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.
Highlights
JUNE 24, 2011 VOLUME 286 NUMBER 25 tion, which in turn mediate a variety of cellular processes, including gene expression, contraction, neurotransmission, and exocytosis [1]
Crystallographic studies of three of these provided the molecular basis for its functional versatility by identifying two protein-protein interactions modules: an Src homology 3 (-SH3) and a guanylate kinase (-GK), which are typically found in members of the membrane-associated guanylate kinase family of scaffolding proteins (8 –10)
Preassociation of CaV2a with a Polypeptide Encoding Binding Motif of ␣1 Pore-forming as the ␣1-interaction domain (AID) Inhibits Endocytosis—We have shown previously that full-length CaV2a is as efficient as the SH3 domain to promote endocytosis of CaV␣1 when association to it is prevented and that it induces internalization of the distantly related Shaker potassium channel [7]
Summary
CDNA Constructs and Recombinant Proteins—The CaV1.2 channel construct and dynamin has been described elsewhere [7]. Cobalt-based agarose beads were coupled to the proteins and incubated with pre-cleared extract from tsA201 cells transfected with a dynamin-encoding plasmid 24 to 36 h earlier. Electrophysiological recordings on CaV1.2-expressing oocytes were performed using the cut-open oocyte technique with a CA-1B amplifier (Dagan Corp., Minneapolis MN) as described [25]. Linear components were eliminated by P/-4 prepulse protocol that consisted of four consecutives pulses a fourth of the amplitude of the test pulse Current traces during these prepulses were added together and substracted from the current traces obtained with the main pulse. Spectral analysis of living tsA210 cells co-expressing CFP- and YFP-tagged CaV2 constructs were performed according to Kobe et al [27]. Quantitative linear unmixing FRET (lux-FRET) analysis in cuvettes and at the single cell level were performed 18 –24 h after transfection as described recently [28, 29]. Proteins were resolved in a gradient polyacrylamide gel (4 –25%) overnight at 4 °C using cathode buffer containing 100 mM histidine, 0.002% Serva Blue G powder (SERVA electrophoresis), pH 8.0, and anode buffer containing 100 mM Tris, pH 8.8
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