Amino Acids in Segment IVS6 and ß-Subunit Interaction Support Distinct Conformational Changes during Cav2.1 Inactivation

Cav2.1 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 ß-subunits regulates Ca2+ entry through Cav2.1. However, the molecular mechanism of this a1-ß-subunit interaction remains unknown. Herein we report the identification of new determinants within segment IVS6 of the a12.1-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 (kinact) ranging from a 1.5-fold slowing in V1818I (kinact = 0.98 ‘ 0.09 s -1 compared with wild type a12.1/a 2-d/ß1a kinact = 1.35 ‘ 0.25 s-1) to a 75-fold acceleration in mutant M1811Q (kinact = 102 ‘ 3 s-1). Coexpression of mutant a 12.1-subunits with ß2a 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 IBa inactivation. Simulations revealed that these two distinct inactivation phenotypes arise from a ß2a-subunit-induced destabilization of the fast-inactivated state. The IVS6- and ß2a-subunit-mediated effects on Cav2.1 inactivation are likely to occur via independent mechanisms.