Proceedings of The Physiological Society
University of Oxford (2011) Proc Physiol Soc 23, PC120
Structure-function studies of the pore domain of the TMEM16 family of Ca2+-activated Cl- channels.
A. Tamuleviciute1, A. Seed1, P. Tammaro1
1. Faculty of Life Sciences, University of Manchester, Manchester, United Kingdom.
TMEM16 channels are a newly described family of anion channels unrelated in primary sequence to any previously described channel protein (Caputo et al., 2008; Schroeder et al., 2008; Yang et al., 2008). Recent studies have shown that TMEM16A and the related TMEM16B mediate many CaCC functions, including salivary exocrine and tracheal secretion, and olfactory transduction (Ferrera et al., 2010). The molecular features involved in controlling chloride conduction in TMEM16 channels, however, are still poorly understood. Hydropathy analysis of the primary sequence of TMEM16A indicates the presence of a re-entrant loop between the transmembrane domains 5 and 6. It has been suggested that the channel selectivity filter and the conduction pathway for anions reside within this area. To examine the functional role of this region, we used a chimeric strategy involving various TMEM16x proteins with different conduction properties. Wild-type (wt) or mutant (chimeric) TMEM16x channels were heterologously expressed in HEK-293T cells and macroscopic currents were recorded with the whole-cell configuration of the patch-clamp technique. The external solution contained (mM): 150 NaCl, 0.1 CaCl2, 10 glucose and 10 Hepes; pH was 7.4. The pipette solution contained (mM): 130 CsCl, 10 EGTA, 1 MgCl2, 10 Hepes and 8 CaCl2 to obtain ~600 nM free [Ca2+]; pH was 7.3. Two-tailed t-tests were used for comparisons. Shifts in the reversal potential of the current caused by replacement of external Cl- with other anions gave the following selectivity sequence for TMEM16A channels based on permeability ratios (P(x)/P(Cl)): SCN- (6.0±0.9) > ClO4- (4.6±0.3) > I- (2.8±0.2) > NO3- (2.4±0.1) > N3- (1.9±0.2) > Cl- (1.0) >> gluconate (0.12±0.04) (n=4-7). The relative conductance was: N3- (8.9±2.4) > SCN- (4.0±0.7) > I- (3.6±0.6) > NO3- (3.5±0.6) > ClO4- (1.6±0.2) > Cl-(1.0) >> gluconate (0.23±0.03) (n=4-7). The relative anion conductance and selectivity sequences for TMEM16B channels were not statistically different from those of TMEM16A. Replacement of TMEM16A re-entrant loop (599-705) with the putative pore loop of TMEM16B gave rise to functional channels with the same degree of selectivity for anions as in TMEM16A channels. Chimeric channels obtained by substituting the re-entrant loop of TMEM16A with the re-entrant loop of TMEM16 proteins with very limited conductance for anions (TMEM16F, TMEM16K) resulted in channels unable to conduct anions. Taken together, these data reinforce the idea that the re-entrant loop contributes to the channel pore. It is likely that the regions within the sequence of the re-entrant loop that are identical between the TMEM16A and TMEM16B channel (599-607, 617-647, 694-705) contain the structural elements involved in controlling anion selectivity.
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