ROLE OF TMEM16A PROTEIN AS A CALCIUM-ACTIVATED CHLORIDE CHANNEL
Paolo Scudieri, Loretta Ferrera, Elvira Sondo, Emanuela Caci, Luis J.V. Galietta
TMEM16A (also known as anoctamin-1), a plasma membrane protein with eight putative transmembrane domains, has been recently identified as an important component of calcium-activated chloride channels (CaCCs) (Ferrera et al., Physiology 25: 357-363, 2010). There are multiple TMEM16A isoforms generated by alternative splicing of at least three segments, (b), (c) and (d). A fourth segment, termed (a) and coding for a large part of TMEM16A amino-terminus may be skipped when an alternative promoter is used.
We are investigating the TMEM16A structure-function relationship by studying the properties of different TMEM16A isoforms, by truncation of the TMEM16A carboxy-terminus, and by introducing point mutations in different regions of the protein.
In our previous work we showed that TMEM16A alternative splicing has a functional meaning. For example, inclusion of segment b, a 22 amino acid long region localized before the first transmembrane domain, results in a variant, the (abc) isoform, that has a lower apparent affinity for calcium relative to the (ac) isoform. More recently, we have also noted that the (abc) channels have also the tendency to inactivate at very high calcium concentrations. Inclusion/skipping of segment (c), that consists of only four amino acids, appears to modulate the voltage-dependent activation of the channel. Instead, skipping of segment (a), a 116 amino acid long N-terminal region, does not grossly alter the properties of the TMEM16A-dependent chloride channels. We have also studied TMEM16A(0), an isoform devoid of all alternative segments. This protein variant generates chloride currents activated by cytosolic calcium but completely lacking voltage-dependent activation.
We also investigated the role of the TMEM16A carboxy-terminus. By mutagenesis, this region was shortened by 6 (Y977X), 30 (E953X), 45 (Q938X), and 136 (D847X) amino acids. Using the YFP-based assay we found that all mutants had activity comparable to that of the wild type protein, with the exception of D847X, which was totally inactive. Immunostaining of HEK-293 transiently transfected cells revealed that the latter mutant is not able to reach the plasma membrane. By whole cell patch clamp recordings in transiently-transfected HEK-293 cells, we studied the properties of Q938X, the active mutant with the largest carboxy-terminal deletion. Q938X seems to be very similar to the wild type protein in terms of calcium sensitivity and voltage-dependence.
Furthermore, we also evaluated the properties of membrane currents elicited by expression of TMEM16B, a close paralog of the TMEM16A protein. As already reported, we found significant differences between the two proteins. In particular, the kinetics of activation and deactivation were much faster for TMEM16B vs. TMEM16A. Comparison between the two proteins at the sequence and functional level will be important to identify the critical domains involved in channel gating by voltage and calcium.