The applicants propose here investigations into the regulation of the numbers and distributions of voltage-gated potassium channels in developing hippocampal pyramidal neurons. Two major threads bind this work: the development of potassium channel subunits comprising the ion channels underlying three voltage-gated potassium currents (IA, ID and IK), and the regulation of these channels by vesicular trafficking.
Specific Aim 1 : Investigate the behaviors of Shal (Kv4)-related subunits during manipulations that affect IA development. Evidence suggests that Shal (Kv4)-related subunits contribute to IA in pyramidal neurons. The investigators will (1) assess the contribution of Kv4-related subunits to IA by antisense oligodeoxynucleotide disruption of Kv4 subunit biosynthesis followed by immunocytochemical localization of Kv4 protein and voltage clamp analysis of IA, and (2) examine how inhibition of IA development by lack of astroglial contact, disruption of intracellular vesicular trafficking, and inhibition of kinases is reflected in the intracellular distribution of subunit-specific immunoreactivity.
Specific Aim 2 : Investigate the roles of Kv1.1, Kv1.2, Kv1.4, Kvb1 and Kvb2 subunits in the currents underlying delayed rectification (ID and IK) and their postnatal maturation. Shaker-related single channels are assemblies of a and b subunits in stoichiometry a4b4. The applicants have observed: (a) a large increase in the intensity of a Kvb1-specific in situ hybridization signal on hippocampal sections during the first three postnatal weeks, and (b) kinetic, amplitude, and pharmacological changes in delayed rectifier currents of cultured neurons during this same period. Could selective expression of these a and b subunits involved in developmental regulation the delayed rectifier currents ID and IK? They will address this question by: (1) parallel immunochemical and electrophysiological analyses of developmental changes in Kv1.1, Kv1.2, Kv1.4, Kvb1, and ID and IK in cultured neurons during the first three postnatal weeks, and (2) antisense depletion of particular a and b subunits followed by electrophysiological assessment of ID and IK to determine (a) the contributions of a subunits to the development of the delayed rectifier currents, and (b) the consequences of b subunit depletion on the distributions of a subunits and their contributions to ID and IK.
Showing the most recent 10 out of 22 publications
