK (TWIK-related acid-sensitive K(+) channels)-type of two pore-domain K(+) channels. mTASK-2, but not TASK-1 or -3, is present in Ehrlich cells and mouse kidney tissue from where the full coding sequences were obtained. Heterologous expression of mTASK-2 cDNA in HEK-293 cells generated K(+) currents in the absence intracellular Ca(2+). Exposure to hypotonicity enhanced mTASK-2 currents and osmotic cell shrinkage led to inhibition. This occurred without altering voltage dependence and with only slight decrease in pK(a) in hypotonicity but no change in hypertonicity. Replacement with other cations yields a permselectivity sequence for mTASK-2 of K(+) > Rb(+) Cs(+) > NH(4)(+) > Na(+) congruent with Li(+), similar to that for the native conductance (I(K, vol)). Clofilium, a quaternary ammonium blocker of I(K, vol), blocked the mTASK-2-mediated K(+) current with an IC(50) of 25 microm. The presence of mTASK-2 in Ehrlich cells, its functional similarities with I(K, vol), and its modulation by changes in cell volume suggest that this two-pore domain K(+) channel participates in the regulatory volume decrease phenomenon.

Cloning, cellular distribution and functional expression of small intestinal epithelium guinea pig ClC-5 chloride channel.

We report the cloning of a guinea pig ClC-5 chloride channel (gpClC-5) from distal small intestinal epithelial cells by RT-PCR and RACE. The transcript is shown to be present in duodenum, jejunum and ileum epithelium by RT-PCR and Northern analysis. This is confirmed by in situ hybridisation which also shows the transcript to be homogeneously distributed in the crypt and villus regions. Expression of gpClC-5 in HEK-293 cells generated markedly outwardly rectified chloride currents with a perm-selectivity sequence of NO(3)(-)>Cl(-)>Br(-)>I(-)>F(-)>gluconate(-). The possible role of gpClC-5 in this epithelial location is discussed.

K+ conductance activated during regulatory volume decrease. The channels in Ehrlich cells and their possible molecular counterpart.

K+ currents activated by hypotonic cell swelling have been studied in Ehrlich ascites tumour cells by the whole-cell recording mode of the patch-clamp technique. K+ together with Cl- currents developed in the absence of added intracellular Ca2+ and with strong buffering of internal Ca2+ in experiments conducted at 37 degrees C. Manipulation of the extracellular medium with other cations suggests a selectivity sequence of K+ > Rb+ > NH4+ > or = Na+ approximately equals Li+ approximately equals Cs+. The current-voltage relationship of the volume-sensitive K+ current was well fitted with the Goldman-Hodgkin-Katz current equation between -130 and 20 mV at both physiological and high K+ extracellular solutions. The class III antiarrhytmic drug clofilium blocked the volume-sensitive K+ current in a voltage-independent manner. Clofilium was also found to be a strong inhibitor of the regulatory volume decrease (RVD) response of Ehrlich cells. The leukotriene D4 (LTD4) can activate the same current in isotonicity, consistent with a role for this compound in the signalling process of volume regulation. It is suggested that K+ channels activated by cell swelling belong to the so-called background K+ channel group. These are voltage-independent channels which underlie the resting potential of many cells and have recently been identified as belonging to a family of K+ channels with two pore domains in tandem (2P-4TM). Preliminary experiments show the presence of the TASK-2 channel, a member of the 2P-4TM family inhibited by acid extracellular pH, in Ehrlich cells and suggest that it might underlie the swelling-induced K+ current.

Splice variants of a ClC-2 chloride channel with differing functional characteristics.

We identified two ClC-2 clones in a guinea pig intestinal epithelial cDNA library, one of which carries a 30-bp deletion in the NH(2) terminus. PCR using primers encompassing the deletion gave two products that furthermore were amplified with specific primers confirming their authenticity. The corresponding genomic DNA sequence gave a structure of three exons and two introns. An internal donor site occurring within one of the exons accounts for the deletion, consistent with alternative splicing. Expression of the variants gpClC-2 and gpClC-2Delta77-86 in HEK-293 cells generated inwardly rectifying chloride currents with similar activation characteristics. Deactivation, however, occurred with faster kinetics in gpClC-2Delta77-86. Site-directed mutagenesis suggests that a protein kinase C-mediated phosphorylation consensus site lost in gpClC-2Delta77-86 is not responsible for the observed change. The deletion-carrying variant is found in most tissues examined, and it appears more abundant in proximal colon, kidney, and testis. The presence of a splice variant of ClC-2 modified in its NH(2)-terminal domain could have functional consequences in tissues where their relative expression levels are different.

K+ currents activated by leukotriene D4 or osmotic swelling in Ehrlich ascites tumour cells.

K+ and Cl- currents activated by hypoosmotic cell swelling (IK,vol and Icl,vol) or after addition of leukotriene D4 (LTD4) to cells in isotonic medium were studied in Ehrlich ascites tumour cells. IK,vol and Icl,vol were not affected by strong buffering of intracellular Ca2+ or by additional removal of extracellular Ca2+. In isotonic media, 5 nmol/l LTD4 activated large K+ but not Cl- currents. The LTD4-activated IK was, as has been shown previously for IK,vol, insensitive to charybdotoxin (ChTX) but was blocked by the antiarrhythmic drug clofilium. The current/voltage (I/V) relation for the LTD4-activated IK was, as recently demonstrated for IK,vol, well fitted by the Goldman-Hodgkin-Katz current equation between -130 mV and 30 mV in both physiological and K+-rich extracellular solutions. LTD4 had no additional effect on the magnitude of IK in Ehrlich cells already activated by the hypoosmotic stimulus. Nevertheless, the onset time for IK after hypoosmotic cell swelling was significantly less in the presence of LTD4. The similar I/V relation, pharmacological sensitivity and lack of additivity suggest that hypoosmotic swelling and addition of LTD4 activate the same K+ channels in Ehrlich cells. The influence of [Ca2+]i appears, however, to differ between IK,vol and the IK activated by LTD4 in that the latter was reduced significantly by strong buffering of [Ca2+]i. This might reflect the involvement of some additional factor in the hypoosmotic activation of K+ channels besides the stimulation mediated by LTD4.

Characterisation of a cell swelling-activated K+-selective conductance of ehrlich mouse ascites tumour cells.

The K+ and Cl- currents activated by hypotonic cell swelling were studied in Ehrlich ascites tumour cells using the whole-cell recording mode of the patch-clamp technique. Currents were measured in the absence of added intracellular Ca2+ and with strong buffering of Ca2+. K+ current activated by cell swelling was measured as outward current at the Cl- equilibrium potential (ECl) under quasi-physiological gradients. It could be abolished by replacing extracellular Na+ with K+, thereby cancelling the driving force. Replacement with other cations suggested a selectivity sequence of K+ > Rb+ > NH4 approximately Na+ approximately Li+; Cs+ appeared to be inhibitory. The current-voltage relationship of the volume-sensitive K+ current was well fitted with the Goldman-Hodgkin-Katz current equation between -130 and +20 mV with a permeability coefficient of around 10(-6) cm s(-1) with both physiological and high-K+ extracellular solutions. The class III antiarrhythmic drug clofilium blocked the volume-sensitive K+ current in a voltage-independent manner with an IC50 of 32 microM. Clofilium was also found to be a strong inhibitor of the regulatory volume decrease response of Ehrlich cells. Cell swelling-activated K+ currents of Ehrlich cells are voltage and calcium insensitive and are resistant to a range of K+ channel inhibitors. These characteristics are similar to those of the so-called background K+ channels. Noise analysis of whole-cell current was consistent with a unitary conductance of 5.5 pS for the single channels underlying the K+ current evoked by cell swelling, measured at 0 mV under a quasi-physiological K+ gradient.

Different efficacy of specific agonists at 5-HT3 receptor splice variants: the role of the extra six amino acid segment.

1. Whole cell voltage clamp electrophysiology and radioligand binding were used to examine the agonist characteristics of the two splice variants of the 5-HT3 receptor which have been cloned from neuronal cell lines. Homo-oligomeric 5-HT3 receptors were examined in HEK 293 cells stably transfected with either long (5-HT3-L) or short (5-HT3-S) receptor subunit DNAs. 2. Functional homo-oligomeric receptors were formed from both subunits, and responses to 5-HT3 receptor agonists (5-hydroxytryptamine (5-HT), 2-methyl 5-HT and m-chlorophenylbiguanide) were qualitatively similar. 3. Maximum currents (Rmax) elicited by the 5-HT3 receptor agonists m-chlorophenylbiguanide (mCPBG) and 2-methyl-5-HT (2-Me-5-HT), as compared to 5-HT, differed in the two splice variants: Rmax mCPBG/Rmax 5-HT values were 0.68+/-0.04 and 0.91+/-0.01 in 5-HT3-L and 5-HT3-S receptors, respectively. Comparable values for 2-Me-5-HT were 0.30+/-0.02 and 0.23+/-0.02. 4. Radioligand binding data showed no difference in affinity of agonist or antagonist binding sites; thus the six amino acid deletion appears to cause differences in agonist efficacy. 5. The role of the 6 amino acid insertion was further investigated by use of site-directed mutagenesis to create two mutant receptors, one where serine 286 was replaced with alanine, and the second where all 6 amino acids were replaced with alanines. 6. Examination of the mutant receptors when stably expressed in HEK 293 cells revealed agonist properties resembling long and not short 5-HT3 receptors. Thus specific amino acids in this region are not responsible for the observed differences. 7. The data show intracellular structure can have significant effects on ligand-gated ion channel function, and suggest that minor changes in structure may be responsible for differences in function observed when ligand-gated ion channel proteins are modulated intracellularly.

Voltage control of calcium transients elicited by caffeine and tetracaine in cultured rat muscle cells.

Cultured hind limb skeletal muscle cells from newborn rats were used to study the effect of caffeine and tetracaine upon intracellular Ca2+ release under voltage or current clamp conditions. Free [Ca2+]i was measured using the fluorescent calcium-sensitive dye Fluo-3. A field containing one or several myotubes was observed with a video camera and image analysis of fluorescence changes was performed. Addition of 100-500 microM tetracaine to the external saline elicited strong fluorescence responses in non-clamped cells, but significantly lower responses in cells clamped at -90 mV. At the same time, tetracaine inhibited voltage induced calcium release. Voltage and tetracaine modulation over the action of caffeine (500 microM) was also observed. Pretreatment of cells with 10 microM nifedipine abolished the caffeine induced fluorescence response in non-clamped cells. These findings suggest that, in cultured muscle cells, calcium release through the caffeine and tetracaine sensitive pathways is controlled by both membrane potential and the dihydropyridine receptor.