Immortalized GnRH neurons express large-conductance calcium-activated potassium channels.
The expression and function of large-conductance Ca2+ -activated K+ (BK) channels in the GT1-7 line of immortalized gonadotropin-releasing hormone (GnRH) neurons was investigated. Ionic currents were recorded using the patch-clamp technique, cytoplasmic free Ca2+ concentration ([Ca2+]i) was monitored using the fluorescent indicator, fura-2, and GnRH secretion was measured by radioimmunoassay. In cell-attached and inside-out patch-clamp recordings, K+ channels with a single-channel conductance of approximately 200 pS were detected. Depolarizing the patch increased the unitary current size and the open probability. In perforated-patch recordings, depolarizing pulses (50 ms) to potentials of -10 to +60 mV from a holding potential of -90 mV elicited outward current with early transient and sustained components. The transient current peaked 2-10 ms after the beginning of each pulse and increased in a voltage-dependent manner. This current was: (1) unaffected by the small-conductance Ca2+ -activated K+ channel blocker, apamin (100 nM); (2) reduced by the BK channel blocker, charybdotoxin (5 nM); (3) abolished by the Ca2+ channel blocker, CdCl2 (25 mu M), and (4) prolonged by the endoplasmic reticulum Ca2+ -ATPase inhibitor, thapsigargin (1 mu M). Based on the single-channel and whole-cell conductances, the number of channels per patch, the patch area, and the surface area of the cell, each GT1-7 cell contains 30-60 BK channels. The functional role of BK channels in GT1-7 cells was evaluated by measuring the effect of charybdotoxin (100 nM) on basal [Ca2+]i and GnRH secretion, as well as on the [Ca2+]i and GnRH secretory responses to gamma-aminobutyric acid (GABA, 100 mu M), an excitatory neurotransmitter in this system. Charybdotoxin had no effect on basal [Ca2+]i or GnRH secretion, or on the GABA-evoked [Ca2+]i and GnRH secretory responses. These results indicate that GT1-7 cells express BK channels; however, the physiological role of BK channels in GT1-7 cells remains elusive.
Spergel
D J
DJ
Endocrinology and Reproduction Research Branch, National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, MD 20892, USA.
Catt
K J
KJ
Rojas
E
E
D000818:Animals; D002118:Calcium / Q000502:physiology*; D002121:Calcium Channel Blockers / Q000494:pharmacology; D000252:Calcium-Transporting ATPases / Q000037:antagonists & inhibitors; D002460:Cell Line; D004791:Enzyme Inhibitors / Q000494:pharmacology; D007987:Gonadotropin-Releasing Hormone / Q000378:metabolism*; D008564:Membrane Potentials / Q000187:drug effects / Q000502:physiology; D051379:Mice; D009474:Neurons / Q000187:drug effects / Q000378:metabolism*; D018408:Patch-Clamp Techniques; D015221:Potassium Channels / Q000187:drug effects / Q000378:metabolism*
D002121:Calcium Channel Blockers; D004791:Enzyme Inhibitors; D015221:Potassium Channels; D007987:Gonadotropin-Releasing Hormone; D000252:Calcium-Transporting ATPases; D002118:Calcium