Endogenous and exogenous Ca2+ buffers differentially modulate Ca2+-dependent inactivation of Ca(v)2.1 Ca2+ channels.
Voltage-gated Ca2+ channels undergo a negative feedback regulation by Ca2+ ions, Ca2+-dependent inactivation, which is important for restricting Ca2+ signals in nerve and muscle. Although the molecular details underlying Ca2+-dependent inactivation have been characterized, little is known about how this process might be modulated in excitable cells. Based on previous findings that Ca2+-dependent inactivation of Ca(v)2.1 (P/Q-type) Ca2+ channels is suppressed by strong cytoplasmic Ca2+ buffering, we investigated how factors that regulate cellular Ca2+ levels affect inactivation of Ca(v)2.1 Ca2+ currents in transfected 293T cells. We found that inactivation of Ca(v)2.1 Ca2+ currents increased exponentially with current amplitude with low intracellular concentrations of the slow buffer EGTA (0.5 mm), but not with high concentrations of the fast Ca2+ buffer BAPTA (10 mm). However, when the concentration of BAPTA was reduced to 0.5 mm, inactivation of Ca2+ currents was significantly greater than with an equivalent concentration of EGTA, indicating the importance of buffer kinetics in modulating Ca2+-dependent inactivation of Ca(v)2.1. Cotransfection of Ca(v)2.1 with the EF-hand Ca2+-binding proteins, parvalbumin and calbindin, significantly altered the relationship between Ca2+ current amplitude and inactivation in ways that were unexpected from behavior as passive Ca2+ buffers. We conclude that Ca2+-dependent inactivation of Ca(v)2.1 depends on a subplasmalemmal Ca2+ microdomain that is affected by the amplitude of the Ca2+ current and differentially modulated by distinct Ca2+ buffers.
Kreiner
Lisa
L
Department of Pharmacology and Center for Neurodegenerative Disease, Emory University School of Medicine, Atlanta, Georgia 30322, USA.
Lee
Amy
A
D000818:Animals; D015153:Blotting, Western; D002021:Buffers; D064026:Calbindins; D002118:Calcium / Q000737:chemistry* / Q000378:metabolism; D020864:Calcium Channels, N-Type / Q000737:chemistry* / Q000378:metabolism; D002460:Cell Line; D003593:Cytoplasm / Q000378:metabolism; D018076:DNA, Complementary / Q000378:metabolism; D004533:Egtazic Acid / Q000031:analogs & derivatives / Q000737:chemistry / Q000494:pharmacology; D004594:Electrophysiology; D005786:Gene Expression Regulation; D006801:Humans; D007700:Kinetics; D008954:Models, Biological; D010320:Parvalbumins / Q000737:chemistry; D018408:Patch-Clamp Techniques; D051381:Rats; D064030:S100 Calcium Binding Protein G / Q000737:chemistry; D014162:Transfection
D002021:Buffers; D064026:Calbindins; D002118:Calcium; D020864:Calcium Channels, N-Type; D018076:DNA, Complementary; D004533:Egtazic Acid; D010320:Parvalbumins; D064030:S100 Calcium Binding Protein G; C025603:1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid