Abstract
Besides opening and closing, high voltage-activated calcium channels transit to a nonconducting inactivated state from which they do not re-open unless the plasma membrane is repolarized. Inactivation is critical for temporal regulation of intracellular calcium signaling and prevention of a deleterious rise in calcium concentration. R-type high voltage-activated channels inactivate fully in a few hundred milliseconds when expressed alone. However, when co-expressed with a particular β-subunit isoform, β(2a), inactivation is partial and develops in several seconds. Palmitoylation of a unique di-cysteine motif at the N terminus anchors β(2a) to the plasma membrane. The current view is that membrane-anchored β(2a) immobilizes the channel inactivation machinery and confers slow inactivation phenotype. β-Subunits contain one Src homology 3 and one guanylate kinase domain, flanked by variable regions with unknown structures. Here, we identified a short polybasic segment at the boundary of the guanylate kinase domain that slows down channel inactivation without relocating a palmitoylation-deficient β(2a) to the plasma membrane. Substitution of the positively charged residues within this segment by alanine abolishes its slow inactivation-conferring phenotype. The linker upstream from the polybasic segment, but not the N- and C-terminal variable regions, masks the effect of this determinant. These results reveal a novel mechanism for inhibiting voltage-dependent inactivation of R-type calcium channels by the β(2a)-subunit that might involve electrostatic interactions with an unknown target on the channel's inactivation machinery or its modulatory components. They also suggest that intralinker interactions occlude the action of the polybasic segment and that its functional availability is regulated by the palmitoylated state of the β(2a)-subunit.
Highlights
Membrane anchoring underlies inhibition of voltage-dependent inactivation (VDI) of calcium channels by the 2a-subunit
Channel complexes bearing 2a SH3-PBLKAla-GK inactivates nearly as fast as the construct lacking this segment (Fig. 4, B and C, and Table 1). These results demonstrate that the basic residues within the PBLK segment are required for conferring a slow inactivation phenotype to nonmembrane-anchored -subunit and suggest the involvement of electrostatic interactions in VDI modulation
Four conclusions can be drawn from this work as follows: (i) there is an additional structural determinant in 2a isoform, the distal polybasic linker segment, besides the N-terminal palmitoylable region, that contributes to slowing down inactivation; (ii) the polybasic segment inhibits inactivation in a membraneanchoring independent manner and the basic residues within are required for this effect; (iii) the linker sequence upstream from the polybasic segment masks the contribution of the latter, and (iv) the effect of the palmitoylated N terminus of the
Summary
Membrane anchoring underlies inhibition of voltage-dependent inactivation (VDI) of calcium channels by the 2a-subunit. The linker upstream from the polybasic segment, but not the N- and C-terminal variable regions, masks the effect of this determinant These results reveal a novel mechanism for inhibiting voltage-dependent inactivation of R-type calcium channels by the 2a-subunit that might involve electrostatic interactions with an unknown target on the channel’s inactivation machinery or its modulatory components. They suggest that intralinker interactions occlude the action of the polybasic segment and that its functional availability is regulated by the palmitoylated state of the 2a-subunit. The exposition of this segment appears to be regulated by the palmitoylated state of the N terminus of 2a
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