Mechanism of Auxiliary Subunit Modulation of Neuronal α1E Calcium Channels

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Abstract
Voltage-gated calcium channels are composed of a main pore-forming α1 moiety, and one or more auxiliary subunits (β, α2δ) that modulate channel properties. Because modulatory properties may vary greatly with different channels, expression systems, and protocols, it is advantageous to study subunit regulation with a uniform experimental strategy. Here, in HEK 293 cells, we examine the expression and activation gating of α1E calcium channels in combination with a β (β1–β4) and/or the α2δ subunit, exploiting both ionic- and gating-current measurements. Furthermore, to explore whether more than one auxiliary subunit can concomitantly specify gating properties, we investigate the effects of cotransfecting α2δ with β subunits, of transfecting two different β subunits simultaneously, and of COOH-terminal truncation of α1E to remove a second β binding site. The main results are as follows. (a) The α2δ and β subunits modulate α1E in fundamentally different ways. The sole effect of α2δ is to increase current density by elevating channel density. By contrast, though β subunits also increase functional channel number, they also enhance maximum open probability (Gmax/Qmax) and hyperpolarize the voltage dependence of ionic-current activation and gating-charge movement, all without discernible effect on activation kinetics. Different β isoforms produce nearly indistinguishable effects on activation. However, β subunits produced clear, isoform-specific effects on inactivation properties. (b) All the β subunit effects can be explained by a gating model in which subunits act only on weakly voltage-dependent steps near the open state. (c) We find no clear evidence for simultaneous modulation by two different β subunits. (d) The modulatory features found here for α1E do not generalize uniformly to other α1 channel types, as α1C activation gating shows marked β isoform dependence that is absent for α1E. Together, these results help to establish a more comprehensive picture of auxiliary-subunit regulation of α1E calcium channels.