Electrophysiological Characterization of Pancreatic Islet Cells in the Mouse Insulin Promoter-Green Fluorescent Protein Mouse

Abstract

We recently reported a transgenic [mouse insulin promoter (MIP)-green fluorescent protein (GFP)] mouse in which GFP expression is targeted to the pancreatic islet beta-cells to enable convenient identification of beta-cells as green cells. The GFP-expressing beta-cells of the MIP-GFP mouse were functionally indistinguishable from beta-cells of normal mice. Here we characterized the ionic channel properties and exocytosis of MIP-GFP mouse islet beta- and alpha-cells. Beta-cells displayed delayed rectifying K+ and high-voltage-activated Ca2+ channels and exhibited Na+ currents only at hyperpolarized holding potential. Alpha-cells were nongreen and had both A-type and delayed rectifier K+ channels, both low-voltage-activated and high-voltage-activated Ca2+ channels, and displayed Na+ currents readily at -70 mV holding potential. Alpha-cells had ATP-sensitive K+ channel (KATP) channel density as high as that in beta-cells, and, surprisingly, alpha-cell KATP channels were more sensitive to ATP inhibition (IC50=0.16+/-0.03 mM) than beta-cell KATP channels (IC50=0.86+/-0.10 mM). Whereas alpha-cells were rather uniform in size [2-4.5 picofarad (pF)], beta-cells varied vastly in size (2-12 pF). Of note, small beta-cells (<4.5 pF) showed little exocytosis, whereas medium beta-cells (5-8 pF) exhibited vigorous exocytosis, but large beta-cells (>8 pF) had weaker exocytosis. We found no correlation between beta-cell size and their Ca2+ channel density, suggesting that Ca2+ influx may not be the cause of the heterogeneity in exocytotic responses. The MIP-GFP mouse therefore offers potential to further explore the functional heterogeneity in beta-cells of different sizes. The MIP-GFP mouse islet is therefore a reliable model to efficiently examine alpha-cell and beta-cell physiology and should greatly facilitate examination of their pathophysiology when the MIP-GFP mice are crossed with diabetic models.

Voltage-dependent K+ channels are positive regulators of alpha cell action potential generation and glucagon secretion in mice and humans

“…However, our observation that Kv2.1/Kv2.2 and BK account for the majority of Kv current in alpha cells suggests that Kv3 channels play only a minor role. Finally, Kv4.3 has been suggested to contribute a rapidly inactivating TEAinsensitive Kv current in mouse alpha cells [3,6,19]. We did not detect such a current in our preparation, and the Kv4.3 inhibitor phrixotoxin had no effect on alpha cell Kv currents in our hands.…”

“…3c, d). Notably, we did not observe the TEA-resistant rapidly inactivating current component reported previously in isolated alpha cells [19] and alpha cells from intact islets [3]. In beta cells, inhibition of Kv currents with TEA augments insulin secretion in a glucose-dependent manner, since Kv channels contribute to action potential repolarisation [11,16,20].…”

“…This discrepancy may relate to differences in cell preparation, cell identification or recording conditions. Previous studies examined alpha cells identified electrophysiologically in intact islets [3] or green fluorescent protein (GFP)-negative single cells from mouse insulin promoter-GFP islets [6,19]. The present study examined isolated cells, which were identified postexperimentation by glucagon immunostaining.…”

Linoleic acid induces Ca2+-induced inactivation of voltage-dependent Ca2+ currents in rat pancreatic β-cells

“…8G0 . 1 pF respectively (Gopel et al 2000, Leung et al 2005. The criterion for b-cell identification in our experiment is that the whole-cell capacitance of the clamped cells is over 6 pF.…”

Presence of functional hyperpolarisation-activated cyclic nucleotide-gated channels in clonal alpha cell lines and rat islet alpha cells

“…The difference might be attributed to the unique biophysical properties of alpha cells. Numerous studies have demonstrated that: (1) alpha cell K ATP channels are more sensitive to ATP inhibition than those in beta cells; thus, the K ATP channels in alpha cells possess intermediate activity under low glucose concentrations [3,11,31,37]; (2) unlike the beta cell Na + channels, which are activated at a more negative membrane potential, the alpha cell Na + channels are active within a physiological range of membrane potentials [14,17,31]; and (3) in addition to the L-type Ca 2+ channels that are the most important in modulating [Ca 2+ ] i in beta cells, the activities of T-type and N-type Ca 2+ channels in alpha cells are critical in the generation of action potentials and control of glucagon secretion [11,14,15,38]. Based on these findings, it has been proposed that glucagon secretion only occurs within a narrow window of intermediate alpha cell K ATP channel activity (e.g.…”