The threshold for action potential generation and the frequency of firing of central neurons depend critically on the cell surface density and the functional properties of sodium channels. Control of the cell surface density and localization of sodium channels is a critical aspect of neuronal function. Previous results show that the Type I and Type II sodium channels are differentially localized in cell bodies and axons of many central neurons and suggest that assembly, cell surface insertion, and differential targeting of sodium channel subtypes to specific locations in neurons all play important roles in this process. In preparation for examination of these issues at the molecular level, we have recently cloned cDNAs encoding the Type I and Type IIA sodium channels and expressed them in mammalian cells. We have also isolated the first cDNA clones encoding the beta1 subunit and shown that it has important effects on the expression and function of sodium channels in Xenopus oocytes. In this project, we will use a combination of cell biological and molecular biological approaches to define the molecular mechanisms underlying these post-transcriptional regulatory processes in central neurons. We will isolate cDNA clones encoding beta2 subunits and cDNA clones encoding any additional beta1 subunit subtypes that are expressed in rat brain and examine their functional properties by co- expression in Xenopus oocytes with Type I and Type IIA alpha subunits. Subunit-specific anti-peptide antibodies will be used to determine which beta1 and beta2 subunits are associated with Type I and Type IIA alpha subunits in rat brain. After determination of the natural subunit combinations that form sodium channels in the brain, we will examine the assembly and cell surface expression of alpha subunits alone or in combination with beta1 and/or beta2 subunits in mammalian cells in culture using the naturally occurring combinations of subunit subtypes. We will extend this work on transfected cells to determine the effect of beta1 and beta2 subunits on expression and function of Type I and Type IIA sodium channels in neurons in cell culture. In order to define the primary structural signals which neurons use to specify targeting of the Type I and Type IIA sodium channels, we will establish conditions to study the differential targeting of epitope-tagged Type I and Type IIA alpha subunits to cell bodies and axons of rat brain neurons in organotypic cultures which maintain normal neuronal morphology, define the amino acid sequences which contain the information for differential targeting of Type I and IIA sodium channel alpha subunits, and determine the role of beta1 and beta2 subunits in differential targeting. Finally, we will examine the role of identified targeting amino acid sequences in alpha subunits and assembly with beta1 and beta2 subunits in control of sodium channel localization and expression in vivo in transgenic mice. Together, these experiments will provide important new information on the mechanisms used by central neurons to control the localization, cell surface density, and functional activity of sodium channels.
Showing the most recent 10 out of 55 publications
