REGULATION OF CALCIUM ACTIVATED POTASSIUM CHANNELS

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The broad and long term objectives are to understand the mechanisms of airway smooth muscle tension control by neurohumoral factors, and to search for cellular or molecular targets at which the treatment of bronchoconstriction may be directed. The specific aims of the research proposal are: (1) Determine if the 6gamma subunit of G protein (G6gamma) has a direct regulatory effect on the activity of Ca2+-activated potassium channel (KCa); (2) Investigate if the 6-adrenoceptor agonist isoproterenol can stimulate KCa activity independent of adenylyl cyclase (AC) and protein kinase A (PKA). The effect of bovine brain G6gamma on airway smooth muscle KCa activity will be examined on inside-out patches. To establish that any regulatory effect of the G6gamma preparation is a specific effect of G6gamma rather than that of other constituents in the preparation, experiments will be conducted to make sure that: boiled G6gamma lacks the effect; the effect can be reversed by excessive bovine brain Galpha-GDP; the buffer used to suspend G6gamma lacks the effect; the effect is not due to contaminating Galpha-GTP. The whole cell configuration of the patch clamp technique will be used to test if isoproterenol stimulates KCa at the cellular level, and whether that stimulation is independent of AC and PKA. Voltage pulses will be applied to cells that have been treated with isoproterenol alone, or plus an AC inhibitor, or plus a PKA inhibitor, or plus a nonhydralyzable ATP analog, or plus a membrane-permeable cAMP antagonist. The identify of the activated current will be determined using the KCa-specific channel antagonist, charybdotoxin.