Gonadotropin-Releasing Hormone Neurons Express K_ATPChannels That Are Regulated by Estrogen and Responsive to Glucose and Metabolic Inhibition

Abstract

Gonadotropin-releasing hormone (GnRH) is released in a pulsatile manner that is dependent on circulating 17␤-estradiol (E2) and glucose concentrations. However, the intrinsic conductances responsible for the episodic firing pattern underlying pulsatile release and the effects of E2 and glucose on these conductances are primarily unknown. Whole-cell recordings from mouse enhanced green fluorescent protein-GnRH neurons revealed that the K ATP channel opener diazoxide induced an outward current that was antagonized by the sulfonylurea receptor 1 (SUR1) channel blocker tolbutamide. Single-cell reverse transcription (RT)-PCR revealed that the majority of GnRH neurons expressed Kir6.2 and SUR1 subunits, which correlated with the diazoxide/tolbutamide sensitivity. Also, a subpopulation of GnRH neurons expressed glucokinase mRNA, a marker for glucose sensitivity. Indeed, GnRH neurons decreased their firing in response to low glucose concentrations and metabolic inhibition. The maximum diazoxide-induced current was approximately twofold greater in E2-treated compared with oil-treated ovariectomized females. In current clamp, estrogen enhanced the diazoxide-induced hyperpolarization to a similar degree. However, based on quantitative RT-PCR, estrogen did not increase the expression of Kir6.2 or SUR1 transcripts in GnRH neurons. In the presence of ionotropic glutamate and GABA A receptor antagonists, tolbutamide depolarized and significantly increased the firing rate of GnRH neurons to a greater extent in E2-treated females. Finally, tolbutamide significantly increased GnRH secretion from the preoptic-mediobasal hypothalamus. Therefore, it appears that K ATP channels and glucokinase are expressed in GnRH neurons, which renders them directly responsive to glucose. In addition, K ATP channels are involved in modulating the excitability of GnRH neurons in an estrogen-sensitive manner that ultimately regulates peptide release.

Kisspeptin Depolarizes Gonadotropin-Releasing Hormone Neurons through Activation of TRPC-Like Cationic Channels

“…In addition to activating TRPC channels, kisspeptin also attenuated a resting (barium and cesium sensitive) Kir current in GnRH neurons. This effect of kisspeptin in GnRH neurons, albeit small, maybe critical because Kir channels (e.g., K ATP channels) are highly expressed in GnRH neurons and clamp the cells in a negative resting state of Ϫ63 mV (Lagrange et al, 1995;Zhang et al, 2007). Therefore, by inhibiting Kir channels along with the Experiments were conducted with cesium-gluconate pipette solution so as to block voltage-gated potassium currents; HEPESbuffered CSF containing 4-AP and cesium was used to block the A-type potassium channel and h channel.…”

“…To characterize the ionic mechanism(s) underlying the kisspeptin-evoked depolarization, the kisspeptin-induced inward currents were recorded at a holding potential of Ϫ60 mV, which closely approximates the resting membrane potential of these neurons, followed by an analysis of the reversal potentials of the kisspeptin-induced currents (Zhang et al, 2007). A saturating The forward primer is listed first, and the reverse primer is listed second.…”

Dopaminergic inhibition of gonadotropin-releasing hormone neurons in the cichlid fish, Astatotilapia burtoni

“…The identity of the potassium channel involved in D2R-mediated inhibition of mammalian and fish GnRH neurons is unknown, although candidates include G-protein-coupled inwardly rectifying and ATP-sensitive channels. These channels both control mammalian GnRH neurons via modulation by multiple peptides (Constantin and Wray, 2016;Rønnekleiv and Kelly, 2013;Zhang et al, 2007), and are linked to D2R modulation in other brain regions (Neusch et al, 2000;Perez et al, 2006;Werner et al, 1996).…”

Kisspeptin inhibits a slow afterhyperpolarization current via protein kinase C and reduces spike frequency adaptation in GnRH neurons

“…Each measurement was repeated 10 times, and the signals were averaged. The mean resting membrane potential of GnRH neurons measured with low-EGTA (25-50 M) internal solution was Ϫ65.9 Ϯ 1.0 mV (n ϭ 17), which is about 3 mV more negative than that measured with high EGTA (11 mM) internal solution (57). The reported membrane potentials have been corrected for the liquid junction potential of 10 mV.…”

“…Whole cell patch recordings were made with a Zeiss Axioskop FS upright microscope equipped with fluorescence (fluorescein isothiocyanate filter set) and infrared differential interference contrast imaging devices. GnRH neurons were identified by the method described in our previous papers (57)(58)(59). Patch pipettes (1.5 mm OD borosilicate glass; A-M Systems) were pulled on a Brown/Flaming puller (model P-97; Sutter Instrument) and were filled with the following internal solution (in mM): 130 KMeSO4, 5 Na2 phosphocreatine, 10 KCl, 1 MgCl2, 0.025 EGTA, 10 HEPES, 2.5 ATP, and 0.25 GTP adjusted to pH 7.3 with KOH; 290 mosm.…”