COMMENTARY
COMMENTARY
Functional reconstitution of a chloride channel bares its soul H. Criss Hartzell1 and Chelsey Chandler Ruppersburg Department of Cell Biology, Emory University School of Medicine, Atlanta, GA 30322
Chloride channels have been called the poor cousins of the aristocratic cation channels because their physiological functions involve “cleaning up the mess after the party” (1). Although Cl− channels are not as glamorous as the Na+, Ca2+, and K+ channels dominating neuronal signaling, their functions are indispensable (2), as exemplified by cholera caused by superactivation of a Cl− channel (CFTR) in the gut and cystic fibrosis caused by insufficiency of the same Cl− channel in the lung. In part, our understanding of these navels of negativity has been slow to ripen because the molecular zoo of Cl− channels is incomplete. Humans have >200 cation channel genes in >20 families, but only four Cl− channel families comprised of a paltry 34 genes were known until recently. This limited sampling has restricted efforts to uncover the essence of Cl− channel pores. Therefore, a sensation occurred when the Cl− channel gene superfamily blossomed in size in 2008 with the addition of a 10-gene family called TMEM16, also called Anoctamin (3–5). TMEM16A and TMEM16B encode subunits of Ca2+-activated Cl− channels (CaCCs) that are widely expressed throughout the body. CaCCs open when intracellular Ca2+ rises, for example following activation of G proteincoupled receptors by certain hormones. CaCCs’ best known function is in epithelial fluid secretion, a role played in parallel with CFTR (Fig. 1). The aqueous portion of secretions such as tears, saliva, milk, and pancreatic juice is produced by cells that actively accumulate Cl− from their blood-side by pumps and transporters, and then release Cl− into the glandular ducts through Cl− channels on the other side of the cell. Cl− efflux drives fluid secretion as Na+ and water follow to maintain charge neutrality and osmotic balance. The same secretory mechanism keeps epithelial surfaces, like the airway, moist. Here, Terashima et al. (6) deliver the gold standard of proof that TMEM16A, also called ANO1, forms the pore through which Cl− traverses the plasma membrane. This is an important discovery because ion channels typically have multiple subunits: the pore-forming α-subunits escort the permeant ion across the plasma membrane and accessory subunits regulate channel www.pnas.org/cgi/doi/10.1073/pnas.1319415110
opening or chaperone the α-subunit to its target. Proving that a subunit forms the ion conduction pathway across the membrane is a first step to probe its “active site” and understand what determines its substrate (Cl−) specificity and orchestrates its opening. Terashima et al. (6) expressed TMEM16A in Sf9 cells, purified it to near-homogeneity, and reconstituted it into liposomes for Cl− flux measurements. The authors found that TMEM16A alone is sufficient to form a functional CaCC that is selective for Cl− over cations and is activated by the appropriate Ca2+ concentrations. Most importantly, additional protein components are not required for its function. The ability to isolate a functional Cl− channel gives hope that structural data, something the Cl− channel field is sorely lacking, will be forthcoming. TMEM16A is not the first ion channel to be functionally reconstituted in this way, so why all of the fuss? The attention may be because TMEM16A discovery has been an odyssey (7). CaCCs are incredibly versatile and perform diverse esoteric functions. CaCCs were first described as mediating the fast block to polyspermy in amphibian oocytes, modulating synaptic transmission in salamander photoreceptors, and effecting epithelial secretion. CaCCs are now also known to regulate contractility in smooth muscle and myoepithelial cells, participate in neuronal excitability and sensory transduction, and have been implicated in cellular proliferation and migration (8–11). Given the widespread importance of CaCCs in physiology and their prospects as therapeutic targets for hypertension, asthma, and other disorders, it is surprising that their molecular identity was discovered only after >30 confusing years. At least three genes have been proposed to encode CaCCs, but each was like Goldilocks trying out Papa Bear’s bed (7). However, TMEM16A and TMEM16B fit just right; the currents they induce closely recapitulate native CaCCs and their knockdown in tissues, such as salivary gland, reduces fluid secretion (3–5). Why was the identity of CaCCs such a hard nut to crack? A major hurdle has been that many Cl− channels look alike biophysically. Unlike cation channels that can be
TMEM16A / ANO1
CBM1
CBM 2
b
Cl-
Apical (lumen) side
+ Na H O 2
Cl-
CaCC
C CFTR
Ca2+
cAMP Cl-
Na
+
Cl-
Cl-
Cl-
AE
NKCC Basal (blood) side
Cl-
+ K
HCO3-
NCC
Cl-
+ Na
Cl-
Na+ H2O
Fig. 1. (Lower) Cl− is loaded into epithelial cells by basal Na+-K+-2Cl− cotransporters (NKCC), anion exchangers (AE), and Na+-Cl− cotransporters (NCC), and released apically by various Cl− channels including CFTR and CaCCs encoded by TMEM16A. (Upper) Black line: revised TMEM16A topology (13); thin green line: re-entrant loop model (3); CBM1, CBM2, b: putative CaM binding domains; star: two glutamic acids that regulate Ca2+ sensitivity.
separated by their ion selectivity (Na+, K+, Ca2+), Cl− channels are relatively anion nonselective, so that one kind of Cl− channel can masquerade as another. Deviant behavior of splice variants and heteromeric channels adds to the confusion. Moreover, pharmacological tools to identify anion channels are persistently primitive. Whereas unlocking the secrets of cation channels benefitted from specific toxins, like bungarotoxin and tetrodotoxin, Cl− channel drugs (including new elixirs from high-throughput screens) are generally small hydrophobic molecules with unknown sites of action and vague specificity (12). The problem is compounded by the fact Author contributions: H.C.H. and C.C.R. wrote the paper. The authors declare no conflict of interest. See companion article on page 19354. 1
To whom correspondence should be addressed. E-mail: criss. [email protected].
PNAS | November 26, 2013 | vol. 110 | no. 48 | 19185–19186
that Cl− channels of untold types are everywhere. This abundance makes it challenging to ascertain if a cDNA encodes a Cl− channel or up-regulates an imposter. The silver standard to decide this question is mutagenesis of amino acids that control a fundamental channel property, like ionic selectivity, but this approach has been less than forthcoming in identifying key residues in TMEM16A. For example, initial reports that mutations in a putative re-entrant loop dramatically altered the cation:anion selectivity (3) have not been confirmed, and it now appears that the transmembrane topology is different from that first predicted (13). For us old “geezers” who lived through the experience of being introduced to a new Cl− channel gene that eventually turned out to be a transcription factor, these inconsistencies conjure up insecurities. Thus, the report by Terashima et al. (6) is a welcome proof that puts to rest any reservations. The work by Terashima et al. (6) opens a door to solving many of the puzzles and controversies that have plagued the CaCC arena. CaCCs are synergistically activated by Ca2+ and membrane potential and— unlike the maxi-K channel that is also Ca2+and voltage-dependent—the behavior of TMEM16A currents at various Ca2+ concentrations differ qualitatively in waveform, rectification, and voltage dependence. This behavior has important physiological consequences, because depending on intracellular Ca2+ concentration, TMEM16A can be switched to either secrete or absorb Cl−, vital for controlling fluid levels at epithelia–air interfaces (14). This distinctive behavior at low and high Ca2+ even led some investigators to entertain the idea of CaCCs being comprised of two interacting channels. The finding that the reconstituted channel is synergistically controlled by Ca2+ and voltage shows that these properties are intrinsic to the TMEM16A protein. However, insight into the mechanisms responsible for this regulation is hindered by lack of homology between TMEM16s and other ion channels and the absence of obvious sequence motifs that might be responsible for these functions. A major conundrum in the CaCC/ TMEM16A field is how the channel is opened by Ca2+ : Does Ca2+ bind directly to the channel or is a separate Ca2+ sensor protein, like calmodulin (CaM), required? Terashima et al. (6) provide overwhelming evidence that CaM is not needed. The
19186 | www.pnas.org/cgi/doi/10.1073/pnas.1319415110
authors show that the TMEM16A protein used to reconstitute Ca2+-activated Cl− fluxes does not contain CaM and that addition of excess CaM to TMEM16A-containing liposomes does not alter Cl− flux. Furthermore, the authors cannot detect binding of TMEM16A to purified CaM. Terashima et al. also demonstrate that mutagenesis of
two glutamates that had previously been suggested as crucial for Ca2+ sensing (13) greatly reduce the Ca2+ sensitivity. These results strengthen suggestions that Ca2+ opens TMEM16A by directly binding to it (13). These data, however convincing, pose a quandary. Three published papers provide a contrary view and show by coimmunoprecipitation and pull-down assays that CaM binds to TMEM16A (15–17). Because such assays intrinsically do not provide information about stoichiometry or affinity, their physiological significance might be questioned. The issue is further befuddled because of divergent notions about the consequences
of CaM binding. Tian et al. (15) propose that a CaM binding site (“b”) in the N terminus is absolutely required for channel activation by Ca2+. In contrast, Vocke et al. (17) conclude that a different CaM binding site (CBM1) is required for, or facilitates, TMEM16A activation, whereas Jung et al. (16) deduce that CaM is not required for Ca2+-dependent activation, although it regulates the anion selectivity of the channel by binding CBM1 and CBM2 (Fig. 1). Having purified TMEM16A protein should help resolve the mechanisms of TMEM16A activation. Another question is the function of TMEM16A paralogs. It remains uncertain whether all TMEM16s are Cl− channels (9). TMEM16F has been reported to be a Cl− channel, a nonselective cation channel, and a phospholipid scramblase translocating phosphatidyl serine between membrane leaflets (18). Other TMEM16 members also have scramblase activities (18) and the Accardi laboratory has shown that a purified TMEM16 homolog from Aspergillus fumigatus is a dualfunction nonselective channel and scramblase (19). Although TMEM16A has not been shown to have scramblase activity, given the high conservation of the TMEM16 family, one cannot help but wonder about the relationship of TMEM16A to lipids, and whether the reason the CaCC field has been likened to a greased pig is precisely because TMEM16A is one.
1 Miller C (2006) ClC chloride channels viewed through a transporter lens. Nature 440(7083):484–489. 2 Duran C, Thompson CH, Xiao Q, Hartzell HC (2010) Chloride channels: Often enigmatic, rarely predictable. Annu Rev Physiol 72:95–121. 3 Yang YD, et al. (2008) TMEM16A confers receptor-activated calcium-dependent chloride conductance. Nature 455(7217): 1210–1215. 4 Caputo A, et al. (2008) TMEM16A, a membrane protein associated with calcium-dependent chloride channel activity. Science 322(5901):590–594. 5 Schroeder BC, Cheng T, Jan YN, Jan LY (2008) Expression cloning of TMEM16A as a calcium-activated chloride channel subunit. Cell 134(6):1019–1029. 6 Terashima H, Picollo A, Accardi A (2013) Purified TMEM16A is sufficient to form Ca2+-activated Cl− channels. Proc Natl Acad Sci USA 110:19354–19359. 7 Hartzell C, Putzier I, Arreola J (2005) Calcium-activated chloride channels. Annu Rev Physiol 67:719–758. 8 Ferrera L, Caputo A, Galietta LJ (2010) TMEM16A protein: A new identity for Ca(2+)-dependent Cl− channels. Physiology (Bethesda) 25(6):357–363. 9 Duran C, Hartzell HC (2011) Physiological roles and diseases of Tmem16/Anoctamin proteins: Are they all chloride channels? Acta Pharmacol Sin 32(6):685–692. 10 Kunzelmann K, et al. (2011) Anoctamins. Pflugers Arch 462(2): 195–208.
11 Huang F, Wong X, Jan LY (2012) International Union of Basic and Clinical Pharmacology. LXXXV: Calcium-activated chloride channels. Pharmacol Rev 64(1):1–15. 12 Verkman AS, Galietta LJ (2009) Chloride channels as drug targets. Nat Rev Drug Discov 8(2):153–171. 13 Yu K, Duran C, Qu Z, Cui YY, Hartzell HC (2012) Explaining calcium-dependent gating of anoctamin-1 chloride channels requires a revised topology. Circ Res 110(7):990–999. 14 Xiao Q, et al. (2011) Voltage- and calcium-dependent gating of TMEM16A/Ano1 chloride channels are physically coupled by the first intracellular loop. Proc Natl Acad Sci USA 108(21): 8891–8896. 15 Tian Y, et al. (2011) Calmodulin-dependent activation of the epithelial calcium-dependent chloride channel TMEM16A. FASEB J 25(3):1058–1068. 16 Jung J, et al. (2013) Dynamic modulation of ANO1/TMEM16A HCO3(−) permeability by Ca2+/calmodulin. Proc Natl Acad Sci USA 110(1):360–365. 17 Vocke K, et al. (2013) Calmodulin-dependent activation and inactivation of anoctamin calcium-gated chloride channels. J Gen Physiol 142(4):381–404. 18 Suzuki J, et al. (2013) Calcium-dependent phospholipid scramblase activity of TMEM16 protein family members. J Biol Chem 288(19):13305–13316. 19 Malvezzi M, et al. (2013) Ca2+-dependent phospholipid scrambling by a reconstituted TMEM16 ion channel. Nat Commun 4:2367.
The work by Terashima et al. opens a door to solving many of the puzzles and controversies that have plagued the CaCC arena.
Hartzell and Ruppersburg
COMMENTARY
Functional reconstitution of a chloride channel bares its soul H. Criss Hartzell1 and Chelsey Chandler Ruppersburg Department of Cell Biology, Emory University School of Medicine, Atlanta, GA 30322
Chloride channels have been called the poor cousins of the aristocratic cation channels because their physiological functions involve “cleaning up the mess after the party” (1). Although Cl− channels are not as glamorous as the Na+, Ca2+, and K+ channels dominating neuronal signaling, their functions are indispensable (2), as exemplified by cholera caused by superactivation of a Cl− channel (CFTR) in the gut and cystic fibrosis caused by insufficiency of the same Cl− channel in the lung. In part, our understanding of these navels of negativity has been slow to ripen because the molecular zoo of Cl− channels is incomplete. Humans have >200 cation channel genes in >20 families, but only four Cl− channel families comprised of a paltry 34 genes were known until recently. This limited sampling has restricted efforts to uncover the essence of Cl− channel pores. Therefore, a sensation occurred when the Cl− channel gene superfamily blossomed in size in 2008 with the addition of a 10-gene family called TMEM16, also called Anoctamin (3–5). TMEM16A and TMEM16B encode subunits of Ca2+-activated Cl− channels (CaCCs) that are widely expressed throughout the body. CaCCs open when intracellular Ca2+ rises, for example following activation of G proteincoupled receptors by certain hormones. CaCCs’ best known function is in epithelial fluid secretion, a role played in parallel with CFTR (Fig. 1). The aqueous portion of secretions such as tears, saliva, milk, and pancreatic juice is produced by cells that actively accumulate Cl− from their blood-side by pumps and transporters, and then release Cl− into the glandular ducts through Cl− channels on the other side of the cell. Cl− efflux drives fluid secretion as Na+ and water follow to maintain charge neutrality and osmotic balance. The same secretory mechanism keeps epithelial surfaces, like the airway, moist. Here, Terashima et al. (6) deliver the gold standard of proof that TMEM16A, also called ANO1, forms the pore through which Cl− traverses the plasma membrane. This is an important discovery because ion channels typically have multiple subunits: the pore-forming α-subunits escort the permeant ion across the plasma membrane and accessory subunits regulate channel www.pnas.org/cgi/doi/10.1073/pnas.1319415110
opening or chaperone the α-subunit to its target. Proving that a subunit forms the ion conduction pathway across the membrane is a first step to probe its “active site” and understand what determines its substrate (Cl−) specificity and orchestrates its opening. Terashima et al. (6) expressed TMEM16A in Sf9 cells, purified it to near-homogeneity, and reconstituted it into liposomes for Cl− flux measurements. The authors found that TMEM16A alone is sufficient to form a functional CaCC that is selective for Cl− over cations and is activated by the appropriate Ca2+ concentrations. Most importantly, additional protein components are not required for its function. The ability to isolate a functional Cl− channel gives hope that structural data, something the Cl− channel field is sorely lacking, will be forthcoming. TMEM16A is not the first ion channel to be functionally reconstituted in this way, so why all of the fuss? The attention may be because TMEM16A discovery has been an odyssey (7). CaCCs are incredibly versatile and perform diverse esoteric functions. CaCCs were first described as mediating the fast block to polyspermy in amphibian oocytes, modulating synaptic transmission in salamander photoreceptors, and effecting epithelial secretion. CaCCs are now also known to regulate contractility in smooth muscle and myoepithelial cells, participate in neuronal excitability and sensory transduction, and have been implicated in cellular proliferation and migration (8–11). Given the widespread importance of CaCCs in physiology and their prospects as therapeutic targets for hypertension, asthma, and other disorders, it is surprising that their molecular identity was discovered only after >30 confusing years. At least three genes have been proposed to encode CaCCs, but each was like Goldilocks trying out Papa Bear’s bed (7). However, TMEM16A and TMEM16B fit just right; the currents they induce closely recapitulate native CaCCs and their knockdown in tissues, such as salivary gland, reduces fluid secretion (3–5). Why was the identity of CaCCs such a hard nut to crack? A major hurdle has been that many Cl− channels look alike biophysically. Unlike cation channels that can be
TMEM16A / ANO1
CBM1
CBM 2
b
Cl-
Apical (lumen) side
+ Na H O 2
Cl-
CaCC
C CFTR
Ca2+
cAMP Cl-
Na
+
Cl-
Cl-
Cl-
AE
NKCC Basal (blood) side
Cl-
+ K
HCO3-
NCC
Cl-
+ Na
Cl-
Na+ H2O
Fig. 1. (Lower) Cl− is loaded into epithelial cells by basal Na+-K+-2Cl− cotransporters (NKCC), anion exchangers (AE), and Na+-Cl− cotransporters (NCC), and released apically by various Cl− channels including CFTR and CaCCs encoded by TMEM16A. (Upper) Black line: revised TMEM16A topology (13); thin green line: re-entrant loop model (3); CBM1, CBM2, b: putative CaM binding domains; star: two glutamic acids that regulate Ca2+ sensitivity.
separated by their ion selectivity (Na+, K+, Ca2+), Cl− channels are relatively anion nonselective, so that one kind of Cl− channel can masquerade as another. Deviant behavior of splice variants and heteromeric channels adds to the confusion. Moreover, pharmacological tools to identify anion channels are persistently primitive. Whereas unlocking the secrets of cation channels benefitted from specific toxins, like bungarotoxin and tetrodotoxin, Cl− channel drugs (including new elixirs from high-throughput screens) are generally small hydrophobic molecules with unknown sites of action and vague specificity (12). The problem is compounded by the fact Author contributions: H.C.H. and C.C.R. wrote the paper. The authors declare no conflict of interest. See companion article on page 19354. 1
To whom correspondence should be addressed. E-mail: criss. [email protected].
PNAS | November 26, 2013 | vol. 110 | no. 48 | 19185–19186
that Cl− channels of untold types are everywhere. This abundance makes it challenging to ascertain if a cDNA encodes a Cl− channel or up-regulates an imposter. The silver standard to decide this question is mutagenesis of amino acids that control a fundamental channel property, like ionic selectivity, but this approach has been less than forthcoming in identifying key residues in TMEM16A. For example, initial reports that mutations in a putative re-entrant loop dramatically altered the cation:anion selectivity (3) have not been confirmed, and it now appears that the transmembrane topology is different from that first predicted (13). For us old “geezers” who lived through the experience of being introduced to a new Cl− channel gene that eventually turned out to be a transcription factor, these inconsistencies conjure up insecurities. Thus, the report by Terashima et al. (6) is a welcome proof that puts to rest any reservations. The work by Terashima et al. (6) opens a door to solving many of the puzzles and controversies that have plagued the CaCC arena. CaCCs are synergistically activated by Ca2+ and membrane potential and— unlike the maxi-K channel that is also Ca2+and voltage-dependent—the behavior of TMEM16A currents at various Ca2+ concentrations differ qualitatively in waveform, rectification, and voltage dependence. This behavior has important physiological consequences, because depending on intracellular Ca2+ concentration, TMEM16A can be switched to either secrete or absorb Cl−, vital for controlling fluid levels at epithelia–air interfaces (14). This distinctive behavior at low and high Ca2+ even led some investigators to entertain the idea of CaCCs being comprised of two interacting channels. The finding that the reconstituted channel is synergistically controlled by Ca2+ and voltage shows that these properties are intrinsic to the TMEM16A protein. However, insight into the mechanisms responsible for this regulation is hindered by lack of homology between TMEM16s and other ion channels and the absence of obvious sequence motifs that might be responsible for these functions. A major conundrum in the CaCC/ TMEM16A field is how the channel is opened by Ca2+ : Does Ca2+ bind directly to the channel or is a separate Ca2+ sensor protein, like calmodulin (CaM), required? Terashima et al. (6) provide overwhelming evidence that CaM is not needed. The
19186 | www.pnas.org/cgi/doi/10.1073/pnas.1319415110
authors show that the TMEM16A protein used to reconstitute Ca2+-activated Cl− fluxes does not contain CaM and that addition of excess CaM to TMEM16A-containing liposomes does not alter Cl− flux. Furthermore, the authors cannot detect binding of TMEM16A to purified CaM. Terashima et al. also demonstrate that mutagenesis of
two glutamates that had previously been suggested as crucial for Ca2+ sensing (13) greatly reduce the Ca2+ sensitivity. These results strengthen suggestions that Ca2+ opens TMEM16A by directly binding to it (13). These data, however convincing, pose a quandary. Three published papers provide a contrary view and show by coimmunoprecipitation and pull-down assays that CaM binds to TMEM16A (15–17). Because such assays intrinsically do not provide information about stoichiometry or affinity, their physiological significance might be questioned. The issue is further befuddled because of divergent notions about the consequences
of CaM binding. Tian et al. (15) propose that a CaM binding site (“b”) in the N terminus is absolutely required for channel activation by Ca2+. In contrast, Vocke et al. (17) conclude that a different CaM binding site (CBM1) is required for, or facilitates, TMEM16A activation, whereas Jung et al. (16) deduce that CaM is not required for Ca2+-dependent activation, although it regulates the anion selectivity of the channel by binding CBM1 and CBM2 (Fig. 1). Having purified TMEM16A protein should help resolve the mechanisms of TMEM16A activation. Another question is the function of TMEM16A paralogs. It remains uncertain whether all TMEM16s are Cl− channels (9). TMEM16F has been reported to be a Cl− channel, a nonselective cation channel, and a phospholipid scramblase translocating phosphatidyl serine between membrane leaflets (18). Other TMEM16 members also have scramblase activities (18) and the Accardi laboratory has shown that a purified TMEM16 homolog from Aspergillus fumigatus is a dualfunction nonselective channel and scramblase (19). Although TMEM16A has not been shown to have scramblase activity, given the high conservation of the TMEM16 family, one cannot help but wonder about the relationship of TMEM16A to lipids, and whether the reason the CaCC field has been likened to a greased pig is precisely because TMEM16A is one.
1 Miller C (2006) ClC chloride channels viewed through a transporter lens. Nature 440(7083):484–489. 2 Duran C, Thompson CH, Xiao Q, Hartzell HC (2010) Chloride channels: Often enigmatic, rarely predictable. Annu Rev Physiol 72:95–121. 3 Yang YD, et al. (2008) TMEM16A confers receptor-activated calcium-dependent chloride conductance. Nature 455(7217): 1210–1215. 4 Caputo A, et al. (2008) TMEM16A, a membrane protein associated with calcium-dependent chloride channel activity. Science 322(5901):590–594. 5 Schroeder BC, Cheng T, Jan YN, Jan LY (2008) Expression cloning of TMEM16A as a calcium-activated chloride channel subunit. Cell 134(6):1019–1029. 6 Terashima H, Picollo A, Accardi A (2013) Purified TMEM16A is sufficient to form Ca2+-activated Cl− channels. Proc Natl Acad Sci USA 110:19354–19359. 7 Hartzell C, Putzier I, Arreola J (2005) Calcium-activated chloride channels. Annu Rev Physiol 67:719–758. 8 Ferrera L, Caputo A, Galietta LJ (2010) TMEM16A protein: A new identity for Ca(2+)-dependent Cl− channels. Physiology (Bethesda) 25(6):357–363. 9 Duran C, Hartzell HC (2011) Physiological roles and diseases of Tmem16/Anoctamin proteins: Are they all chloride channels? Acta Pharmacol Sin 32(6):685–692. 10 Kunzelmann K, et al. (2011) Anoctamins. Pflugers Arch 462(2): 195–208.
11 Huang F, Wong X, Jan LY (2012) International Union of Basic and Clinical Pharmacology. LXXXV: Calcium-activated chloride channels. Pharmacol Rev 64(1):1–15. 12 Verkman AS, Galietta LJ (2009) Chloride channels as drug targets. Nat Rev Drug Discov 8(2):153–171. 13 Yu K, Duran C, Qu Z, Cui YY, Hartzell HC (2012) Explaining calcium-dependent gating of anoctamin-1 chloride channels requires a revised topology. Circ Res 110(7):990–999. 14 Xiao Q, et al. (2011) Voltage- and calcium-dependent gating of TMEM16A/Ano1 chloride channels are physically coupled by the first intracellular loop. Proc Natl Acad Sci USA 108(21): 8891–8896. 15 Tian Y, et al. (2011) Calmodulin-dependent activation of the epithelial calcium-dependent chloride channel TMEM16A. FASEB J 25(3):1058–1068. 16 Jung J, et al. (2013) Dynamic modulation of ANO1/TMEM16A HCO3(−) permeability by Ca2+/calmodulin. Proc Natl Acad Sci USA 110(1):360–365. 17 Vocke K, et al. (2013) Calmodulin-dependent activation and inactivation of anoctamin calcium-gated chloride channels. J Gen Physiol 142(4):381–404. 18 Suzuki J, et al. (2013) Calcium-dependent phospholipid scramblase activity of TMEM16 protein family members. J Biol Chem 288(19):13305–13316. 19 Malvezzi M, et al. (2013) Ca2+-dependent phospholipid scrambling by a reconstituted TMEM16 ion channel. Nat Commun 4:2367.
The work by Terashima et al. opens a door to solving many of the puzzles and controversies that have plagued the CaCC arena.
Hartzell and Ruppersburg
