Altered biochemical properties of transient receptor potential vanilloid 6 calcium channel by peptide tags.
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-TRPV6(WT)) showed much lower levels of aggregate-like bands in Western blots than those of Myc-TRPV6(WT). By contrast, the glycosylation level was higher with Myc-TRPV6(WT) than that with YFP-TRPV6(WT). We additionally demonstrate that peptide tags affect the protein integrity of TRPV6 channels. Myc-TRPV6(WT) was expressed as an intact channel, whereas the pore mutants Myc-TRPV6(D542A) and Myc-TRPV6(D542K) 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-TRPV6(WT). However, regardless of the peptide tag used, TRPV6(D542A) and TRPV6(D542K) electrophysiologically inhibited TRPV6(WT) 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.
Park
Eun-Jung
EJ
Department of Physiology, Seoul National University College of Medicine, Korea.
Kim
Byung-Joo
BJ
Kim
Su-Hwa
SH
Kim
Sung-Young
SY
Sung
Tae-Sik
TS
Chae
Hong-Gu
HG
Kim
Sang Jeong
SJ
Kim
Jun
J
Park
Hyun Ho
HH
So
Insuk
I
Jeon
Ju-Hong
JH
D015153:Blotting, Western; D002118:Calcium / Q000378:metabolism; D015220:Calcium Channels / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D002460:Cell Line; D000939:Epitopes / Q000737:chemistry; D016259:Genes, myc; D006031:Glycosylation; D006801:Humans; D008024:Ligands; D008164:Luminescent Proteins / Q000737:chemistry; D009154:Mutation; D010455:Peptides / Q000378:metabolism*; D010957:Plasmids; D011506:Proteins / Q000378:metabolism; D050916:TRPV Cation Channels / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D014162:Transfection
D015220:Calcium Channels; D000939:Epitopes; D008024:Ligands; D008164:Luminescent Proteins; D010455:Peptides; D011506:Proteins; D050916:TRPV Cation Channels; C422827:TRPV6 protein, human; D002118:Calcium