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Ca²⁺-Activated K⁺ Channels in Gonadotropin-Releasing Hormone-Stimulated Mouse Gonadotrophs

Division of Endocrinology, Department of Internal Medicine, School of Medicine, University of California, Davis, California 95616

Address all correspondence and requests for reprints to: Dennis W. Waring, Ph.D., Division of Endocrinology, Department of Internal Medicine, University of California, One Shields Avenue, Davis, California 95616. E-mail: dwwaring{at}ucdavis.edu.

GnRH receptor activation elicits release of intracellular Ca²⁺, which leads to secretion and also activates Ca²⁺-activated ion channels underlying membrane voltage changes. The predominant Ca²⁺-activated ion channels in rat and mouse gonadotrophs are Ca²⁺-activated K⁺ channels. To establish the temporal relationship between GnRH-induced changes in intracellular [Ca²⁺] ([Ca²⁺]_i) and membrane current (I_m), and to identify specific Ca²⁺-activated K⁺ channels linking GnRH-induced increase in [Ca²⁺]_i to changes in plasma membrane electrical activity, we used single female mouse gonadotrophs in the perforated patch configuration of the patch-clamp technique, which preserves signaling pathways. Simultaneous measurement of [Ca²⁺]_i and I_m in voltage-clamped gonadotrophs revealed that GnRH stimulates an increase in [Ca²⁺]_i that precedes outward I_m, and that activates two kinetically distinct currents identified, using specific toxin inhibitors, as small conductance Ca²⁺-activated K⁺ (SK) current (I_SK) and large (big) conductance voltage- and Ca²⁺-activated K⁺ (BK) current (I_BK). We show that the apamin-sensitive current has an IC₅₀ of 69 pM, consistent with the SK2 channel subtype and confirmed by immunocytochemistry. The magnitude of the SK current response to GnRH was attenuated by 17β-estradiol (E₂) pretreatment. Iberiotoxin, an inhibitor of BK channels, completely blocked the residual apamin-insensitive outward I_m, substantiating that I_BK is a component of the GnRH-induced outward I_m. In contrast to its suppression of I_SK, E₂ pretreatment augmented peak I_BK. SK or BK channel inhibition modulated GnRH-stimulated LH secretion, implicating a role for these channels in gonadotroph function. In summary, in mouse gonadotrophs the GnRH-stimulated increase in [Ca²⁺]_i activates I_SK and I_BK, which are differentially regulated by E₂ and which may be targets for E₂ positive feedback in LH secretion.