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Understanding the mechanism responsible for γ 6 ‐dependent modulation of low voltage activated Ca 2+ channels by exploring physical interactions
Author(s) -
Garcia Thomas,
Best Philip
Publication year - 2009
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.23.1_supplement.998.27
Subject(s) - hek 293 cells , biotinylation , protein subunit , immunoprecipitation , chemistry , mutant , biophysics , western blot , microbiology and biotechnology , transmembrane domain , membrane , biochemistry , biology , receptor , gene
The γ 6 subunit of voltage dependent Ca 2+ channels is known to decrease Ca 2+ current when co‐expressed with the low voltage activated Ca 2+ channel subunit α3.1. This effect of γ 6 is specific to current density as there is no change in the biophysical properties of the channels when measured at the macroscopic level. Previous studies using chimeric and mutant γ subunits indicate that the first transmembrane domain (TM1) of γ 6 and more specifically a GxxxA motif within it are critical for γ 6 's inhibitory function. In this study, cell surface membrane protein biotinylation, purification, and quantitative western blot demonstrate that the functional effect γ 6 confers upon Cav3.1 current is not due to a change in the plasma membrane expression of α3.1. Furthermore, co‐immunoprecipitation assay demonstrates a physical association of γ 6 and α3.1 in both HEK cells and atrial myocytes. Subsequently, we engineered chimeric and mutant subunits of γ 6 to identify potential interaction site(s). Results from these experiments indicate the functionally critical domain and motif of γ 6 are not required for γ 6 interaction with α3.1. Further studies are required to determine the region(s) in γ 6 involved in interaction with α3.1, as well as the γ 6 interaction site within α3.1. Supported by a GIA from the AHA.