Membrane-associated guanylate kinase-like properties of beta-subunits required for modulation of voltage-dependent Ca2+ channels

Abstract

High-voltage-activated Ca2+ channels regulate diverse functions ranging from muscle contraction to synaptic transmission. Association between auxiliary beta- and distinct pore-forming alpha1-subunits is obligatory for forming functional high-voltage-activated Ca2+ channels, yet the structural determinants underlying this interaction remain poorly understood. Recently, homology modeling of Ca(2+)-channel beta1b-subunit identified src homology 3 (SH3) and guanylate kinase (GK) motifs in a tandem arrangement reminiscent of the membrane-associated guanylate kinase (MAGUK) class of scaffolding proteins. However, direct evidence for MAGUK-like properties and their functional implications in beta-subunits is lacking. Here, we show a functional requirement for both SH3 and GK domains in beta2a. Point mutations in either the putative beta2a SH3 or GK domains severely blunted modulation of recombinant L-type channels, showing the importance of both motifs for a functional alpha1-beta interaction. Coexpression of these functionally deficient beta2a-SH3 and GK mutants rescued WT currents, demonstrating trans complementation similar to that observed in MAGUKs. Truncated "hemi-beta2a" subunits, containing either the SH3 or GK domain, were ineffective on their own, but reconstituted WT currents when coexpressed. Moreover, the SH3 and GK domains were found to interact in vitro. These findings reveal MAGUK-like properties in beta-subunits that are critical for alpha1-subunit modulation, revise current models of alpha1-beta association, and predict new physiological dimensions of beta-subunit function.

Fig. 2.

Mutations in β_2a-SH3 and GK motifs disrupt function. (A) Domain structures for β_2a and PSD-95. The β-subunit D4 region contains the BID, important for α₁ modulation. Sequence alignment shows homology between β_2a and PSD-95 in their SH3 domains. ^* denotes a conserved leucine residue critical for MAGUK structure–function; #s denote a trio of residues critical for α₁–β binding. (B)(Left) Exemplar current from channels reconstituted with the SH3 mutant, β_2a[L93P] (black). (Right) G–V curves for SH3 mutant, β_2a[L93P] (□) or GK mutant, β_2a[P234R] (○). (C) Functional rescue by trans complementation of SH3 and GK mutants. Exemplar current and G–V curve for channels reconstituted with β_2a[L93P] and β_2a[P234R] (▪). (D) Comparison of tail-current density evoked by +100-mV test pulse (J₁₀₀) in channels reconstituted with different combinations of WT or mutant β_2a-subunits. (E) Q_ON comparison. For D and E, ^*, P < 0.001, P < 0.05; #, P = 0.39; and ##, P = 0.55 compared with WT β_2a. (F) Comparison of normalized tail-activation curves (G/G₁₀₀). WT β_2a data (gray) are shown for reference.

Fig. 1.

Effects of β_2a on recombinant L-type channels reconstituted in HEK 293 cells. (A) (Left) Voltage protocol (Upper) and exemplar current (Lower) from a cell transfected with α_1C-GFP alone (no β). Here and throughout, exemplar currents were evoked by a 20-ms test pulse to +50 mV from a holding potential of -100 mV; tail currents were analyzed at -50 mV repolarization. (Right) Population tail-activation (G–V) curve for no-β channels constructed from tail-current density (J_tail). (B) Exemplar current and G–V curve for cotransfection of α_1C-GFP and β_2a-subunits (▪). No β (○) is plotted for comparison; note difference in scale. (C) Exemplar ON-gating currents (Upper) and population ON-gating charge (Q_ON, Lower). ^*, P < 0.001; number of cells in parentheses. (D) Plot of tail-current density at the reversal potential (J_rev) vs. Q_ON for channels with β_2a (▪) or no β (○). Lines are determined from linear regression of β_2a (black) or no β (gray). (Inset) Detail for no β near the origin.

Fig. 4.

β_2a function restored by hemi-β_2a fragments. (A) Mapping of putative SH3 and GK motifs onto β-subunit domain structure. Hemi-β_2a constructs included either the SH3 (NSH3) or GK domain (GKC). (B)(Left) Exemplar current from channels reconstituted with GKC (black). (Right) G–V curves for channels reconstituted with NSH3 (○) or GKC (□). (C) Exemplar current and G–V curve from channels reconstituted with NSH3 and GKC (black; ▪). WT β_2a data (gray) are shown for reference. (D) Comparison of J₁₀₀ in channels reconstituted with hemi-β_2a combinations. (E) Q_ON comparison. In D and E, ^*, P < 0.001; #, P = 0.38; and ##, P = 0.58 compared with WT β_2a; number of cells is in parentheses. (F) G/G₁₀₀ comparison.

Fig. 3.

β_2a-subunit SH3 and GK domains interact. (A) Coomassie blue-stained SDS/PAGE of purified Xpress-tagged NSH3 and V5-tagged GKC. (B) GKC pull-down of NSH3, and anti-express immunoblot (Xpr). Lane 1, purified Xpr-NSH3; lane 2, purified V5-GKC; lanes 3 and 4, pull-down of Xpress-NSH3 in the absence or presence of immobilized V5-GKC, respectively.

Fig. 5.

Conceptualization of β-structural determinants for modulation of α₁-subunits. (A) In WT β, intramolecular SH3 and GK interaction could form a functional unit that modulates α₁-subunits (one-site model; Left). By contrast, SH3 and GK domains could interact with independent binding sites on α₁ (two-site model; Right). (B) SH3 and GK mutants are functionally deficient. However, trans complementation could reconstitute a functional unit (one-site model; Left) or separately provide WT SH3 and GK domains to accommodate independent binding sites (two-site model; Right). (C) Hemi-β_2a-subunits could operate similarly.