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.

Agency
National Institute of Health (NIH)
Institute
National Institute of Neurological Disorders and Stroke (NINDS)
Type
First Independent Research Support & Transition (FIRST) Awards (R29)
Project #
5R29NS029804-02
Application #
3478366
Study Section
Physiology Study Section (PHY)
Project Start
1991-07-20
Project End
1996-06-30
Budget Start
1992-07-01
Budget End
1993-06-30
Support Year
2
Fiscal Year
1992
Total Cost
Indirect Cost
Name
University of Pittsburgh
Department
Type
Schools of Medicine
DUNS #
053785812
City
Pittsburgh
State
PA
Country
United States
Zip Code
15213
Fomina, A F; Levitan, E S; Takimoto, K (1996) Dexamethasone rapidly increases calcium channel subunit messenger RNA expression and high voltage-activated calcium current in clonal pituitary cells. Neuroscience 72:857-62
Takimoto, K; Levitan, E S (1996) Altered K+ channel subunit composition following hormone induction of Kv1.5 gene expression. Biochemistry 35:14149-56
Fass, D M; Levitan, E S (1996) Bay K 8644 reveals two components of L-type Ca2+ channel current in clonal rat pituitary cells. J Gen Physiol 108:1-11
Fass, D M; Levitan, E S (1996) L-type Ca2+ channels access multiple open states to produce two components of Bay K 8644-dependent current in GH3 cells. J Gen Physiol 108:13-26
Fomina, A F; Levitan, E S (1995) Three phases of TRH-induced facilitation of exocytosis by single lactotrophs. J Neurosci 15:4982-91
Levitan, E S; Gealy, R; Trimmer, J S et al. (1995) Membrane depolarization inhibits Kv1.5 voltage-gated K+ channel gene transcription and protein expression in pituitary cells. J Biol Chem 270:6036-41
Takimoto, K; Gealy, R; Levitan, E S (1995) Multiple protein kinases are required for basal Kv1.5 K+ channel gene expression in GH3 clonal pituitary cells. Biochim Biophys Acta 1265:22-8
Takimoto, K; Gealy, R; Fomina, A F et al. (1995) Inhibition of voltage-gated K+ channel gene expression by the neuropeptide thyrotropin-releasing hormone. J Neurosci 15:449-57
Kramer, R H; Mokkapatti, R; Levitan, E S (1994) Effects of caffeine on intracellular calcium, calcium current and calcium-dependent potassium current in anterior pituitary GH3 cells. Pflugers Arch 426:12-20
Takimoto, K; Levitan, E S (1994) Glucocorticoid induction of Kv1.5 K+ channel gene expression in ventricle of rat heart. Circ Res 75:1006-13

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