Anion permeation in calcium-activated chloride channels formed by TMEM16A from Xenopus tropicalis

Abstract

Calcium-activated chloride channels (CaCC) formed by anoctamin1/TMEM16A subunits are ubiquitously expressed, and these channels are known to prevent polyspermy in amphibian oocytes. Here, we describe a TMEM16A clone isolated from Xenopus tropicalis oocytes (xtTMEM16A) and how the anion permeation properties are modified in single-site mutants of the ion pore. The anion permeability sequence was SCN(-) > I(-) > Br(-) > Cl(-) > gluconate (relative permeabilities 5.6:3.0:2.1:1:0.2, respectively). Dose-response curves indicated that the voltage-dependent half-maximal concentration for Ca(2+) activation (K d of the Hill equation at +100 mV) was 120 nM in normal external Cl(-), whereas it was displaced leftward to 75 nM Ca(2+), when I(-) replaced Cl(-). The I(-):Cl(-) mole fraction (MF) of the external solution was varied in order to gain insight into the permeation mechanism of the pore. No anomaly in MF behavior was observed for conductance, but it was observed for current reversal potential, which deviated from the prediction of the Goldman-Hodgkin-Katz equation. Mutations of positively charged amino acids in the pore, R646 and R761, to glutamate resulted in reduction of the relative permeability to I(-). Data from the wild type and mutants could be well fitted by a three-barrier, two-site permeation model. This suggests a multi-ion pore with at least two binding sites for anions, with R646 mole fraction closer to the extracellular membrane surface--being important for the stability of both sites--and R761--located deeper within the membrane--mainly affecting the innermost binding site. Considerations of xtTMEM16A putative pore region topology are discussed in the light of two alternative topological models of the protein.

Study of permeation and blocker binding in TMEM16A calcium-activated chloride channels

“…This strongly suggests that A-9-C binds approximately midway across the plasma membrane. This observation is consistent with our previous proposal 6 of an anion-binding site in the permeation pathway which is affected by the same mutations that affect the A-9-C block. As will be discussed below in more detail, this may indicate that blockers bind to the same sites as permeant anions, thus hindering anion permeation by binding persistently to these structures.…”

“…10 Two anion-binding sites with electrical distances of 0.3 and 0.5-0.6 are likely to be present in the ion permeation pathway. 6 Based on modeling analysis of the effects of mutation R646E, we concluded that this residue is important for the conformation of both binding sites. On the other hand, analysis of R761 points out that this residue must be involved in the conformation of the deeper binding site.…”

“…Note that only the results from these 3 mutants and from the WT are presented here; for details about the behavior of R646E, R761E and the DM, please refer to the earlier study. 6 As shown in Figure 3, the behavior of the K613E and K628E mutants drifted apart from that of the wildtype channels, whereas the R630E mutant did not. Fit to the data with the Three-Barriers Two-Wells permeation model indicates that the innermost well is the one most affected in K613E, whereas the outermost well is most affected in K628E.…”

“…In our previous report, 6 we observed that some pore mutationsnamely R646E, R761E, and the DM R646ECR761E-may affect the dependence of the reversal potential (Er) on the I ¡ : Cl ¡ mole fraction (MF; Er vs. MF curve). Through such MF experiments, we have shown anomalous Cl ¡ and I ¡ permeation behavior.…”

“…5 In this context, we continued our study of residues which could be important for the determination of the properties of the pore of Xenopus tropicalis TMEM16A (xtTMEM16A). We have previously reported 6 that positively charged residues R646 and R761 are important for ion permeation. Further examination suggested that residues in the TM5 transmembrane domain should also contribute significantly to the properties of the pore.…”