Every cell in the body is surrounded by a barrier called the cell membrane. There are, however, a number of ways that molecules can pass through this membrane to either enter or leave the cell.

Calcium-activated channels are a group of proteins that are embedded within the cell membrane and that allow different ions to pass through the membrane. These proteins are involved in a number of processes in a variety of tissues, for example in the gut, lungs and nervous system. A family of proteins called TMEM16 includes a number of calcium-activated channels that have been recently identified. However, it is not clear how these TMEM16 channel proteins detect the calcium ions that cause them to open.

Two ideas have been suggested: the calcium ions might be detected by a protein called calmodulin, which then forces the channel to open; alternatively, the calcium ions might be detected by the channel protein itself. Tien, Peters et al. have now tested both of these ideas by focusing on a calcium-activated channel protein called TMEM16A, which allows chloride ions to pass through membranes.

The possible role of calmodulin was tested in several ways, such as by preventing it from binding to the TMEM16A protein or from binding to calcium. However, none of these changes affected the opening of the channel; so Tien, Peters et al. concluded that calmodulin is not involved in these channels being activated by calcium ions.

Next, Tien, Peters et al. tested specific parts of the TMEM16A channel protein itself to see if they were involved in calcium detection instead. Proteins are made from smaller building blocks called amino acids, and it is known that some amino acids are more likely to bind to calcium ions than others. There are 38 of these amino acids in the TMEM16A channel that are also found in other members of the TMEM16 family in both fruit flies and mammals. Tien, Peters et al. found that replacing five of these with other amino acids made the channel less sensitive to calcium. Further experiments suggested that four of these five amino acids are clustered at the site where a calcium ion might bind to the TMEM16A channel protein, which suggests that the protein itself can detect calcium directly.

The next challenge will be to understand how calcium ions binding to the site on the TMEM16A channel protein can cause the channel to open to allow the chloride ions to pass through.

Transmembrane proteins of the TMEM16 family include a number of calcium-dependent ion channels, such as the TMEM16A (Caputo et al., 2008; Schroeder et al., 2008; Yang et al., 2008b) and TMEM16B (Schroeder et al., 2008; Stöhr et al., 2009) calcium-activated chloride channels (CaCCs) and the TMEM16F (Yang et al., 2012) small conductance calcium-activated nonselective cation (SCAN) channel. These proteins have been implicated in a wide range of physiological activities (Hartzell et al., 2009; Kunzelmann et al., 2011; Berg et al., 2012; Scudieri et al., 2012; Huang et al., 2012a) including the control of fluid secretion in epithelia (Romanenko et al., 2010; Huang et al., 2012b), the regulation of membrane excitability in neurons (Billig et al., 2011; Huang et al., 2012c), the scrambling of membrane lipids in platelets (Suzuki et al., 2010; Yang et al., 2012), and the metastasis of certain cancers (Katoh, 2003; Duvvuri et al., 2012). Despite their important physiological roles, the mechanism by which calcium activates these proteins is still extensively debated.

Based on studies of calcium-activated potassium channels (K_Cas) (Maylie et al., 2004; Salkoff et al., 2006), two models have been proposed to account for the calcium sensitivity of TMEM16A-CaCC (Figure 1A). In one model resembling that of the small-conductance K_Ca (SK) channels (Xia et al., 1998; Schumacher et al., 2001), calcium ions interact with calmodulin (CaM), and the physical association of calmodulin with TMEM16A is required to transduce the free energy of ion–protein interaction into movements of the channel gate (Tian et al., 2011; Vocke et al., 2013). In a second model similar to that of the large-conductance K_Ca (BK) channels (Schreiber and Salkoff, 1997; Shi et al., 2002; Xia et al., 2002; Wu et al., 2010; Yuan et al., 2010), calcium binds directly to TMEM16A, and this interaction drives channel activation (Yu et al., 2012, 2014; Terashima et al., 2013).

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In this study, we performed a series of experiments designed to clarify the mechanism by which TMEM16A-CaCCs are gated by calcium. First, with multiple lines of evidence, we confirmed that CaM is not required for TMEM16A-CaCC activation. Then, we performed a comprehensive alanine mutagenesis screen of all highly conserved acidic residues in TMEM16A that are predicted to be accessible to the cytoplasm. Based on our measurements of these mutants' apparent calcium sensitivity in inside-out excised patches, we identify four acidic residues (including two acidic residues that were recently implicated in the calcium-dependent activation of TMEM16A-CaCCs [Yu et al., 2012]) that satisfy several criteria characteristic of calcium-binding sites. Substitution of each of these residues with various amino acids shifts the apparent sensitivity of TMEM16A-CaCC for different divalent cations, and cysteine residues at these sites can be cross-linked by oxidizing intracellular solutions. Our results thus demonstrate that direct binding of calcium to TMEM16A triggers channel activation independently of calmodulin, identify novel interaction sites between calcium ions and TMEM16A, and lay the groundwork for future studies examining the mechanism of calcium-dependent TMEM16 channel activation.

Calcium is the physiological stimulus that activates calcium-activated chloride channels (CaCCs). Studying how calcium activates the newly discovered TMEM16A-CaCC channel will help us understand its physiological functions. Thus far, the molecular nature of the TMEM16A calcium sensor has been under extensive debate. One theory asserts that a ubiquitous calcium sensing protein, calmodulin (CaM), is responsible for calcium activation of the CaCC (Tian et al., 2011; Vocke et al., 2013), resembling the mechanism of the small-conductance K_Ca (SK) channels. A competing theory asserts that calcium ions can directly bind to the channel itself without the involvement of CaM (Yu et al., 2012, 2014; Terashima et al., 2013), analogous to the mechanism of the large-conductance K_Ca (BK) channels. To settle this debate and to advance the molecular understanding of how calcium activates TMEM16A, we performed a comprehensive screen of evolutionarily conserved intracellular acidic residues in the TMEM16A protein and mapped its calcium sensor to a region between transmembrane segments 5 and 7. By manipulating the charge, size, and reactivity of these side chains, we characterized the mutant channels' sensitivity and selectivity for different divalent cations and showed that four critical acidic residues (E698, E701, E730, and D734) in this region are situated at a solvent-accessible site that likely interacts with calcium in the cytoplasm during channel activation.

In contrast to previous studies of TMEM16A calcium-dependent activation based on whole-cell recordings, we used the inside-out patch clamp to quantify the apparent calcium sensitivity (EC₅₀) of the channel in this study. Monitoring channel opening in response to calcium applied to the inside-out patch and comparing channel EC₅₀ values is a more direct measurement of calcium-dependent channel activation compared to the methods used in other studies (Jung et al., 2013; Terashima et al., 2013; Vocke et al., 2013). Although recent electrophysiological studies of TMEM16A-CaCCs using the whole-cell patch clamping configuration provided valuable insights toward understanding channel activation (Yu et al., 2012; Jung et al., 2013; Vocke et al., 2013), the difficulty in temporally and spatially controlling intracellular calcium concentrations inherent to the whole-cell patch clamp method renders the quantification of calcium sensitivity difficult. By using inside-out patch recordings to accurately assess channel sensitivity to divalent cations and exploiting differences in the coordination chemistry of hard and soft ligands to deduce the functional role of individual acidic residues, our study based on a mutagenesis screen of acidic residues provides a comprehensive survey of the potential high affinity calcium binding sites within the TMEM16A protein.

Our study is in agreement with the conclusions of Terashima et al. (2013) and Yu et al. (2014) that calmodulin is not involved in the calcium-dependent activation of TMEM16A channels. Similar to these studies, we were unable to detect alterations of TMEM16A channel activity by manipulating calmodulin function. Moreover, mutations of four putative calcium binding residues E698, E701, E730, and D734 differentially shifted the EC₅₀ of channel activation to different calcium concentrations depending on the size and charge of the amino acid side chains, suggesting that these residues are likely involved in binding calcium. Furthermore, the differences in the way these mutations affect channel sensitivity to the hard calcium and strontium ions and the softer cadmium ions suggest that these mutations directly impact channel–ion interactions rather than acting via an allosteric mechanism. It remains possible that CaM may physically interact with TMEM16A channels under certain conditions as evidenced by previous biochemical studies (Jung et al., 2013; Vocke et al., 2013). Nevertheless, this interaction, if present, is not responsible for the calcium-dependent activation of TMEM16A-CaCC channels.

It seems likely that calcium directly binds to and activates TMEM16A-CaCC channels in a fashion reminiscent of the calcium activation mechanism of the large conductance, calcium-activated BK potassium channel, one of the most well-characterized calcium-activated ion channels. Both channels contain calcium binding sites that are composed of multiple intracellular acidic residues, and substitution mutagenesis studies of both channels reveal that side chain properties are critical to the binding of calcium and other metal ions such as strontium, barium, and cadmium. Removing carboxylate groups from side chains dramatically reduces or eliminates the ability of both channels to bind calcium, while preserving carboxylate groups with charge-conserving mutations tends to partially rescue calcium sensitivity.

Structural and functional characterizations of BK channels over the past three decades have generated a wealth of information that provides valuable inspiration to the study of TMEM16A-CaCC channels. There are three distinct metal binding sites in the pore-forming subunit of BK channels (Schreiber and Salkoff, 1997; Shi et al., 2002; Xia et al., 2002). Two high affinity binding sites (micromolar) correspond to the calcium bowl in the RCK2 domain (Schreiber and Salkoff, 1997) and the acidic residue D367 in the RCK1 domain (Shi et al., 2002; Xia et al., 2002; Zhang et al., 2010), and a low affinity site (millimolar) that can nonspecifically bind various divalent cations corresponds to residues from the membrane spanning domain and the cytosolic RCK1 domain (Shi et al., 2002; Xia et al., 2002; Yang et al., 2008a). These three metal ion binding sites act relatively independently to activate BK channels as mutating any one of them has a minimal impact on the other two sites. Extensive mutagenesis along with characterization of divalent cation selectivity has elucidated the properties of the different metal ion binding sites (Zeng et al., 2005; Zhou et al., 2012). Micromolar calcium and strontium ions can activate the channel through both the calcium bowl and the RCK1 high affinity sites but not through the low affinity site. Micromolar cadmium only activates the channel via the RCK1 high affinity site; even 300 μM cadmium cannot act on the calcium bowl site to activate the channel. In contrast, 200 μM barium has no effect on the RCK1 site (Zhou et al., 2012). Instead, barium ions activate the channel through the calcium bowl site with five times lower affinity than calcium. On the other hand, millimolar concentrations of all these divalent cations can activate the channel through the low affinity metal ion binding site (Zeng et al., 2005).

For TMEM16A-CaCC, the apparent calcium (EC₅₀: 1.0 ± 0.1 μM) and strontium (EC₅₀: 7.7 ± 0.3 μM) sensitivities are similar to that of the high affinity cation binding sites in BK channels. However, the TMEM16A channel is much less sensitive to barium (EC₅₀: 330 ± 30 μM) and cadmium (EC₅₀: 940 ± 80 μM) than BK channels, which responds to 10 μM barium and cadmium via the calcium bowl and the RCK1 high affinity site, respectively. This difference suggests that the high affinity calcium binding site(s) in the TMEM16A channel might be less effective in binding barium and cadmium ions due to their less optimal coordination chemistry. Alternatively, barium and/or cadmium might activate the TMEM16A channel via an unknown low affinity cation binding site, which remains latent under physiological conditions. This scenario seems plausible as none of our high affinity site mutations abolishes cadmium activation (Figure 6H). The identity of this putative low affinity, nonselective cation binding site and its potential involvement in TMEM16A channel activation require further study.

By following up on our comprehensive alanine mutagenesis with multiple point mutations of the putative calcium binding residues and testing their effects on channel activation by different divalent cations, we were able to take advantage of the well-studied chemical properties of divalent cation coordination to analyze these putative calcium binding sites in detail. After these candidate acidic residues were positively identified, conservative mutations preserving either the negative charge (glutamate to aspartate or vice versa) or the approximate side chain volume (glutamate to glutamine, or aspartate to asparagine) were expected to have relatively minor effects on calcium coordination in actual binding sites, considering that either the electrostatic or steric side chain properties would be preserved under those conditions. Conversely, introduction of positively charged arginine side chain would have been expected to produce much more drastic effects. Indeed, this pattern was observed for four of the five acidic residues identified in the mutagenesis screen. The calcium sensitivity of E698, E701 and D734 was drastically reduced by arginine substitutions, in contrast to the milder effects caused by conservative substitutions. Mutations of E730 followed the same pattern consistent with a divalent ion binding site, though with generally less drastic effects. The E650R mutant, on the other hand, retained the highest sensitivity to calcium of all substitutions at that site, whereas conservative substitutions greatly impacted calcium sensitivity. These results suggest that E650 is unlikely to interact directly with a calcium ion, at least not in the same manner as those of the other four, and we have thus excluded it from our proposed model of the primary calcium interaction site (Figure 7C). Given the possibility that E650 may be involved in the transduction of conformational changes associated with calcium binding to trigger the channel's gating machinery, it will be important to further scrutinize its functional role in future studies.

Our analyses using different divalent cations to activate various TMEM16A mutants are also consistent with the notion that E698, E701, E730 and D734, but not E650, are involved in direct interactions with strontium. Strontium is a divalent ion with chemical properties similar to calcium, as it has two electrons in its outer valence shell and is considered a ‘hard’ ligand (Rubin, 1963; Dudev and Lim, 2003). Although strontium has a slightly larger ionic radius than calcium, it has been shown previously to robustly activate TMEM16A channels (Yuan et al., 2013). The effects of side chain substitutions at those four sites on calcium and strontium sensitivity showed similar trends. In contrast, E650 mutants were generally insensitive to strontium activation, and the mutant displaying the highest sensitivity was again E650R, in keeping with our findings regarding calcium activation.

In addition to strontium, we also employed cadmium as an alternative TMEM16A activator. Cadmium is among the transition metals and acts as a ‘soft’ ligand, but has an ionic radius nearly identical to that of calcium (Sóvágó and Várnagy, 2013). Here, we found that while the wildtype channel was far less sensitive to cadmium ions than calcium ions, the apparent cadmium sensitivities of the mutant channels were insensitive to the side chain manipulations and remain in the submillimolar to millimolar range. In our view, there are at least two plausible explanations for this phenomenon. Firstly, as cadmium ions can be adequately coordinated by fewer negatively charged groups, alterations of just one of the interacting acidic residues may have less drastic effects on cadmium coordination. Alternately, as mentioned previously, it is possible that a second, lower affinity and/or less calcium-selective binding site may exist in the channel that can more readily bind cadmium following mutation in the higher affinity site identified in this study. At present, our results do not allow us to distinguish between these two possibilities, particularly as structural and functional studies of TMEM16A gating are still in their infancy. Nevertheless, the identification of residues forming a high affinity binding site, and our finding that cadmium can activate TMEM16A mutants rendered insensitive to calcium, provide key tools for the future study of channel gating processes downstream of TMEM16A’s high affinity binding of divalent cations.

Unlike the high affinity sites of BK channels that are located at the large cytosolic C-terminus (Salkoff et al., 2006), the high affinity calcium binding residues (E698, E701, E730, and D734) of the TMEM16A channels are between the putative transmembrane (TM) segments 5 and 7 (Figure 7C). This is consistent with recent findings (Yu et al., 2012; Scudieri et al., 2013) that the third intracellular loop (between TM6′ and TM7, Figure 7C, Model B) is important for the calcium sensitivity of the TMEM16A and TMEM16B CaCC channels. Previous studies (Yang et al., 2008b; Yu et al., 2012) have proposed two different models of membrane topology for TMEM16A. The earlier model (Model A in Figure 7C) includes a long extracellular or reentrant loop between TM5 and TM6″ (Yang et al., 2008b), while a subsequent model (Model B in Figure 7C) proposes that the peptide between TM5 and TM6″ in Model A actually includes one transmembrane segment (TM6′) (Yu et al., 2012). Our current study provides new insight into the membrane topology of this region by showing that residues 701 and 730 are accessible to the intracellular solvent and are in close proximity such that a disulfide bridge can form between E701C and E730C to impact calcium activation of the channel. This functional study implies that the hydrophobic region spanning 28 residues (including the putative TM6″) between E701 and E730 likely forms a short reentrant loop (Model C in Figure 7C), bringing together these four acidic residues at both ends of this loop to coordinate calcium ions. Further structural information is needed to validate this model.

In summary, our comprehensive survey of evolutionarily conserved acidic residues has identified several critical residues in the TMEM16A-CaCC that are responsible for its activation by calcium. Our study provides further evidence that the TMEM16A-CaCC channel directly interacts with intracellular calcium without involving CaM. Interestingly, these putative calcium binding acidic residues are highly conserved among proteins in the TMEM16 family, some of which have been shown to be important in cellular processes ranging from mucus secretion (Huang et al., 2012b) to blood coagulation (Suzuki et al., 2010; Yang et al., 2012) in mammals and host defense in Drosophila (Wong et al., 2013). The identification of these evolutionarily conserved acidic residues that bind calcium in the TMEM16A-CaCC channel will contribute towards a general understanding of the molecular mechanisms of calcium-activated TMEM16 channels as well as their cellular functions in response to calcium signaling in various cell types and organisms.

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Author details

1. Jason Tien

   Department of Physiology, University of California, San Francisco, San Francisco, United States

   Contribution

   JT, Tested the divalent cation effects on these residues, Performed the crosslinking experiment, Analyzed the data, Conducted mutagenesis, Wrote the manuscript

   Contributed equally with

   Christian J Peters

   Competing interests

   The authors declare that no competing interests exist.

2. Christian J Peters

   Department of Physiology, University of California, San Francisco, San Francisco, United States

   Contribution

   CJP, Tested the divalent cation effects on these residues, Analyzed the data, Wrote the manuscript

   Contributed equally with

   Jason Tien

   Competing interests

   The authors declare that no competing interests exist.

3. Xiu Ming Wong

   Graduate Program in Chemistry and Chemical Biology, University of California, San Francisco, San Francisco, United States

   Contribution

   XMW, Tested calmodulin effects

   Competing interests

   The authors declare that no competing interests exist.

4. Tong Cheng

   1. Department of Physiology, University of California, San Francisco, San Francisco, United States
   2. Department of Biochemistry and Biophysics, University of California, San Francisco, San Francisco, United States
   3. Howard Hughes Medical Institute, University of California, San Fransisco, San Fransisco, United States

   Contribution

   TC, Conducted mutagenesis

   Competing interests

   The authors declare that no competing interests exist.

5. Yuh Nung Jan

   1. Department of Physiology, University of California, San Francisco, San Francisco, United States
   2. Department of Biochemistry and Biophysics, University of California, San Francisco, San Francisco, United States
   3. Howard Hughes Medical Institute, University of California, San Fransisco, San Fransisco, United States

   Contribution

   YNJ, Conceived and designed the study

   Competing interests

   The authors declare that no competing interests exist.

6. Lily Yeh Jan

   1. Department of Physiology, University of California, San Francisco, San Francisco, United States
   2. Department of Biochemistry and Biophysics, University of California, San Francisco, San Francisco, United States
   3. Howard Hughes Medical Institute, University of California, San Fransisco, San Fransisco, United States

   Contribution

   LYJ, Conceived and designed the study, Analyzed the data, Wrote the manuscript

   For correspondence

   Lily.Jan@ucsf.edu

   Competing interests

   The authors declare that no competing interests exist.

7. Huanghe Yang

   1. Department of Physiology, University of California, San Francisco, San Francisco, United States
   2. Department of Biochemistry and Biophysics, University of California, San Francisco, San Francisco, United States
   3. Howard Hughes Medical Institute, University of California, San Fransisco, San Fransisco, United States

   Contribution

   HY, Conceived and designed the study, Tested calmodulin effects, Conducted the screen with alanine mutations and identified/verified the key residues for calcium binding, Analyzed the data, Wrote the manuscript

   For correspondence

   huanghe.yang@ucsf.edu

   Competing interests

   The authors declare that no competing interests exist.

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