The goal of this proposal is to determine the mechanism and consequence of hormone-dependent regulation of voltage-gated potassium channel expression. The approach taken will be to use biophysical and molecular genetic methods to study steroid hormone induction of potassium channels in clonal pituitary cells. This model system may reveal mechanisms employed by many neurotransmitters, hormones and drugs. The disruption of normal channel expression could induce aberrant electrical activity that provokes seizures in the brain and arrhythmic beating of the heart. Thus, elucidating the mechanisms of regulation of ion channel expression may be essential for understanding and treating epilepsy and heart disease. Dexamethasone, a glucocorticoid hormone agonist, increases both the steady state concentration of mRNA encoding a voltage-gated potassium channel [Kvl] and the voltage-gated potassium current [Ik(i)] in GH3 pituitary tumor cells.
The specific aims of this proposal are to test the hypotheses that the dexamethasone-induced increase in Ik(i): 1. is mediated by glucocorticoid receptor-stimulated gene expression. Kvl mRNA will be measured with Northern blots while channel activity will be measured with whole cell and cell-attached patch clamp recording. Inhibitors of transcription and protein synthesis and glucocorticoid receptor agonists and antagonists will be tested for effects on induction of potassium channel expression. 2. is directly caused by the increase in Kvl mRNA Kvl gene expression in GH3 cells or oocytes will be inhibited with antisense oligonucleotides or elevated by overproduction of Kvl mRNA. These conditions will determine if changing Kvl mRNA levels is necessary and sufficient for dexamethasone-mediated induction of voltage-gated potassium current. 3. alters basal and neurotransmitter-evoked cellular electrical activity. The effect of dexamethasone on modulation of potassium, calcium and sodium channels by neuropeptides (e.g. somatostatin, thyrotropin releasing hormone) will be examined with whole cell perforated patch recording. Likewise, the effect of the steroid on spontaneous and transmitter-induced action potentials will be measured. These studies will appraise the impact of potassium channel induction by dexamethasone on cell excitability.
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