Kv2.1 ablation alters glucose-induced islet electrical activity, enhancing insulin secretion

Abstract

Voltage-gated potassium currents (Kv), primarily due to Kv2.1 channels, are activated by glucose-stimulated pancreatic beta cell depolarization, but the exact role (or roles) of this channel in regulating insulin secretion remains uncertain. Here we report that, compared with controls, Kv2.1 null mice have reduced fasting blood glucose levels and elevated serum insulin levels. Glucose tolerance is improved and insulin secretion is enhanced compared to control animals, with similar results in isolated islets in vitro. Isolated Kv2.1(-/-) beta cells have residual Kv currents, which are decreased by 83% at +50 mV compared with control cells. The glucose-induced action potential (AP) duration is increased while the firing frequency is diminished, similar to the effect of specific toxins on control cells but substantially different from the effect of the less specific blocker tetraethylammonium. These results reveal the specific role of Kv2.1 in modulating glucose-stimulated APs of beta cells, exposing additional important currents involved in regulating physiological insulin secretion.

Fig. 1

Kv2.1 knockout animals show aberrant glucose homeostasis. (A) Cartoon of the Kv2.1 targeted gene disruption and the resulting disrupted protein structure in grey versus WT in black. (B) PCR products from homozygous knockout, −/−, heterozygous, +/−, and control, +/+, animals with primers specific to the surrounding Kv2.1 gene disruption sequence and one primer specific to the targeting sequence. (C) Southern blot probed with a Kv2.1 specific probe run with genomic DNA from control,+/+, and targeted, +/−, embryonic stem cells cut with an endonuclease that removes part of the Kv2.1 sequence and the targeting cassette. (D) Western blot run with Kv2.1−/− brain extracts lanes 1–4, control brain extracts lanes 5–7, Kv2.1−/− islets lane 8, and control islets lane 9. (E) Glucose tolerance test on Kv2.1−/−, grey line, and control, black line, animals. Serum insulin levels from Kv2.1−/−, grey line, and control, black line, animals are shown in the inset. n= 10 each, * P<0.05. (F) Islet insulin secretion during the 7mM glucose treatment, boxed above, the islets are in 2mM glucose previous to stimulation, + SDEV (n=10 islet sets and 3 insulin assays each).

Fig. 2

Kv2.1−/− β-cells have significantly reduced Kv currents. (A) Control β-cell Kv currents recorded in voltage clamp with voltage steps from −80 to +80 in 10mV increments. (B) Control β-cell Kv-currents 10 minutes post 100nM HaTx. (C) Control β-cell 10 minutes post 100nM ScTx-1. (E) Kv2.1−/− β-cell Kv-currents. (F) Current density vs Voltage plots for β-cells recorded in steps from −80mV to the indicated voltage +/− SEM’s (n>7 for each condition).

Fig. 3

Kv2.1−/− islets have increased glucose induced action potential duration with decreased frequency. (A) Control islet electrical activity in response to 14mM glucose; inset shows the action potentials from the indicated segment of activity, horizontal grey bar. (B) Kv2.1−/− islet electrical activity in response to 14mM glucose; inset shows the action potentials from the indicated segment of activity, horizontal grey bar. (C) Control islet electrical activity in response to 6.5mM glucose; inset shows the action potentials from the indicated segment of activity, horizontal grey bar. (D) Kv2.1−/− islet electrical activity in response to 6.5mM glucose; inset shows the action potentials from the indicated segment of activity, horizontal grey bar.

Fig. 4

Control islets treated with ScTx-1 show similar bursting to untreated Kv2.1 islets. (A) Control islet electrical activity recorded in 14mM glucose and treated with 100nM ScTx-1, black bar. Insets show islet action potentials expanded during 14mM glucose alone or in combination with 100nM ScTx treatment. (B) Kv2.1 islet electrical activity recorded in 14mM glucose and treated with 100nM ScTx-1, black bar. Insets show islet action potentials expanded during 14mM glucose alone or in combination with 100nM ScTx treatment. (C–F) Representative fast acquisition calcium traces recorded with mouse islets loaded with Fluo-4 and incubated with 200uM Tolbutamide in 2mM glucose from control (C) and Kv2.1−/− (D) animals and from the same control (E) and Kv2.1−/− (F) islet four minutes post 25nM ScTx-1.