Loretta Ferrera

TMEM16A, A Membrane Protein Associated with Calcium-Dependent Chloride Channel Activity

Calcium-dependent chloride channels are required for normal electrolyte and fluid secretion, olfactory perception, and neuronal and smooth muscle excitability. The molecular identity of these membrane proteins is still unclear. Treatment of bronchial epithelial cells with interleukin-4 (IL-4) causes increased calcium-dependent chloride channel activity, presumably by regulating expression of the corresponding genes. We performed a global gene expression analysis to identify membrane proteins that are regulated by IL-4. Transfection of epithelial cells with specific small interfering RNA against each of these proteins shows that TMEM16A, a member of a family of putative plasma membrane proteins with unknown function, is associated with calcium-dependent chloride current, as measured with halide-sensitive fluorescent proteins, short-circuit current, and patch-clamp techniques. Our results indicate that TMEM16A is an intrinsic constituent of the calcium-dependent chloride channel. Identification of a previously unknown family of membrane proteins associated with chloride channel function will improve our understanding of chloride transport physiopathology and allow for the development of pharmacological tools useful for basic research and drug development.

Regulation of TMEM16A chloride channel properties by alternative splicing

Expression of TMEM16A protein is associated with the activity of Ca2+-activated Cl− channels. TMEM16A primary transcript undergoes alternative splicing. thus resulting in the generation of multiple isoforms. We have determined the pattern of splicing and assessed the functional properties of the corresponding TMEM16A variants. We found three alternative exons, 6b, 13, and 15, coding for segments of 22, 4, and 26 amino acids, respectively, which are differently spliced in human organs. By patch clamp experiments on transfected cells, we found that skipping of exon 6b changes the Ca2+ sensitivity by nearly 4-fold, resulting in Cl− currents requiring lower Ca2+ concentrations to be activated. At the membrane potential of 80 mV, the apparent half-effective concentration decreases from 350 to 90 nm when the segment corresponding to exon 6b is excluded. Skipping of exon 13 instead strongly reduces the characteristic time-dependent activation observed for Ca2+-activated Cl− channels at positive membrane potentials. This effect was also obtained by deleting only the second pair of amino acids corresponding to exon 13. Alternative splicing appears as an important mechanism to regulate the voltage and Ca2+ dependence of the TMEM16A-dependent Cl− channels in a tissue-specific manner.

The anoctamin family: TMEM16A and TMEM16B as calcium‐activated chloride channels

The Ca2+‐activated Cl− channels (CaCCs) are involved in a variety of physiological functions, such as transepithelial anion transport, smooth muscle contraction and olfaction. Recently, the question of the molecular identity of CaCCs has apparently been resolved with the identification of TMEM16A protein (also known as anoctamin‐1). Expression of TMEM16A is associated with the appearance of Ca2+‐ and voltage‐dependent Cl− currents with properties similar to those of native CaCCs. The putative structure of TMEM16A consists of eight transmembrane domains, with both the amino‐ and the carboxy‐terminus protruding in the cytosol. TMEM16A is also characterized by the existence of different protein variants generated by alternative splicing. A close paralogue of TMEM16A, TMEM16B (anoctamin‐2), is also associated with CaCC activity, although with different properties. The TMEM16B‐dependent channels require higher intracellular Ca2+ concentrations and have faster activation and deactivation kinetics. Expression of other anoctamins is devoid of detectable channel activity. These proteins, such as TMEM16F (anoctamin‐6), may have different functions.

Drastic reduction of the slow gate of human muscle chloride channel (ClC‐1) by mutation C277S

1 Single channel measurements suggest that the human muscle chloride channel ClC‐1 presumably has a double barrelled structure, with a fast single protopore gate and a slow common pore gate similar to that of ClC‐0, the chloride channel from Torpedo. The single point mutation C212S has been shown to abolish the slow gating of ClC‐0 locking the slow gate in the open state. In order to test the hypothesis that the slow gating process found in ClC‐1 corresponds to the well characterised slow gate found in ClC‐0 we investigated the gating effects in ClC‐1 of the homologous mutation corresponding to C212S, C277S. 2 We found that the mutation C277S strongly reduced the slow component of macroscopic gating relaxations at negative and at positive voltages. 3 Time constants of the fast gating relaxations were not affected by the mutation but the minimal open probability of the fast gate at negative voltages was slightly reduced to 0.08 compared with the WT value of 0.22. 4 Additionally, we characterised the block of WT ClC‐1 and mutant C277S by the S(—) enantiomer of CPB (2‐(p‐chlorophenoxy) butyric acid), and found that the block is practically unaffected by the mutation suggesting that CPB does not interact with the slow gate of ClC‐1. 5 We conclude that the slow and fast gating processes of ClC‐1, respectively, reflect the slow common pore gate and the single protopore gate of the double‐barrelled ClC‐1 channel.

A minimal isoform of the TMEM16A protein associated with chloride channel activity

TMEM16A protein, also known as anoctamin-1, has been recently identified as an essential component of Ca2+-activated Cl− channels. We previously reported the existence of different TMEM16A isoforms generated by alternative splicing. In the present study, we have determined the functional properties of a minimal TMEM16A protein. This isoform, called TMEM16A(0), has a significantly shortened amino-terminus and lacks three alternative segments localized in the intracellular regions of the protein (total length: 840 amino acids). TMEM16A(0) expression is associated with Ca2+-activated Cl− channel activity as measured by three different functional assays based on the halide-sensitive yellow fluorescent protein, short-circuit current recordings, and patch-clamp technique. However, compared to a longer isoform, TMEM16(abc) (total length: 982 amino acids), TMEM16A(0) completely lacks voltage-dependent activation. Furthermore, TMEM16A(0) and TMEM16A(abc) have similar but not identical responses to extracellular anion replacement, thus suggesting a difference in ion selectivity and conductance. Our results indicate that TMEM16A(0) has the basic domains required for anion transport and Ca2+-sensitivity. However, the absence of alternative segments, which are present in more complex isoforms of TMEM16A, modifies the channel gating and ion transport ability.

Two open states and rate‐limiting gating steps revealed by intracellular Na+ block of human KCNQ1 and KCNQ1/KCNE1 K+ channels

KCNQ1, the first member of a new K+ channel family, associates with the small KCNE1 subunit to form the slow cardiac delayed rectifier current, IKs. Mutations in both genes encoding these channels lead to cardiac arrhythmia. We studied the block by intracellular Na+ of human homomeric KCNQ1 (homomers) and heteromeric KCNQ1/KCNE1 (heteromers) expressed in CHO cells (Chinese hamster ovary cell line) using whole‐cell patch recording. In the nominal absence of extracellular K+ and with 65 mm intracellular K+, the replacement of 65 mm intracellular N‐methyl‐d‐glucamine (NMDG+) by 65 mm Na+ induced a decay of outward (K+) currents through homomers after maximal activation reminiscent of an inactivation process. The decay had a time constant in the hundreds of milliseconds range. The inactivation process of homomers was, however, not directly dependent on [Na+]i, as evidenced by unaltered biphasic deactivation at negative voltages. An instantaneous voltage‐dependent Na+ block of homomers was revealed using tail current protocols with activating prepulses that saturated the gating processes of the channel. The instantaneous block was partially relieved at very large positive voltages (≥ 60 mV) and in 20 mm extracellular K+. The instantaneous block of homomers was much less pronounced if the tail currents were measured after short activating prepulses, demonstrating the presence of (at least) two open states: a first, relatively [Na+]i‐insensitive and a subsequent [Na+]i‐sensitive open state; the current decay reflects the transition between the two open states. Heteromers exhibited a very similar instantaneous block by Na+i independently of the prepulse duration. Heteromers did not show a Na+i‐induced current decay. Our results demonstrate the presence of two open states of KCNQ1 channels with different [Na+]i sensitivities. The rate‐limiting step of homomeric KCNQ1 gating at positive voltages is the transition between these two open states. The rate‐limiting step of the gating of KCNQ1/KCNE1 channels appears to be the entry into the first open state.

Ca2+‐Activated Cl− Channels

Ca(2+)-activated Cl(-) channels (CaCCs) are plasma membrane proteins involved in various important physiological processes. In epithelial cells, CaCC activity mediates the secretion of Cl(-) and of other anions, such as bicarbonate and thiocyanate. In smooth muscle and excitable cells of the nervous system, CaCCs have an excitatory role coupling intracellular Ca(2+) elevation to membrane depolarization. Recent studies indicate that TMEM16A (transmembrane protein 16 A or anoctamin 1) and TMEM16B (transmembrane protein 16 B or anoctamin 2) are CaCC-forming proteins. Induced expression of TMEM16A and B in null cells by transfection causes the appearance of Ca(2+)-activated Cl(-) currents similar to those described in native tissues. Furthermore, silencing of TMEM16A by RNAi causes disappearance of CaCC activity in cells from airway epithelium, biliary ducts, salivary glands, and blood vessel smooth muscle. Mice devoid of TMEM16A expression have impaired Ca(2+)-dependent Cl(-) secretion in the epithelial cells of the airways, intestine, and salivary glands. These animals also show a loss of gastrointestinal motility, a finding consistent with an important function of TMEM16A in the electrical activity of gut pacemaker cells, that is, the interstitial cells of Cajal. Identification of TMEM16 proteins will help to elucidate the molecular basis of Cl(-) transport.

Upregulation of TMEM16A protein in bronchial epithelial cells by bacterial pyocyanin

Induction of mucus hypersecretion in the airway epithelium by Th2 cytokines is associated with the expression of TMEM16A, a Ca2+-activated Cl- channel. We asked whether exposure of airway epithelial cells to bacterial components, a condition that mimics the highly infected environment occurring in cystic fibrosis (CF), also results in a similar response. In cultured human bronchial epithelial cells, treatment with pyocyanin or with a P. aeruginosa culture supernatant caused a significant increase in TMEM16A function. The Ca2+-dependent Cl- secretion, triggered by stimulation with UTP, was particularly enhanced by pyocyanin in cells from CF patients. Increased expression of TMEM16A protein and of MUC5AC mucin by bacterial components was demonstrated by immunofluorescence in CF and non-CF cells. We also investigated TMEM16A expression in human bronchi by immunocytochemistry. We found increased TMEM16A staining in the airways of CF patients. The strongest signal was observed in CF submucosal glands. Our results suggest that TMEM16A expression/function is upregulated in CF lung disease, possibly as a response towards the presence of bacteria in the airways.

Pharmacological analysis of epithelial chloride secretion mechanisms in adult murine airways

Defective epithelial chloride secretion occurs in humans with cystic fibrosis (CF), a genetic defect due to loss of function of CFTR, a cAMP-activated chloride channel. In the airways, absence of an active CFTR causes a severe lung disease. In mice, genetic ablation of CFTR function does not result in similar lung pathology. This may be due to the expression of an alternative chloride channel which is activated by calcium. The most probable protein performing this function is TMEM16A, a calcium-activated chloride channel (CaCC). Our aim was to assess the relative contribution of CFTR and TMEM16A to chloride secretion in adult mouse trachea. For this purpose we tested pharmacological inhibitors of chloride channels in normal and CF mice. The amplitude of the cAMP-activated current was similar in both types of animals and was not affected by a selective CFTR inhibitor. In contrast, a CaCC inhibitor (CaCCinh-A01) strongly blocked the cAMP-activated current as well as the calcium-activated chloride secretion triggered by apical UTP. Although control experiments revealed that CaCCinh-A01 also shows inhibitory activity on CFTR, our results indicate that transepithelial chloride secretion in adult mouse trachea is independent of CFTR and that another channel, possibly TMEM16A, performs both cAMP- and calcium-activated chloride transport. The prevalent function of a non-CFTR channel may explain the absence of a defect in chloride transport in CF mice.

Phenylquinoxalinone CFTR activator as potential prosecretory therapy for constipation

&NA; Constipation is a common condition for which current treatments can have limited efficacy. By high‐throughput screening, we recently identified a phenylquinoxalinone activator of the cystic fibrosis transmembrane conductance regulator (CFTR) chloride channel that stimulated intestinal fluid secretion and normalized stool output in a mouse model of opioid‐induced constipation. Here, we report phenylquinoxalinone structure‐activity analysis, mechanism of action, animal efficacy data in acute and chronic models of constipation, and functional data in ex vivo primary cultured human enterocytes. Structure‐activity analysis was done on 175 phenylquinoxalinone analogs, including 15 synthesized compounds. The most potent compound, CFTRact‐J027, activated CFTR with EC50 ˜ 200 nM, with patch‐clamp analysis showing a linear CFTR current‐voltage relationship with direct CFTR activation. CFTRact‐J027 corrected reduced stool output and hydration in a mouse model of acute constipation produced by scopolamine and in a chronically constipated mouse strain (C3H/HeJ). Direct comparison with the approved prosecretory drugs lubiprostone and linaclotide showed substantially greater intestinal fluid secretion with CFTRact‐J027, as well as greater efficacy in a constipation model. As evidence to support efficacy in human constipation, CFTRact‐J027 increased transepithelial fluid transport in enteroids generated from normal human small intestine. Also, CFTRact‐J027 was rapidly metabolized in vitro in human hepatic microsomes, suggesting minimal systemic exposure upon oral administration. These data establish structure‐activity and mechanistic data for phenylquinoxalinone CFTR activators, and support their potential efficacy in human constipation.