We have been interested in the role of K+ channels in neurons in general and in their role during hypoxia in particular. Some of the work that we have recently done pertains to the BK(Ca) channel in neocortical cells of mice. We have shown that the BK(Ca) present in these cells (sensitive to voltage, Ca, insensitive to charybdotoxin and Iberiotoxin, with a conductance of about 210 pS) is markedly inhibited by low 02 states in the cell-attached configuration, but not in the excised patch. Since 1) it is well recognized that the depolarization-induced by hypoxia can play an important role in neuronal cell fate, and 2) it is likely that the BK(Ca) channels we are studying are at least partially responsible for this depolarization, we propose 3 specific hypotheses/specific aims to study these channels and understand how they sense 02 lack and the role they play in hypoxic responses in both brainstem and neocortical neurons in mice and humans. These are: a) BK(Ca) channels play an important role in the hypoxia-induced depolarization of neocortical (NEO) and brainstem (hypoglossal, XII) neurons in mice and in NEO neurons of humans; the role of these channels increases with age in early mouse ontogeny. b) The hypoxia-induced inhibition of these BK(Ca) channels is secondary to changes in cytosolic factors such as phosphorylation, redox potential and intracellular pH and not related to a direct effect of 02 (or lack thereof) on the channel itself. c) BK(Ca) channels have binding domains that are essential for the cellular response to hypoxia. In order to address these hypotheses, we use single channel and whole-cell recordings, cell lines transfected with BK channel subunits, RT-PCR and in-situ hybridization to determine the BK(Ca) channel structure-function relationships. Since we have shown that K+ channel activity represents some of the early events in hypoxia, and since these events are important in setting the stage for subsequent events that lead to survival or injury of central neurons, we believe that this work can have far reaching implications on our understanding of, and possibly treatment for, hypoxic injury, stroke and epilepsy.
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