The objective of the proposed experiments is to determine the role of glycosylation in the functioning of the Kv1.1 potassium channel. Previous studies by the investigator have indicated an important effect of glycosylation on voltage dependence and kinetics of activation. On the bases of studies involving changes in extracellular calcium, the modulation of function by specific glycosylations were proposed to occur through changes in surface potential and through effects on the voltage sensor. Now the investigator proposes to identify the contributions of individual glycosylation sites on the voltage dependence through continued mutation and expression of Kv1.1 alpha subunits. Functional and biochemical measurements will be used to determine consequences of decreasing the charge of identified sialidations on channel gating and toxin binding. Divalent sensitivity will be taken as a measure of the contributions by surface potential to gating. In complementary studies the functional effects of increasing the channels silidation state on channel gating, voltage dependence and divalent sensitivity will be tested. These experiments form the bases for hypothesizing that channel glycosylation represents an important mechanism of postranslational modulation of ion channel function.
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