Amino acids in segment IVS6 and β-subunit interaction support distinct conformational changes during Cav2.1 inactivation

Ca(v)2.1 mediates voltage-gated Ca2+ entry into neurons and the release of neurotransmitters at synapses of the central nervous system. An inactivation process that is modulated by the auxiliary beta -subunits regulates Ca2+ entry through Ca(v)2.1. However, the molecular mechanism of this alpha (1)-beta -subunit interaction remains unknown. Herein we report the identification of new determinants within segment IVS6 of the alpha (1)2.1-subunit that markedly influence channel inactivation. Systematic substitution of residues within IVS6 with amino acids of different size, charge, and polarity resulted in mutant channels with rates of fast inactivation (k(inact)) ranging from a 1.5-fold slowing in V1818I (k(inact) = 0.98 +/- 0.09 s(-1) compared with wild type alpha (1)2.1/alpha (2)-delta/beta (1a) k(inact) = 1.35 +/- 0.25 -1) to a 75-fold acceleration in mutant M1811Q (k(inact) 102 +/- 3 s(-1)). Coexpression of mutant alpha (1)2.1-subunits with beta (2a) resulted in two different phenotypes of current inactivation: 1) a pronounced reduction in the rate of channel inactivation or 2) an attenuation of a slow component in I-Ba inactivation. Simulations revealed that these two distinct inactivation phenotypes arise from a beta (2a)-subunit-induced destabilization of the fast-inactivated state. The IVS6- and beta (2a)-subunit-mediated effects on Ca(v)2.1 inactivation are likely to occur via independent mechanisms.