Normal function of the brain depends on the regulated secretion of neurotransmitters. The primary trigger for neurosecretion at both central and peripheral synapses is Ca2+ influx through neuronal voltage-gated Ca2+ channels. The gating behavior of Ca2+ channels is strongly modulated by phosphorylation, but the most basic molecular details of this modulation are unclear. For example, two important protein subunits of Ca2+ channels, the alpha-1 and beta subunits, contain within their primary sequences numerous consensus phosphorylation sites. However, it is not known which of these subunits must be phosphorylated to result in modulation of channel gating. Furthermore, specific amino acids on the channel proteins that undergo physiologically important phosphorylation have not been identified. Such information is needed in order to understand channel modulation and neuronal function. The proposed project seeks to identify which Ca2+ channel subunits must be phosphorylated to produce channel modulation, and also seeks to identify specific, physiologically important phosphorylation sites on those channel subunits involved in channel modulation. In the proposed experiments, cDNAs encoding the alpha-1, alpha-2 and beta, subunits of neuronal class A, class B and class E Ca2+ channels will be expressed in a mammalian cell line. The biophysical properties and phosphorylation-dependent modulation of the expressed channels will be characterized using whole-cell and single-channel patch clamp techniques. Molecular genetic techniques will be used to construct phosphorylation-site mutations in neuronal Ca2+ channel alpha-1 subunits; these mutant alpha-1 subunits will lack one or more consensus sites for phosphorylation by protein kinases. An additional goal of the project is to further characterize the pharmacological sensitivities of cloned neuronal Ca2+ channels expressed in HEK cells; this part of the project will help to establish the identity of cloned Ca2+ channels with Ca2+ channels endogenously expressed in neurons. The proposed research has the potential to contribute significant new insights into the fundamental molecular processes underlying Ca2+ channel modulation and physiology. Such information may ultimately be useful in understanding and treatment of human diseases caused by abnormalities in voltage-gated Ca2+ channels.
