Ca(2+) influx through voltage-gated channels initiates the exocytotic fusion of synaptic vesicles to the plasma membrane. Here we show that RIM binding proteins (RBPs), which associate with Ca(2+) channels in hair cells, photoreceptors, and neurons, interact with alpha(1D) (L type) and alpha(1B) (N type) Ca(2+) channel subunits. RBPs contain three Src homology 3 domains that bind to proline-rich motifs in alpha(1) subunits and Rab3-interacting molecules (RIMs). Overexpression in PC12 cells of fusion proteins that suppress the interactions of RBPs with RIMs and alpha(1) augments the exocytosis triggered by depolarization. RBPs may regulate the strength of synaptic transmission by creating a functional link between the synaptic-vesicle tethering apparatus, which includes RIMs and Rab3, and the fusion machinery, which includes Ca(2+) channels and the SNARE complex.

The ␤ subunits of voltage-gated Ca 2؉ channels are known to be regulators of the channels' gating properties. Here we report a striking additional function of a ␤ subunit. Screening of chicken cochlear and brain cDNA libraries identified ␤4c, a short splice variant of the ␤4 subunit. Although ␤4c occurs together with the longer isoforms ␤4a or ␤4b in the brain, eye, heart, and lung, the cochlea expresses exclusively ␤4c. The association of ␤4c with the Ca 2؉ -channel ␣1 subunit has slight but significant effects on the kinetics of channel activation and inactivation. Yeast two-hybrid and biochemical assays revealed that ␤4c interacts directly with the chromo shadow domain of chromobox protein 2͞heterochromatin protein 1␥ (CHCB2͞HP1␥), a nuclear protein involved in gene silencing and transcriptional regulation. Coexpression of this protein specifically recruits ␤4c to the nuclei of mammalian cells. Furthermore, ␤4c but not ␤4a dramatically attenuates the genesilencing activity of chromobox protein 2͞heterochromatin protein 1␥. The ␤4c subunit is therefore a multifunctional protein that not only constitutes a portion of the Ca 2؉ channel but also regulates gene transcription.