Kreiner Lisa, Lee Amy
Department of Pharmacology and Center for Neurodegenerative Disease, Emory University School of Medicine, Atlanta, Georgia 30322, USA.
J Biol Chem. 2006 Feb 24;281(8):4691-8. doi: 10.1074/jbc.M511971200. Epub 2005 Dec 22.
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.
电压门控Ca2+通道受到Ca2+离子的负反馈调节，即Ca2+依赖性失活，这对于限制神经和肌肉中的Ca2+信号很重要。尽管Ca2+依赖性失活的分子细节已得到表征，但对于可兴奋细胞中该过程如何被调节却知之甚少。基于先前的发现，即Ca(v)2.1（P/Q型）Ca2+通道的Ca2+依赖性失活受到强细胞质Ca2+缓冲的抑制，我们研究了调节细胞Ca2+水平的因素如何影响转染的293T细胞中Ca(v)2.1 Ca2+电流的失活。我们发现，在低细胞内浓度的慢缓冲剂EGTA（0.5 mM）下，Ca(v)2.1 Ca2+电流的失活随电流幅度呈指数增加，但在高浓度的快Ca2+缓冲剂BAPTA（10 mM）下则不然。然而，当BAPTA的浓度降至0.5 mM时，Ca2+电流的失活明显大于同等浓度的EGTA，这表明缓冲动力学在调节Ca(v)2.1的Ca2+依赖性失活中的重要性。将Ca(v)2.1与EF手型Ca2+结合蛋白小白蛋白和钙结合蛋白共转染，显著改变了Ca2+电流幅度与失活之间的关系，其方式与作为被动Ca2+缓冲剂的行为所预期的不同。我们得出结论，Ca(v)2.1的Ca2+依赖性失活取决于受Ca2+电流幅度影响并由不同Ca2+缓冲剂差异调节的质膜下Ca2+微区。
