Direct inhibition of P/Q-type voltage-gated Ca2+ channels by Gem does not require a direct Gem/Cavbeta interaction.
The Rem, Rem2, Rad, and Gem/Kir (RGK) family of small GTP-binding proteins potently inhibits high voltage-activated (HVA) Ca(2+) channels, providing a powerful means of modulating neural, endocrine, and muscle functions. The molecular mechanisms of this inhibition are controversial and remain largely unclear. RGK proteins associate directly with Ca(2+) channel beta subunits (Ca(v)beta), and this interaction is widely thought to be essential for their inhibitory action. In this study, we investigate the molecular underpinnings of Gem inhibition of P/Q-type Ca(2+) channels. We find that a purified Gem protein markedly and acutely suppresses P/Q channel activity in inside-out membrane patches, that this action requires Ca(v)beta but not the Gem/Ca(v)beta interaction, and that Gem coimmunoprecipitates with the P/Q channel alpha(1) subunit (Ca(v)alpha(1)) in a Ca(v)beta-independent manner. By constructing chimeras between P/Q channels and Gem-insensitive low voltage-activated T-type channels, we identify a region encompassing transmembrane segments S1, S2, and S3 in the second homologous repeat of Ca(v)alpha(1) critical for Gem inhibition. Exchanging this region between P/Q and T channel Ca(v)alpha(1) abolishes Gem inhibition of P/Q channels and confers Ca(v)beta-dependent Gem inhibition to a chimeric T channel that also carries the P/Q I-II loop (a cytoplasmic region of Ca(v)alpha(1) that binds Ca(v)beta). Our results challenge the prevailing view regarding the role of Ca(v)beta in RGK inhibition of high voltage-activated Ca(2+) channels and prompt a paradigm in which Gem directly binds and inhibits Ca(v)beta-primed Ca(v)alpha(1) on the plasma membrane.
Fan
Mingming
M
Department of Biological Sciences, Columbia University, New York, NY 10027, USA.
Buraei
Zafir
Z
Luo
Huai-Rong
HR
Levenson-Palmer
Rose
R
Yang
Jian
J
D000595:Amino Acid Sequence; D000818:Animals; D015153:Blotting, Western; D020865:Calcium Channels, P-Type / Q000235:genetics / Q000378:metabolism / Q000502:physiology*; D020907:Calcium Channels, Q-Type / Q000235:genetics / Q000378:metabolism / Q000502:physiology*; D002460:Cell Line; D002462:Cell Membrane / Q000187:drug effects* / Q000502:physiology; D005260:Female; D006801:Humans; D047468:Immunoprecipitation; D007700:Kinetics; D008564:Membrane Potentials / Q000187:drug effects; D008954:Models, Biological; D008969:Molecular Sequence Data; D020559:Monomeric GTP-Binding Proteins / Q000235:genetics / Q000378:metabolism / Q000494:pharmacology*; D009865:Oocytes / Q000187:drug effects / Q000378:metabolism / Q000502:physiology; D011485:Protein Binding; D021122:Protein Subunits / Q000235:genetics / Q000378:metabolism / Q000502:physiology; D017386:Sequence Homology, Amino Acid; D014982:Xenopus laevis
D020865:Calcium Channels, P-Type; D020907:Calcium Channels, Q-Type; D021122:Protein Subunits; D020559:Monomeric GTP-Binding Proteins