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Elevations in pancreatic α-cell intracellular Ca2+ ([Ca2+] i) lead to glucagon (GCG) secretion. Although glucose inhibits GCG secretion, how lactate and pyruvate control α-cell Ca2+ handling is unknown. Lactate enters cells through monocarboxylate transporters (MCTs) and is also produced during glycolysis by lactate dehydrogenase A (LDHA), an enzyme expressed in α-cells. As lactate activates ATP-sensitive K+ (K ATP) channels in cardiomyocytes, lactate may also modulate α-cell K ATP. Therefore, this study investigated how lactate signaling controls α-cell Ca2+ handling and GCG secretion. Mouse and human islets were used in combination with confocal microscopy, electrophysiology, GCG immunoassays, and fluorescent thallium flux assays to assess α-cell Ca2+ handling, V m , K ATP currents, and GCG secretion. Lactate-inhibited mouse (75 ± 25%) and human (47 ± 9%) α-cell [Ca2+] i fluctuations only under low-glucose conditions (1 mM) but had no effect on β- or δ-cells [Ca2+] i. Glyburide inhibition of K ATP channels restored α-cell [Ca2+] i fluctuations in the presence of lactate. Lactate transport into α-cells via MCTs hyperpolarized mouse (14 ± 1 mV) and human (12 ± 1 mV) α-cell V m and activated K ATP channels. Interestingly, pyruvate showed a similar K ATP activation profile and α-cell [Ca2+] i inhibition as lactate. Lactate-induced inhibition of α-cell [Ca2+] i influx resulted in reduced GCG secretion in mouse (62 ± 6%) and human (43 ± 13%) islets. These data demonstrate for the first time that lactate entry into α-cells through MCTs results in K ATP activation, V m hyperpolarization, reduced [Ca2+] i , and inhibition of GCG secretion. Thus, taken together, these data indicate that lactate either within α-cells and/or elevated in serum could serve as important modulators of α-cell function. Image 1 • Lactate reduces islet α-cell Ca2+ entry under low glucose conditions. • Lactate does not alter β- or δ-cell Ca2+ handling under low glucose conditions. • Lactate enters islet α-cells through monocarboxylate transporters. • Lactate hyperpolarizes islet α-cell membrane potential by activating K ATP channels. • Lactate reduces mouse and human islet glucagon secretion. [ABSTRACT FROM AUTHOR]

Kir7.1 is an inwardly rectifying potassium channel with important roles in the regulation of the membrane potential in retinal pigment epithelium, uterine smooth muscle, and hypothalamic neurons. Regulation of G protein-coupled inwardly rectifying potassium (GIRK) channels by G protein-coupled receptors (GPCRs) via the G protein βγ subunits has been well characterized. However, how Kir channels are regulated is incompletely understood. We report here that Kir7.1 is also regulated by GPCRs, but through a different mechanism. Using Western blotting analysis, we observed that multiple GPCRs tested caused a striking reduction in the complex glycosylation of Kir7.1. Further, GPCR-mediated reduction of Kir7.1 glycosylation in HEK293T cells did not alter its expression at the cell surface but decreased channel activity. Of note, mutagenesis of the sole Kir7.1 glycosylation site reduced conductance and open probability, as indicated by single-channel recording. Additionally, we report that the L241P mutation of Kir7.1 associated with Lebers congenital amaurosis (LCA), an inherited retinal degenerative disease, has significantly reduced complex glycosylation. Collectively, these results suggest that Kir7.1 channel glycosylation is essential for function, and this activity within cells is suppressed by most GPCRs. The melanocortin-4 receptor (MC4R), a GPCR previously reported to induce ligand-regulated activity of this channel, is the only GPCR tested that does not have this effect on Kir7.1. [ABSTRACT FROM AUTHOR]

ATP-sensitive potassium (KATP) channels play a key role in mediating glucose-stimulated insulin secretion by coupling metabolic signals to β-cell membrane potential. Loss of KATP channel function due to mutations in ABCC8 or KCNJ11, genes encoding the sulfonylurea receptor 1 (SUR1) or the inwardly rectifying potassium channel Kir6.2, respectively, results in congenital hyperinsulinism. Many SUR1 mutations prevent trafficking of channel proteins from the endoplasmic reticulum to the cell surface. Channel inhibitors, including sulfonylureas and carbamazepine, have been shown to correct channel trafficking defects. In the present study, we identified 13 novel SUR1 mutations that cause channel trafficking defects, the majority of which are amenable to pharmacological rescue by glibenclamide and carbamazepine. By contrast, none of the mutant channels were rescued by KATP channel openers. Cross-linking experiments showed that KATP channel inhibitors promoted interactions between the N terminus of Kir6.2 and SUR1, whereas channel openers did not, suggesting the inhibitors enhance intersubunit interactions to overcome channel biogenesis and trafficking defects. Functional studies of rescued mutant channels indicate that most mutants rescued to the cell surface exhibited WT-like sensitivity to ATP, MgADP, and diazoxide. In intact cells, recovery of channel function upon trafficking rescue by reversible sulfonylureas or carbamazepine was facilitated by theKATP channel opener diazoxide. Our study expands the list of KATP channel trafficking mutations whose function can be recovered by pharmacological ligands and provides further insight into the structural mechanism by which channel inhibitors correct channel biogenesis and trafficking defects. [ABSTRACT FROM AUTHOR]