Regulation of intrinsic excitability in hippocampal neurons by activity-dependent modulation of the KV2.1 potassium channel

Abstract

KV2.1 is the prominent somatodendritic sustained or delayed rectifier voltage-gated potassium (KV) channel in mammalian central neurons, and is a target for activity-dependent modulation via calcineurin-dependent dephosphorylation. Using hanatoxin-mediated block of KV2.1 we show that, in cultured rat hippocampal neurons, glutamate stimulation leads to significant hyperpolarizing shifts in the voltage-dependent activation and inactivation gating properties of the KV2.1-component of delayed rectifier K+ (IK) currents. In computer models of hippocampal neurons, these glutamate- stimulated shifts in the gating of the KV2.1-component of IK lead to a dramatic suppression of action potential firing frequency. Current-clamp experiments in cultured rat hippocampal neurons showed glutamate stimulation induced a similar suppression of neuronal firing frequency. Membrane depolarization also resulted in similar hyperpolarizing shifts in the voltage-dependent gating properties of neuronal IK currents, and suppression of neuronal firing. The glutamate-induced effects on neuronal firing were eliminated by hanatoxin, but not by dendrotoxin-K, a blocker of KV1.1-containing channels. These studies together demonstrate a specific contribution of modulation of KV2.1 channels in the activity-dependent regulation of intrinsic neuronal excitability.

Fig. 1

Glutamate treatment alters steady-state activation and inactivation properties of I_K in cultured rat hippocampal neurons. (A) Representative whole-cell current recordings obtained with the depicted pulse protocol before and after the extracellular perfusion of 10 μM glutamate for 10 min. The interpulse interval between each pulse was 20 sec. (B) Current-voltage relationship of peak currents obtained from the experiments in panel (A). (C) Voltage-dependent activation and steady-state inactivation curves for I_K from control and glutamate-treated neurons. (D) Time-course of recovery from steady-state inactivation of I_K currents from neurons before and after the extracellular perfusion of 10 μM glutamate for 10 min. The holding potential was −80 mV.

Fig. 2

Ca²⁺/calcineurin-dependent modulation of the half-maximal voltage-dependent activation (G_1/2) and steady-state inactivation (Vi_1/2) potentials of I_K currents from cultured rat hippocampal neurons with different drug treatments. Glutamate (10 μM), AP-V (5 μM), Ionomycin (1 μM), Okadaic acid (OA, 0.1 μM), and cadmium chloride (CdCl₂, 1 μM) were applied extracellularly, whereas cyclosporin A (CsA; 1 μM) and cyclophilin A (CypA; 1 μM) were applied intracellularly by dissolving in the pipette solution. Data are presented as mean ± SEM (n = 5, 5, 5, 6, 7, 5, 4, 6, 4, 4, and 4 for Control, Glu, Glu+APV, Glu[Ca²⁺-free], Ionomycin, Glu+CsA+CypA, Glu+OA, Glu+CsA+CypA+OA, Depol.[−20 mV], Depol.[−20 mV]+CdCl₂, and Depol.[−20 mV]+CsA+CypA respectively). Asterisks indicate significant difference in the G_1/2 potentials (*) and Vi_1/2 potentials (**) as compared to respective values under control conditions (p<0.05).

Fig. 3

Block of Kv2.1 currents by hanatoxin (HaTx) eliminates the glutamate-induced shifts in the voltage-dependent gating properties of I_K. (A) Representative whole-cell current recordings from HEK293 cells stably expressing recombinant rat Kv2.1 channels, from a holding potential of −100 mV with 10 mV incremental depolarizing potentials to +80 mV before and after extracellular application of 100 nM HaTx for 10 min. (B–C) Current density plot (B) and steady-state activation curves (C) of recordings obtained from experiments as shown in panel (A). Data are presented as mean ± SEM. (D–E) Glutamate-induced shifts in I_K are specific to currents carried by Kv2.1 channels. (D) Representative whole-cell current recordings obtained from a cultured rat hippocampal neuron following the pulse protocol depicted in Fig. 1A, before and after 10 min superfusion of 100 nM hanatoxin (HaTx), followed by superfusion of 10 μM glutamate along with 100 nM HaTx. (E) Steady-state activation and inactivation curves for I_K recorded from neurons as shown in panel (A). The voltage-dependent biophysical parameters are detailed in Table 1.

Fig. 4

Glutamate-induced alterations in the activation and inactivation properties of Kv2.1/I_K lead to decreased action potential firing frequency in model hippocampal neurons. (A) Action potential firing patterns in an idealized hippocampal pyramidal neuron. Firing patterns were obtained by injecting 75 pA (upper panel) and 150 pA (lower panel) current from a membrane potential of −70 mV into model hippocampal pyramidal neurons using Kv2.1 functional parameters obtained from control or glutamate-treated neurons. Parameters of all other ion channels were kept constant. (B) Kv2.1 current in control (thin black line) and glutamate-stimulated (thick grey line) model neurons invoked by action potential waveforms obtained from a 150 pA current injection. (C) Frequency-intensity plot of action potential spikes generated by the hippocampal pyramidal neuron model upon step-wise current injections.

Fig. 5

Glutamate treatment of cultured rat hippocampal neurons leads to suppression of spontaneous firing. (A) Representative traces of spontaneous firing patterns in cultured hippocampal neurons for a period of 17 min with continuous bath perfusion of ACSF (upper panel), and with 10 μM glutamate application in ACSF for 10 min following an initial ACSF perfusion for 2.5 min and post-glutamate ACSF wash of 4.5 min (bottom panel). (B) Quantitation of spontaneous firing before and after glutamate treatment as shown in panel (A). Data are presented as mean ± SEM of the number of spontaneous spikes per minute, before and after glutamate treatment (n = 4). *indicates significant difference in the number of spontaneous spikes (p<0.05).

Fig. 6

Decreased action potential firing in cultured rat hippocampal neurons upon glutamate-treatment is dependent on Kv2.1. (A, D) Representative traces of action potential firing in cultured hippocampal neurons upon injection of 20 pA (upper panel) and 100 pA (lower panel) currents, respectively, over a period of 1 sec, and before and after the treatment of 10 μM glutamate (A) or 5 μM glutamate (D) for 10 min, followed by wash-out for 2 min. (B, E) Representative traces of action potential firing patterns in cultured hippocampal neurons upon superfusion of 100 nM HaTx for 10 min (before clamping) with injection of 20 pA (upper panel) and 100 pA (lower panel) currents, respectively, over a period of 1 sec, and before and after the treatment with 10 μM (B) or 5 μM (E) glutamate for 10 min, followed by wash-out for 2 min. (C, F) Frequency-intensity plots of action potential spikes generated upon step-wise current injections from experiments in (C) panels (A–B), and (F) panels (D–E). Data are presented as mean ± SEM (n = 5 and 3 for experiments from panels A & D and panels B & E respectively).

Fig. 7

Effects of dendrotoxin-kappa (DTX3) on action potential firing in cultured hippocampal pyramidal neurons without or with glutamate treatment. (A) Representative traces of action potential firing in cultured hippocampal pyramidal neurons pre-treated with 1 μM DTXκ for 10 min, upon injection of 20 pA (upper panel) and 100 pA (lower panel) currents, respectively, over a period of 1 sec, and before and after treatment of 5 μM glutamate along with 1 μM DTXκ for 10 min. (B) Frequency-intensity plot of action potential spikes generated upon step-wise current injections from experiments in panel (a). Frequency-intensity relationship from untreated and 5 μM glutamate-treated neurons as shown in Fig. 6F are plotted alongside for comparison. Data are presented as mean ± SEM (n = 3).

Fig. 8

Membrane depolarization in cultured rat hippocampal neurons leads to suppression of action potential firing frequencies. (A) Representative trace of spontaneous firing in a cultured hippocampal neuron for a period of 18 min with membrane depolarization to −20 mV for 10 min following an initial holding period of 3 min, and post-depolarization period of 5 min, with continuous perfusion of ACSF. (B) Quantitation of spontaneous spikes before and after membrane depolarization to −20 mV as shown in panel (A). Data are presented as mean ± SEM of the number of spontaneous spikes per minute, before and after membrane depolarization (n = 3). *indicates significant difference in the number of spontaneous spikes (p<0.05). (C, D) Decreased action potential firing in cultured hippocampal neurons upon membrane depolarization (−20 mV). (C) Representative traces of action potential firing patterns in cultured hippocampal neurons upon injection of 20 pA (upper panel) and 100 pA (lower panel) currents, respectively, over a period of 1 sec, and before and after membrane depolarization (−20 mV) for 10 min. (D) Frequency-intensity plot of action potential spikes generated upon step-wise current injections from experiments in panel (C). Data are presented as mean ± SEM (n = 3). (E) Representative trace of spontaneous firing patterns in a cultured hippocampal neuron for a period of ≈8 min with episodic membrane depolarization to −20 mV for 30 sec interspersed with 1 min intervals of resting potential (≈ −63 mV), with continuous perfusion of ACSF. (F) Quantitation of spontaneous spikes under resting conditions before and after episodic membrane depolarization to −20 mV as shown in panel (E). Data are presented as mean ± SEM of the number of spontaneous spikes per minute, from every 1 min resting interval (n = 2).