Progress in the previous award period provides strong evidence that channel sialylation plays a role in the gating behavior of sodium channels. Specifically, it has been shown that the removal of sialic acids from channels results in a reduction in the voltage and calcium sensitivity of sodium channel gating. The manner of these effects suggest that sialic acids influence sodium channel gating through an electrostatic mechanism. In this renewal application, this basic hypothesis and its biological significance will be addressed by exploring two questions: 1) What is the actual magnitude of the effect of sialylation on sodium channel gating in vivo? 2) Is variable channel sialylation a potential means of controlling sodium channel gating behavior in vivo? The specific aims proposed are as follows: 1) To determine the effects of increased sialylation on the gating of sodium channels expressed in transfected cell lines. These studies are intended as a further test of the hypothetical electrostatic role of sialic acid in channel gating by complementing previous experiments that removed sialic acid. In these experiments, channel sialylation will be increased through the chimeric addition of glycosylation sites to channels, sialylation of both wild-type and chimeric channels will be increased through expression in cell lines transfected with cloned sialytransferases. 2) To characterize the gating of glycosylation-deficient rSkM1 mutants expressed in skeletal muscle fibers. The gating of mutants of rSkM1 will be determined when these glycosylation-site deletion mutants are transfected into rat skeletal muscle fibers. These studies will provide a direct assessment of the actual contribution of channel sialylation to channel gating in vivo. 3) To investigate whether correlative variations in channel sialylation and gating occur for a given channel isoform in the nervous system and during development. Through the use of isoform-specific antibodies, the sialylation levels of channels will be determined in different parts of the adult and developing nervous system. Variations of such sialylation levels will be correlated with the gating properties of brain sodium channels using the CHO cell expression system developed in Aim #1. Further, the molecular basis for such variations will be addressed through study of the expression of endogenous sialyltransferases. Thus these experiments will determine whether variable sialylation is a potential channel modulation mechanism in the nervous system. These studies are relevant to the understanding of neuromuscular dysfunction that is present in disorders of calcium and glycoprotein metabolism, particularly those involving genetic defects of glycosylation. Furthermore, the project may provide insight into changes in electrical excitability that occur in aging brain, Alzheimer s disease, and epilepsy.
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