2009 Volume 32 Issue 10 Pages 1790-1794
Ion channels are commonly expressed in recombinant forms with peptide tags, which facilitates their molecular and electrophysiological studies. However, peptide tags may alter ion channel properties. Here we describe the differential effect of peptide tags on the biochemical properties of transient receptor potential vanilloid 6 (TRPV6) channels. Yellow fluorescent protein (YFP)-tagged wild-type TRPV6 (YFP-TRPV6WT) showed much lower levels of aggregate-like bands in Western blots than those of Myc-TRPV6WT. By contrast, the glycosylation level was higher with Myc-TRPV6WT than that with YFP-TRPV6WT. We additionally demonstrate that peptide tags affect the protein integrity of TRPV6 channels. Myc-TRPV6WT was expressed as an intact channel, whereas the pore mutants Myc-TRPV6D542A and Myc-TRPV6D542K were observed to be partially fragmented. By contrast, all YFP-tagged channels were intact, although the YFP-tagged pore mutants were less glycosylated than YFP-TRPV6WT. However, regardless of the peptide tag used, TRPV6D542A and TRPV6D542K electrophysiologically inhibited TRPV6WT which indicates that all pore mutants are equivalent electrophysiologically, not biochemically. Thus, our findings suggest that peptide tags can produce unintended biochemical changes of ion channels which highlight the necessity of careful biochemical evaluation to clarify the roles of ion channels.
