Homodimerization of the Src homology 3 domain of the calcium channel β-subunit drives dynamin-dependent endocytosis

Abstract

Voltage-dependent calcium channels constitute the main entry pathway for calcium into excitable cells. They are heteromultimers formed by an α(1) pore-forming subunit (Ca(V)α(1)) and accessory subunits. To achieve a precise coordination of calcium signals, the expression and activity of these channels is tightly controlled. The accessory β-subunit (Ca(V)β), a membrane associated guanylate kinase containing one guanylate kinase (β-GK) and one Src homology 3 (β-SH3) domain, has antagonistic effects on calcium currents by regulating different aspects of channel function. Although β-GK binds to a conserved site within the α(1)-pore-forming subunit and facilitates channel opening, β-SH3 binds to dynamin and promotes endocytosis. Here, we investigated the molecular switch underlying the functional duality of this modular protein. We show that β-SH3 homodimerizes through a single disulfide bond. Substitution of the only cysteine residue abolishes dimerization and impairs internalization of L-type Ca(V)1.2 channels expressed in Xenopus oocytes while preserving dynamin binding. Covalent linkage of the β-SH3 dimerization-deficient mutant yields a concatamer that binds to dynamin and restores endocytosis. Moreover, using FRET analysis, we show in living cells that Ca(V)β form oligomers and that this interaction is reduced by Ca(V)α(1). Association of Ca(V)β with a polypeptide encoding the binding motif in Ca(V)α(1) inhibited endocytosis. Together, these findings reveal that β-SH3 dimerization is crucial for endocytosis and suggest that channel activation and internalization are two mutually exclusive functions of Ca(V)β. We propose that a change in the oligomeric state of Ca(V)β is the functional switch between channel activator and channel internalizer.

FIGURE 1.

Wild-type β-SH3 domain but not β-SH3 C113A forms dimers. A, schematic and ribbon representation of Ca_Vβ structure in complex with the highly conserved β-binding site of Ca_Vα₁ (AID, helix shown in gray) (Protein Data Bank code 1TOJ). SH3, shown in gray, and GK, shown in black, modulate different channel functions. The two N-terminal cysteine residues that undergo palmitoylation in Ca_Vβ_2a variant used in this study are marked by zigzag lines in the sketch at the top of the panel. B, oligomeric state of wild-type β-SH3 determined by BN-PAGE. Lanes 1–4 were loaded with β-SH3, and lanes 5 and 6 were loaded with β-SH3 concatamer. To induce dissociation into lower-order oligomeric states, including monomers, samples were treated as indicated at the bottom of the panel as described under “Experimental Procedures.” C, size-exclusion chromatography profile (Superdex 200 10/30 column, GE Healthcare) of β-SH3 C113A and β-SH3 concatamer. The corresponding profile for wild-type β-SH3 has been shown elsewhere (7), and its peak, corresponding to the monomeric fraction, is denoted here with an open arrowhead. The elution volume of molecular weight standards are indicated above the chromatogram. Numbers correspond to molecular masses in kDa. Vo, void, 67.0 (albumin); 43.0 (ovalbumin); and 13.7 (ribonuclease). The adjacent panel shows the same proteins resolved onto a reducing SDS-PAGE: lane 1, molecular weight standards; lane 2, wild-type β-SH3; lane 3, β-SH3 C113A; and lane 4, β-SH3 concatamer. D, oligomeric state of β-SH3 C113A determined by BN-PAGE. Lanes 1–3 were loaded with β-SH3 C113A, and lane 4 was loaded with β-SH3 concatamer. Samples were treated as indicated.

FIGURE 2.

β-SH3 C113A dimerization-deficient mutant preserves its association with dynamin but loses the endocytic capability. A, Western blot of a pull-down assay using His-tagged wild-type and C113A β-SH3 as bait. Lysates from cells expressing dynamin were incubated with either β-SH3 (lane 1) or β-SH3 C113A (lane 2) precoupled to cobalt beads or with cobalt beads alone (lane 3). Bound proteins were detected with anti-dynamin antibody. B, a schematic describing the experimental protocol for CMBI assay. 1, Xenopus oocytes are first injected with Ca_Vα₁-encoding cRNA; 2, after a few days, when the channels are already expressed at the plasma membrane, oocytes are reinjected with the test protein; and 3, a few hours later, gating currents are recorded. C, representative gating currents recordings and CMBI assay results from Xenopus oocytes expressing Ca_V1.2 alone and following injection of either β-SH3 or β-SH3 C113A. The shaded area under the gating current represents the total amount of charges moved during the voltage step and equals the number of channels (N) times the number of charges displaced per channel (q). The bar graph shows N × q values from oocytes expressing the indicated channel-protein combinations; Ca_V1.2 alone (120 ± 13 pC, n = 14), Ca_V1.2+β-SH3 (25 ± 8 pC, n = 12), and Ca_V1.2+β-SH3 C113A (108 ± 7 pC, n = 17). Average N × q values of Ca_V1.2+β-SH3 oocytes were significantly smaller that for Ca_V1.2 alone (p < 2.0 × 10⁻⁶) or Ca_V1.2+β-SH3 C113A (p < 1.3 × 10⁻⁸) according to a two-tailed t test.

FIGURE 3.

Covalently linking two molecules of β-SH3 C113A dimerization-deficient mutant restores the endocytic capability. A, Western blot (WB) of a pulldown assay was done as described in the legend to Fig. 2 but instead used concatameric versions of β-SH3 and β-SH3 C113A. Lysate from cells expressing dynamin were incubated with either β-SH3 concatamer (lane 1) or β-SH3 C113A concatamer (lane 2) or with cobalt beads alone (lane 3). B, CMBI assay results from Xenopus oocytes expressing Ca_V1.2 alone and in combination with β-SH3 either as a concatamer or non-covalently linked. The bar graph shows N × q values from oocytes expressing the indicated channel-protein combinations; Ca_V1.2 alone (145. ± 8 pC, n = 9), Ca_V1.2+β-SH3 concatamer (15 ± 4 pC, n = 6), and Ca_V1.2+β-SH3 (39 ± 7 pC, n = 8). Average N × q values were significantly smaller in the presence of β-SH3 (p < 5.7 × 10⁻⁹) or β-SH3 concatamer (p < 2.4 × 10⁻⁸) compared with Ca_V1.2 alone. Values for N × q between monomer and concatamers were also different but to a smaller degree (p < 0.04). C, same as B, but from Xenopus oocytes expressing Ca_V1.2 alone and in combination with either a β-SH3 C113A concatamer or β-SH3 C113A. The bar graph shows average N × q values for the indicated channel-protein combinations; Ca_V1.2 alone (143 ± 18 pC, n = 11), Ca_V1.2+β-SH3 C113A concatamer (44 ± 5 pC, n = 9), or Ca_V1.2+β-SH3 C113A (136 ± 13 pC, n = 15). Average N × q values were significantly smaller for Ca_V1.2+β-SH3 C113A concatamer than for Ca_V1.2 alone (p < 4.2 × 10⁻⁶) or Ca_V1.2+β-SH3 C113A (p < 2.9 × 10⁻⁵) according to a two-tailed t test.

FIGURE 4.

Membrane-targeted Ca_Vβ_2a also forms dimers. A, fluorescence emission spectra of living tsA210 cells expressing the indicated CFP- and YFP-tagged Ca_Vβ_2a constructs either separately or together (co-transfection). Mix corresponds to the emission spectra of a mixture of single transfected cells (mix). Emission spectra were collected at excitation wavelength λ_exc = 440 nm. a.u., arbitrary units. B, distribution and quantitative pixel-based FRET analysis of CFP-and YFP-tagged Ca_Vβ_2a in living tsA210 cells. Scale bar, 5 μm. Ef_D, apparent FRET efficiency, mem, membrane-localized Ca_Vβ_2a; intra, intracellular-localized Ca_Vβ_2a. Data represent mean values ± S.E. (n = 6). C, BN-PAGE analysis of Ca_Vβ_2a. Lanes 1–4 were loaded with Ca_Vβ_2a and lanes 5 and 6 with the Ca_Vβ_2a concatamer. In the absence of SDS, the Ca_Vβ_2a concatamer is poorly resolved (lane 5). Samples were treated as indicated at the bottom of the panel. D, BN-PAGE analysis of Ca_Vβ_2a C113A. Lanes 1–4 were loaded with Ca_Vβ_2a C113A and lanes 5 and 6 were loaded with the Ca_Vβ_2a concatamer.

FIGURE 5.

Association of Ca_Vβ_2a with the AID motif abolishes channel internalization and impairs dimerization. A, bar graph showing average N × q values for the indicated channel-protein combinations. Ca_V1.2 ΔAID alone (200 ± 29 pC, n = 14) was significantly larger than following injection of either Ca_Vβ_2a wild-type (40 ± 11 pC, n = 8) or C113A mutant (58 ± 9 pC, n = 18). B, bar graph showing average N × q values for the following: Ca_V1.2 ΔAID alone (119 ± 13 pC, n = 16) and after injection of Ca_Vβ_2a either preincubated with GST (23 ± 4 pC, n = 11) or with GST-AID (104 ± 15 pC, n = 17). C, FRET efficiencies for Ca_Vβ_2a-CFP and Ca_Vβ_2a-YFP proteins coexpressed in the absence (7.6 ± 0.3%) and presence (3.7 ± 0.3%) of α₁ pore-forming subunit. FRET experiments were repeated three times with consistent results.

FIGURE 6.

Schematic representation of channel activation and channel internalization by Ca_Vβ. The model illustrates that two different oligomeric states of the protein fulfils two opposing effects on calcium currents. Ca_Vβ binds as a monomer to the AID site of Ca_Vα₁ through the GK domain (shown in black) and activates calcium currents. Dissociation of Ca_Vβ weakens intramolecular SH3-GK interactions, making both domains available for further interactions, including dimerization. Association of Ca_Vβ dimers with dynamin, via its SH3 domain (shown in gray), promotes internalization of the channel complex and inhibition of calcium currents through Ca_Vα₁. It remains to be determined what triggers dissociation of the β-subunit from its traditional Ca_Vα₁ partner. Other ligand proteins may promote the interaction of Ca_Vβ with dynamin.