Mediation of neuronal apoptosis by Kv2.1-encoded potassium channels

Abstract

Cellular K+ efflux is a requisite event in the unfolding of apoptosis programs across many types of cells and death-inducing stimuli; however, the molecular identities of the ion channels mediating this key event have remained undefined. Here, we show that Kv2.1-encoded K+ channels are responsible for the expression of apoptosis in cortical neurons in vitro. Transient expression of two different dominant-negative forms of this subunit in neurons completely eliminated the enhancement of K+ currents that normally accompanies the cell death process. Importantly, neurons deficient in functional Kv2.1-encoded K+ channels were protected from oxidant and staurosporine-induced apoptosis. Finally, Chinese hamster ovary cells, which do not express endogenous voltage-gated K+ channels, became substantially more sensitive to apoptosis after transient expression of wild-type Kv2.1. These results suggest that Kv2.1-encoded K+ channels are necessary for the apoptotic signaling cascade in mammalian cortical neurons in culture and are sufficient for increasing the susceptibility to apoptogens in a nonexcitable cell.

Figure 1.

Kv2.1 DN expression in cortical neurons. a, Left, Representative whole-cell recordings obtained from GFP-negative (blue traces) and GFP-positive (greentraces) neurons 24, 30, and 48 hr after transfection with Kv2.1N216, evoked by a voltage step from -70 to +35 mV. Calibration: 5nA, 25msec. Right, Current—voltage relationship of a single GFP-positive (green) and GFP-negative cell (blue) 48 hr after transfection. b, Time course of Kv2.1N216 expression, detected by a decrease in K⁺ current evoked by a voltage step from -70 to 5 mV in a total of 23 GFP-positive cells (top, green). Blue line indicates average K⁺ current density obtained from GFP-negative cells (n = 21) on the same coverslips. Bottom panel, Mean (± SEM) K⁺ current densities in GFP-negative (blue; n = 10) and GFP-positive (green; n = 8) neurons (^*p = 0.0005; t test) 48–54 hr after transfection. c, Representative K⁺ currents in GFP-negative and GFP-positive neurons obtained 30 hr (top traces) and 48 hr (bottom traces) after transfection with Kv2.1W365C/Y380T, evoked by sequential voltage steps to +35 mV from -70 mV. Inset, Mean K⁺ current densities of GFP-negative (n = 7) and GFP-positive (n = 10) neurons 48–54 hr after transfection (p < 0.0001; t test). Calibration: 2 nA, 25 msec.

Figure 2.

Kv2.1 DN expression blocks apoptosis-related enhancement of K ⁺ currents. a, Top, Whole-cell currents evoked by a single voltage step from -70 to 5 mV from GFP-negative (blue) and GFP-positive (green) neurons in cultures transfected with Kv2.1N216 4 hr after treatment with either vehicle or 100 μm DTDP (10min). Bottom, Mean ± SEM current densities from GFP-negative and GFP-positive cells obtained 4 hr after treatment with vehicle (0.001% DMSO) or DTDP. Calibration: 7 nA, 20 msec (^*p < 0.001; ANOVA, Bonferoni). B, Results from identical experiment using the Kv2.1 pore mutant (^*p < 0.001).

Figure 3.

Kv2.1 mediates apoptosis. a, Neurons were transfected with a luciferase reporter and Kv2.1 N216. Treatment with vehicle or 100 μm DTDP (10min) and 200 μm NMDA or NMDA + 10 μm MK801 (overnight) was performed at 24 and 48 hr after transfection; luciferase activity was measured 24 hr later. Shown are mean (± SD) luciferase activity values (in counts per second; CPS) for single experiments performed in quadruplicate (^*p < 0.05; t test). Expression of Kv2.1N216 at 48 hr protects against DTDP but not NMDA toxicity. Inset depicts the mean (± SEM) viability, expressed as a percentage of control, for three independent experiments (^*p ≤ 0.05, Mann—Whitney one-sample test vs 100% viability). b, Similar results were observed for neurons transfected with Kv2.1W365C/Y380T and treated overnight with 0.5 μm staurosporine. Inset depicts the mean viability for four independent experiments (^*p ≤ 0.05). c, Expression of Kv2.2W373C/Y388T is not neuroprotective. Inset depicts the mean viability for three independent experiments (^*p ≤ 0.05). d, CHO cells were transfected with GFP along with NR1 a (mock), EGFP-Kv1.4, or Kv2.1 and treated with DTDP (5 μm, 5 min), DTDP, and BAF (50 μm) or vehicle 24 hr after transfection. Twenty-four hours after treatment, GFP-positive cells were counted (3 coverslips per group). Percentage viability was determined by comparing DTDP and vehicle-treated groups (^*#p < 0.01; ANOVA, Tukey). Inset, K⁺ currents in CHO cells transfected with EGFP-Kv1.4 (left) or GFP and Kv2.1(right), evoked by incremental voltage steps to +20mV from -70mV. Calibration: 2nA, 25msec.