Temperature and redox state dependence of native Kv2.1 currents in rat pancreatic beta-cells

Abstract

In pancreatic beta-cells, voltage-dependent K(+) (Kv) channels repolarise glucose-stimulated action potentials. Kv channels are therefore negative regulators of Ca(2+) entry and insulin secretion. We have recently demonstrated that Kv2.1 mediates the majority of beta-cell voltage-dependent outward K(+) current and now investigate the function of native beta-cell Kv2.1 channels at near-physiological temperatures (32-35 degrees C). While beta-cell voltage-dependent outward K(+) currents inactivated little at room temperature, both fast-inactivation (111.5 +/- 14.3 ms) and slow-inactivation (1.21 +/- 0.12 s) was observed at 32-35 degrees C. Kv2.1 mediates the fast-inactivating current observed at 32-35 degrees C, since it could be selectively ablated by expression of a dominant-negative Kv2.1 construct (Kv2.1N). The surprising ability of Kv2.1N to selectively remove the fast-inactivating component, together with its sensitivity to tetraethylammonium (TEA), demonstrate that this component is not mediated by the classically fast-inactivating and TEA-resistant channels such as Kv1.4 and 4.2. Increasing the intracellular redox state by elevating the cytosolic NADPH/NADP(+) ratio from 1/10 to 10/1 increased the rates of both fast- and slow-inactivation. In addition, increasing the intracellular redox state also increased the relative contribution of the fast-inactivation component from 38.8 +/- 2.1 % to 55.9 +/- 1.8 %. The present study suggests that, in beta-cells, Kv2.1 channels mediate a fast-inactivating K(+) current at physiological temperatures and may be regulated by the metabolic generation of NADPH.

Figure 1. β-Cell voltage-dependent outward K⁺ currents at 22 and 32–35 °C

Currents were elicited by depolarisation from −70 mV in 20 mV increments to +70 mV. A, representative traces. B, comparison of the voltage dependence of steady-state conductance at 22 °C (•) with that at 32–35 °C for peak (□) and steady-state (○) conductance. C, voltage dependence of steady-state inactivation for currents elicited at 22 °C (▪) and peak current at 32–35 °C (□).

Figure 2. Activation and inactivation kinetics of β-cell voltage-dependent outward K⁺ currents at 22 and 32–35 °C

Currents were elicited by depolarisation from −70 to +30 mV. A, currents recorded from the same cell during a temperature ramp from 23.3 to 32.5 °C followed by application of 10 mm TEA (representative of 3 experiments). B, representative activation curves. Curves were fitted to a single-exponential function to derive activation time constants, shown on the right. C, representative traces for 8 s depolarising pulses. D, inactivation curves were fitted to a single-exponential (22 °C) or double-exponential (32–35 °C) function to derive time constants of inactivation. Currents inactivated with a single, slow time constant at 22 °C and with two time constants at 32–35 °C, fast and slow. All current traces shown are normalised to peak current. ***P < 0.001 compared with 22 °C.

Figure 3. The fast-inactivating component of β-cell voltage-dependent outward K⁺ currents at 32–35 °C is mediated by Kv2.1

Currents were elicited by depolarisation from −70 mV in 20 mV increments to +70 mV (A) or by a single depolarisation to +30 mV from −70 mV (C). A, representative traces for cells infected with AdGFP or AdKv2.1N at 22 and 32–35 °C. B, conductance-voltage relationships at 32–35 °C for peak (squares) and steady-state (circles) currents from AdGFP-infected (filled symbols) and AdKv2.1N-infected (open symbols) cells. *P < 0.05 and ***P < 0.001 compared with control peak currents. C, representative traces for AdGFP- and AdKv2.1N-infected cells. D, inactivation curves were fitted to a single-exponential (AdKv2.1N) or double-exponential (AdGFP) function to derive time constants of inactivation.

Figure 4. The inactivation properties of β-cell Kv currents can be modulated by continuous perfusion and the cytoplasmic NADPH/NADP⁺ ratio

Currents were elicited by depolarisation from −70 to +30 mV. All recordings were obtained at 32–35 °C. A, representative traces, normalised to peak current, for currents recorded during static incubation (Static) and during continuous perfusion of extracellular solution (Perfusion). B, continuous perfusion significantly reduced the relative contribution of the fast-inactivation component. C, representative traces, normalised to peak current, for cells with cytoplasmic NADPH/NADP⁺ ratios of 1/10, 1/1 and 10/1. In D-F, the contribution of the fast-inactivation component to total inactivation (D), fast-inactivation time constant (τ_f, E) and slow-inactivation time constant (τ_s, F) are shown for each ratio. *P < 0.05 and ***P < 0.001 compared with Static (B) or with 1/10 (D-F).