Background: Alternative splicing can result in proteins with distinct subcellular distributions and functions. Results: Three ClC-3 splice variants are expressed in the mammalian brain with different subcellular localizations, but identical transport properties. Conclusion: Differences in the subcellular localization of ClC-3 splice variants suggest diverse cellular functions. Significance: The existence of multiple splice variants needs to be considered when studying cellular functions of ClC-3.

High voltage-gated calcium channels enable calcium entry into cells in response to membrane depolarization. Association of the auxiliary ␤-subunit to the ␣-interaction-domain in the pore-forming ␣ 1 -subunit is required to form functional channels. The ␤-subunit belongs to the membrane-associated guanylate kinase class of scaffolding proteins containing a Src homology 3 and a guanylate kinase domain. Although the latter is responsible for the high affinity binding to the ␣-interaction domain, the functional significance of the Src homology 3 domain remains elusive. Here, we show that injection of isolated ␤-subunit Src homology 3 domain into Xenopus laevis oocytes expressing the ␣ 1 -subunit reduces the number of channels in the plasma membrane. This effect is reverted by coexpressing ␣ 1 with a dominant-negative mutant of dynamin, a GTPase involved in receptor-mediated endocytosis. Full-length ␤-subunit also down-regulates voltage-gated calcium channels but only when lacking the ␣-interaction domain. Moreover, isolated Src homology 3 domain and the full-length ␤-subunit were found to interact in vitro with dynamin and to internalize the distantly related Shaker potassium channel. These results demonstrate that the ␤-subunit regulates the turnover of voltagegated calcium channels and other proteins in the cell membrane. This effect is mediated by dynamin and depends on the association state of the ␤-subunit to the ␣ 1 -pore-forming subunit. Our findings define a novel function for the ␤-subunit through its Src homology 3 domain and establish a link between voltage-gated calcium channel activity and the cell endocytic machinery.Cellular processes including muscle contraction, endocrine secretion, synaptic transmission, and gene expression (1), depend on the regulated influx of calcium through voltagegated calcium channels (VGCCs).3 VGCCs are multiprotein complexes containing a pore-forming subunit (Ca V ␣ 1 ) and a variable number of auxiliary subunits. Association of the auxiliary ␤-subunit (Ca V ␤) to a site shared by all Ca V ␣ 1 , the so-called ␣-interaction domain (AID), is mandatory to form a fully functional VGCC. Homology modeling (2) and the recent high resolution crystal structures of three Ca V ␤ isoforms (3-5) identified Ca V ␤ as a novel member of the membrane-associated guanylate kinase class of scaffolding proteins containing a Src homology 3 (SH3) and a guanylate kinase (GK) domain (Fig. 1A). As shown by the crystal structure of Ca V ␤ complexed to AID, the Ca V ␤-GK binds to the AID, whereas Ca V ␤-SH3 interacts with GK. Although SH3 domains are known to mediate protein-protein interactions by binding to proline-rich motifs in ligand proteins (6), no interactions mediated by the Ca V ␤-SH3 have been described yet. Moreover, the functional integrity of Ca V ␤-SH3 domain is uncertain since the residues homologous to the ones critical for binding PXXP motifs in canonical SH3 modules are occluded in the crystal structure of Ca V ␤. Intriguingly, canine and human cardiac cells express splicing varian...

Background: Enhancement of L-type calcium channel trafficking by ␤-subunits (Ca V ␤) remains unclear. Results: Ca V ␤ associates directly with actin filaments. In cardiomyocytes, overexpression of Ca V ␤ 2 stimulates L-type currents without altering their voltage dependence of activation. Actin cytoskeleton disruption inhibited channel up-regulation. Conclusion: Ca V ␤ anchors the channel to actin filaments for plasma membrane delivery. Significance: Novel insights into L-type channel anterograde trafficking are presented.

Voltage-dependent calcium channels constitute the main entry pathway for calcium into excitable cells. They are heteromultimers formed by an ␣ 1 pore-forming subunit (Ca V ␣ 1 ) and accessory subunits. To achieve a precise coordination of calcium signals, the expression and activity of these channels is tightly controlled. The accessory ␤-subunit (Ca V ␤), a membrane associated guanylate kinase containing one guanylate kinase (␤-GK) and one Src homology 3 (␤-SH3) domain, has antagonistic effects on calcium currents by regulating different aspects of channel function. Although ␤-GK binds to a conserved site within the ␣ 1 -pore-forming subunit and facilitates channel opening, ␤-SH3 binds to dynamin and promotes endocytosis. Here, we investigated the molecular switch underlying the functional duality of this modular protein. We show that ␤-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 ␤-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 ␤ form oligomers and that this interaction is reduced by Ca V ␣ 1 . Association of Ca V ␤ with a polypeptide encoding the binding motif in Ca V ␣ 1 inhibited endocytosis. Together, these findings reveal that ␤-SH3 dimerization is crucial for endocytosis and suggest that channel activation and internalization are two mutually exclusive functions of Ca V ␤. We propose that a change in the oligomeric state of Ca V ␤ is the functional switch between channel activator and channel internalizer.Voltage-dependent calcium channels link membrane depolarization to transient increases in cytosolic calcium concentration, which in turn mediate a variety of cellular processes, including gene expression, contraction, neurotransmission, and exocytosis (1). To direct the signal to a specific subset of an extensive family of effectors, cells are endowed with a complex network of regulators that constrain the timing and spreading of the calcium increase. The subset of voltage-dependent calcium channels that are activated by strong depolarization, also called high voltage-activated (HVA) 3 channels, are heteromultimers consisting of a central pore-forming subunit (Ca V ␣ 1 ) that associates with one or more accessory subunits. Among them the accessory ␤-subunit (Ca V ␤) has traditionally been recognized as one of the most important modulator of HVA channels (1-3). More recently, it has been shown that the same subunit mediates several other cellular processes (for review, see Ref. 4), including regulation of insulin secretion (5), gene transcription (6) and endocytosis (7). Four Ca V ␤ isoforms (Ca V ␤ 1 to Ca V ␤ 4 ) have been cloned from different nonallelic genes. Crystallographic studies of three of these provided the molecular basis for its functional versatility by identify...