A-type K+ channels encoded by Kv4.2, Kv4.3 and Kv1.4 differentially regulate intrinsic excitability of cortical pyramidal neurons

Abstract

Rapidly activating and rapidly inactivating voltage-gated A-type K+ currents, IA, are key determinants of neuronal excitability and several studies suggest a critical role for the Kv4.2 pore-forming α subunit in the generation of IA channels in hippocampal and cortical pyramidal neurons. The experiments here demonstrate that Kv4.2, Kv4.3 and Kv1.4 all contribute to the generation of IA channels in mature cortical pyramidal (CP) neurons and that Kv4.2-, Kv4.3- and Kv1.4-encoded IA channels play distinct roles in regulating the intrinsic excitability and the firing properties of mature CP neurons. In vivo loss of Kv4.2, for example, alters the input resistances, current thresholds for action potential generation and action potential repolarization of mature CP neurons. Elimination of Kv4.3 also prolongs action potential duration, whereas the input resistances and the current thresholds for action potential generation in Kv4.3−/− and WT CP neurons are indistinguishable. In addition, although increased repetitive firing was observed in both Kv4.2−/− and Kv4.3−/− CP neurons, the increases in Kv4.2−/− CP neurons were observed in response to small, but not large, amplitude depolarizing current injections, whereas firing rates were higher in Kv4.3−/− CP neurons only with large amplitude current injections. In vivo loss of Kv1.4, in contrast, had minimal effects on the intrinsic excitability and the firing properties of mature CP neurons. Comparison of the effects of pharmacological blockade of Kv4-encoded currents in Kv1.4−/− and WT CP neurons, however, revealed that Kv1.4-encoded IA channels do contribute to controlling resting membrane potentials, the regulation of current thresholds for action potential generation and repetitive firing rates in mature CP neurons.

Figure 1. Deletion of Kv4.2 or Kv4.3 increases firing rates in layer 5 CP neurons

Repetitive firing was evoked in mature layer 5 CP neurons in acute slices prepared from WT, Kv4.2^−/− or Kv4.3^−/− animals in response to prolonged (500 ms) depolarizing current injections of varying amplitudes. A, representative voltage records are shown; the injected current amplitudes are illustrated under the voltage records. B, mean ± SEM numbers of action potentials evoked during 500 ms current injections are plotted as a function of the amplitudes of the injected currents. The input–output curves (number of spikes vs. injected current amplitude) for Kv4.2^−/− (n = 30) and Kv4.3^−/− (n = 21) CP neurons were significantly (P < 0.001) different from the curve for WT (n = 20) CP neurons. Bonferroni post hoc analysis further revealed that the responses of Kv4.2^−/− and Kv4.3^−/− CP neurons to depolarizing current injections of small and large amplitudes were distinct. ‡†Values measured in Kv4.2^−/− or Kv4.3^−/− were significantly (‡P < 0.01; †P < 0.05) different from those determined in WT cells.

Figure 2. Simultaneous loss of Kv4.2 and Kv4.3 reveals two distinct repetitive firing patterns in CP neurons

A and B, representative voltage recordings from Kv4.2^−/−/Kv4.3^−/− CP neurons during prolonged (500 ms) depolarizing current injections of varying amplitudes are illustrated. A, repetitive firing of single action potentials was observed in the majority (∼60%) of Kv4.2^−/−/Kv4.3^−/− CP neurons. B, in the remaining (∼40%) Kv4.2^−/−/Kv4.3^−/− CP neurons (Kv4.2^−/−/Kv4.3^−/− doublets), however, action potential doublets were observed. The inset shows the third spike doublet on an expanded time scale. C, mean ± SEM numbers of action potentials evoked in Kv4.2^−/−/Kv4.3^−/− CP neurons (A) during 500 ms current injections are plotted as a function of the amplitudes of the injected currents. Mean ± SEM numbers of action potentials in Kv4.2^−/− and Kv4.3^−/− CP neurons are replotted from Fig. 1C for comparison purposes. The input–output curve (number of spikes vs. injected current amplitude) for Kv4.2^−/−/Kv4.3^−/− (n = 16) CP neurons were significantly (P < 0.001) different from those of Kv4.2^−/− (n = 30) or Kv4.3^−/− (n = 21) CP neurons.

Figure 3. Targeted deletion of Kv4.2 or Kv4.3 differentially affects the resting membrane properties of layer 5 CP neurons

A, representative action potentials, evoked in response to brief (5 ms) current injections in WT, Kv4.2^−/−, Kv4.3^−/− and Kv4.2^−/−/Kv4.3^−/− CP neurons are illustrated. The insets show each action potential on an expanded time scale. The amplitudes of the injected currents are given under the voltage records. Resting and active membrane properties were analysed in individual WT (n = 24), Kv4.2^−/− (n = 30), Kv4.3^−/− (n = 21) and Kv4.2^−/−/Kv4.3^−/− (n = 15) CP neurons, and mean ± SEM values are presented in B–E. V_m, resting membrane potential; R_in, input resistance; I_thr, current threshold for action potential generation; Width, action potential duration at half-maximum (50%) repolarization. *‡†Values measured in Kv4.2^−/−, Kv4.3^−/− or Kv4.2^−/−/Kv4.3^−/− cells were significantly (*P < 0.001; ‡P < 0.01; †P < 0.05) different from those determined in WT cells. In addition, values in Kv4.2^−/−/Kv4.3^−/− cells were significantly ( P < 0.001; P < 0.01; P < 0.05) different from those in Kv4.3^−/− cells, and #values in Kv4.2^−/−/Kv4.3^−/− cells were significantly (#P < 0.001) different from Kv4.2^−/− cells.

Figure 4. TEA markedly prolongs action potential waveforms in Kv4.2^−/− CP neurons

A, representative action potential waveforms evoked by brief (5 ms) current injections in WT, Kv4.2^−/− and Kv4.3^−/− layer 5 CP neurons in the presence of 3 mm TEA. B, action potential widths at half-maximum (50%) repolarization in individual WT (n = 10), Kv4.2^−/− (n = 11) and Kv4.3^−/− (n = 8) CP neurons in the presence or absence of 3 mm TEA were normalized to the average action potential widths measured in neurons of the same genotype in the absence of TEA; mean ± SEM action potential widths at half-maximum are presented. TEA significantly (*P < 0.001; ‡P < 0.01) increased action potential durations in WT, Kv4.2^−/− and Kv4.3^−/− CP neurons. In addition, the magnitude of the effect of TEA on action potential durations was significantly (P < 0.001) larger in Kv4.2^−/− than in either WT or Kv4.3^−/− CP neurons.

Figure 5. Intrinsic properties of Kv1.4^−/− and WT CP neurons are similar

A, representative recordings from WT and Kv1.4^−/− CP neurons in response to prolonged (500 ms) depolarizing current injections of varying amplitudes are shown. The mean ± SEM numbers of action potentials evoked during 500 ms current injections in WT (n = 24) and Kv1.4^−/− (n = 13) CP neurons are similar at all current injection amplitudes. B, representative single action potential waveforms evoked in response to brief (5 ms) depolarizing current injections in WT and Kv1.4^−/− CP neurons. The mean ± SEM action potential width at half-maximum was significantly (^‡P < 0.01) shorter in Kv1.4^−/− (n = 13) than in WT (n = 24) CP neurons (see text).

Figure 6. Ba²⁺ blockade of Kv4-encoded I_A reveals a functional role for Kv1.4 in regulating the excitability of layer 5 CP neurons

A, representative voltage recordings from WT and Kv1.4^−/− CP neurons in the presence of 400 μm Ba²⁺ in response to prolonged (500 ms) depolarizing current injections are illustrated. Repetitive firing of single action potentials was evoked in the majority (∼60%) of WT and in ∼50% of Kv1.4^−/− CP neurons in the presence of Ba²⁺ (see text). B, the mean ± SEM numbers of action potentials evoked in WT and Kv1.4^−/− CP neurons in the presence and absence of Ba²⁺ are plotted as a function of the amplitudes of the injected currents; mean ± SEM numbers of action potentials in WT and Kv1.4^−/− CP neurons in the absence of Ba²⁺ are replotted from Fig. 5A for comparison. Firing rates in WT (n = 10) and Kv1.4^−/− (n = 6) CP neurons in the presence of Ba²⁺ were significantly (P < 0.001) higher than in WT (n = 24) and Kv1.4^−/− (n = 13) CP neurons in the absence of Ba²⁺. In addition, in the presence of Ba²⁺, mean ± SEM firing rates were significantly (P < 0.001) higher in Kv1.4^−/− than in WT CP neurons. C, representative single action potentials evoked in response to brief (5 ms) depolarizing current injections in WT and Kv1.4^−/− CP neurons in the presence of 400 μm Ba²⁺. D, in the presence of Ba²⁺, the mean ± SEM current threshold for action potential generation (I_thr) was significantly (P < 0.001) lower in Kv1.4^−/− (n = 10) than in WT (n = 6) CP neurons, whereas the mean ± SEM action potential widths at half-maximum were not significantly different.