Glucose-mediated inhibition of calcium-activated potassium channels limits α-cell calcium influx and glucagon secretion

Abstract

Pancreatic α-cells exhibit oscillations in cytosolic Ca2+ (Ca2+c), which control pulsatile glucagon (GCG) secretion. However, the mechanisms that modulate α-cell Ca2+c oscillations have not been elucidated. As β-cell Ca2+c oscillations are regulated in part by Ca2+-activated K+ (Kslow) currents, this work investigated the role of Kslow in α-cell Ca2+ handling and GCG secretion. α-Cells displayed Kslow currents that were dependent on Ca2+ influx through L- and P/Q-type voltage-dependent Ca2+ channels (VDCCs) as well as Ca2+ released from endoplasmic reticulum stores. α-Cell Kslow was decreased by small-conductance Ca2+-activated K+ (SK) channel inhibitors apamin and UCL 1684, large-conductance Ca2+-activated K+ (BK) channel inhibitor iberiotoxin (IbTx), and intermediate-conductance Ca2+-activated K+ (IK) channel inhibitor TRAM 34. Moreover, partial inhibition of α-cell Kslow with apamin depolarized membrane potential ( Vm) (3.8 ± 0.7 mV) and reduced action potential (AP) amplitude (10.4 ± 1.9 mV). Although apamin transiently increased Ca2+ influx into α-cells at low glucose (42.9 ± 10.6%), sustained SK (38.5 ± 10.4%) or BK channel inhibition (31.0 ± 11.7%) decreased α-cell Ca2+ influx. Total α-cell Ca2+c was similarly reduced (28.3 ± 11.1%) following prolonged treatment with high glucose, but it was not decreased further by SK or BK channel inhibition. Consistent with reduced α-cell Ca2+c following prolonged Kslow inhibition, apamin decreased GCG secretion from mouse (20.4 ± 4.2%) and human (27.7 ± 13.1%) islets at low glucose. These data demonstrate that Kslow activation provides a hyperpolarizing influence on α-cell Vm that sustains Ca2+ entry during hypoglycemic conditions, presumably by preventing voltage-dependent inactivation of P/Q-type VDCCs. Thus, when α-cell Ca2+c is elevated during secretagogue stimulation, Kslow activation helps to preserve GCG secretion.

KATP channel blockers control glucagon secretion by distinct mechanisms: A direct stimulation of α-cells involving a [Ca2+]c rise and an indirect inhibition mediated by somatostatin

“…Surprisingly, it does not seem to involve GIRK channels since TPNQ did not reverse the effect of exogenous SST-14 or the effect of endogenous SST. An alternative candidate for the hyperpolarizing effect of SST is the big conductance K + (BK) channel ( Kcnma1 ) and because it is expressed by α-cells [ 57 , 58 , 96 ], it contributes to the mechanisms controlling glucagon secretion [ 97 ], and is activated by SST in neurons and pituitary cells [ 98 , 99 ]. Identification of the channel responsible for the [Ca 2+ ] c lowering effect of SST is, however, beyond the scope of this study.…”

Lactate activation of α-cell KATP channels inhibits glucagon secretion by hyperpolarizing the membrane potential and reducing Ca2+ entry

“…Intracellular Ca 2+ ([Ca 2+ ] i ) imaging was conducted as previously described using epifluorescent or confocal microscopy with Ca 2+ dye (Fura-2-acetoxymethyl ester [AM]) [ 46 ] or genetic indicators (GCaMP3 or GCaMP6s) [ 47 ]. The mouse islets and human non-β-cells were loaded with Fura-2 AM (2 μM) for 25 min at 37 °C in 5% CO 2 .…”

“…RFP-positive mouse α-cells were patched in extracellular solution (EC) containing (in mM) 140 NaCl, 3.6 KCl, 0.5 MgSO 4 , 1.5 CaCl 2 , 0.5 NaH 2 PO 4 , 5 NaHCO 3 , and 10 HEPES (pH 7.35 with NaOH) supplemented with 1 mM glucose and treatments as indicated in the figure legends, with sucrose added as needed to match the osmolarity. Whole-cell currents were measured as a function of the applied membrane voltage (voltage ramp from −120 to 60 mV) in voltage-clamp mode using an Axopatch 200B amplifier with pCLAMP10 software (Molecular Devices, San Jose, CA, USA) every 15 s starting immediately after a whole-cell configuration was established between patch pipettes and α-cells as previously described [ 43 , 46 , 50 ]. This recording protocol was repeated until the whole-cell currents reached a plateau that corresponded to the maximum K ATP current activation.…”

Mechanisms of the Regulation and Dysregulation of Glucagon Secretion

“…According to this hypothesis, glucagon secretion requires the closure of most of these channels, allowing limited K + efflux to alter the membrane potential to a range that permits the opening of voltage-dependent Na + channels; the resulting Na + influx causes subsequent opening of P/Q type voltage-gated calcium channels (VDCCs); calcium influx through these VDCCs is coupled to fusion of glucagon vesicles with the plasma membrane, resulting in glucagon secretion; and too low or too high ATP levels induce excessive opening or closure of the K-ATP channels, respectively, leading to the inhibition of this pathway [ 7 – 9 ]. K + channels activated by intracellular calcium (calcium activated K + channels) were recently found to contribute to glucagon secretion and were suggested to be useful in limiting voltage-dependent inhibition of P/Q type VDCCs during prolonged periods of low glucose [ 10 ]. Nevertheless, this K-ATP channel hypothesis is not fully accepted [ 7 , 11 ].…”