Synaptic Targenting of Ca2+ Channels in Hippocampus Neurons

In the central nervous system Ca2+ channels play important roles in the conductance, synaptic transmission, and in the integration of electrical signals. To accomplish these diverse functions neurons express a great variety of Ca2+ channels in distinct membrane compartments. This differential subcellular localization of Ca2+ channels requires cellular mechanisms for their transport and immobilization in specific target membranes. Because the mechanisms and signals for this "targeting" process of Ca2+ channels in neurons are still unknown, we propose to study the differential distribution of Ca2+ channels in hippocampus neurons and to identify the targeting signals within the primary structure of voltage-gated Ca2+ channels. The voltage-gated Ca2+ channels are a family of proteins with similar structure and amino acid sequence. Because different members of this channel family are localized in distinct neuronal compartments, such channels must carry the information for their differential targeting within their non-homologous amino acid sequences. These targeting signals can be identified by studying the targeting properties of Ca2+ channel chimeras - recombinant channels composed of parts from two isoforms with different targeting characteristics - heterologously expressed in cultured hippocampal neurons. This goal will be achieved by: (1) establishing a neuronal culture system suitable for heterologous expression of Ca2+ channels and the analysis of their targeting properties; (2) the characterization of the subcellular expression patterns of endogenous Ca2+ channels in cultured hippocampus neurons; (3) analyzing the targeting properties of recombinant Ca2+ channel isoforms heterologously expressed in these neurons; (4) generation and expression of channel chimeras, and analyzing which part of the chimeras carries the targeting signal by progressive restriction of the critical sequence and subsequently by substitution of individual residues within the predicted targeting motif; (5) transferring the targeting information from a Ca2+ channel onto an unrelated membrane protein; and finally (6) identifying the binding partner which anchors the channel in the target membrane compartment. To find out where in neurons specific Ca2+ channels are expressed and how they are targeted into synaptic compartments are important questions in cellular neuroscience. To answer them will represent a significant step forward in understanding how the second messenger Ca2+ can serve so many different functions in our nervous system.