Bidirectional control of BK channel open probability by CAMKII and PKC in medial vestibular nucleus neurons

Abstract

Large conductance K(+) (BK) channels are a key determinant of neuronal excitability. Medial vestibular nucleus (MVN) neurons regulate eye movements to ensure image stabilization during head movement, and changes in their intrinsic excitability may play a critical role in plasticity of the vestibulo-ocular reflex. Plasticity of intrinsic excitability in MVN neurons is mediated by kinases, and BK channels influence excitability, but whether endogenous BK channels are directly modulated by kinases is unknown. Double somatic patch-clamp recordings from MVN neurons revealed large conductance potassium channel openings during spontaneous action potential firing. These channels displayed Ca(2+) and voltage dependence in excised patches, identifying them as BK channels. Recording isolated single channel currents at physiological temperature revealed a novel kinase-mediated bidirectional control in the range of voltages over which BK channels are activated. Application of activated Ca(2+)/calmodulin-dependent kinase II (CAMKII) increased BK channel open probability by shifting the voltage activation range towards more hyperpolarized potentials. An opposite shift in BK channel open probability was revealed by inhibition of phosphatases and was occluded by blockade of protein kinase C (PKC), suggesting that active PKC associated with BK channel complexes in patches was responsible for this effect. Accordingly, direct activation of endogenous PKC by PMA induced a decrease in BK open probability. BK channel activity affects excitability in MVN neurons and bidirectional control of BK channels by CAMKII, and PKC suggests that cellular signaling cascades engaged during plasticity may dynamically control excitability by regulating BK channel open probability.

Fig. 1.

Activation of large conductance K⁺ (BK) channels during action-potential firing. A: double patch-clamp recordings were performed on medial vestibular nucleus (MVN) somata to characterize the activity of BK channels during action potential firing. Top trace show spontaneous action potential firing in an MVN neuron recorded in whole cell current clamp mode, and bottom trace shows simultaneous cell-attached recording in voltage clamp mode in which an active large amplitude single channel is detected. B: example overlay of action potential waveform and typical channel activity, showing a channel that opens during the falling phase of action potentials, as well as in the interspike interval.

Fig. 2.

Biophysical characteristics of BK channels in MVN neurons. A: inside-out patches were pulled from MVN neurons in slices. The presence of a BK channel became apparent upon excision of the patch in standard artificial cerebrospinal fluid. After washing in a high K⁺ solution with 1 μM free Ca²⁺, the current-voltage relationship of BK channels was determined by voltage clamping the patch at different potentials. Reversal potential for K⁺ (EK) was set at 0 mV. B: mean current-voltage relationship of MVN BK channels in 1 μM Ca²⁺, from which a single channel conductance of 267 ± 5 pS (n = 25) was calculated. C: voltage and Ca²⁺ dependence of MVN BK channels, showing that an increasing Ca²⁺ concentration shifts the voltage dependence towards more hyperpolarized potentials. Voltage of half-maximal activation was 101 ± 5 (n = 4) in 0.1 μM Ca²⁺, −58 ± 2 (n = 18) in 1 μM Ca²⁺, and −66 ± 8 (n = 4) in 10 μM Ca²⁺. Slope factor of the voltage dependence in 0.1, 1, and 10 μM Ca²⁺ was 11 ± 2 mV (n = 4), 15 ± 1 mV (n = 18), and 15 ± 2 mV (n = 4), respectively.

Fig. 3.

Activated Ca²⁺/calmodulin-dependent kinase II (CAMKII) increases the open probability of BK channels. A, left: an example of BK channel activity is shown recorded at −50 mV in a 1 μM Ca²⁺ solution in control condition and after 10 min of bath application of activated CAMKII (500 U/ml). A, right: within patch comparisons showed a highly significant effect of CAMKII on channel open probability [from 0.35 ± 0.08 before αCAMKII (●) to 0.67 ± 0.05 after CAMKII (○); n = 11; P = 0.002] B: example of the voltage dependence of a BK channel before (●) and after application of activated CAMKII (○). Active CAMKII induced a mean shift of 56 ± 10 mV (n = 6; P = 0.002) towards more hyperpolarized potentials, with a small change in the slope factor of 4.6 ± 0.8 mV (n = 6; P = 0.05). C: control recordings of active BK channels in time showed a slight, but nonsignificant, run-up in BK channel open probability [from 0.65 ± 0.04 control early (●) to 0.70 ± 0.05 control late (○); n = 8; P = 0.08]. **P < 0.01.

Fig. 4.

Dephosphorylation of BK channels in inside-out patches does not affect open probability. To test for the presence of any endogenously active CAMKII bound to BK channels in inside-out patches, we bath applied alkaline phosphatase to (60–80 U/ml) to channels recorded at −30 mV. Alkaline phosphatase had little effect on BK channel open probability [from 0.82 ± 0.03 before alkaline phosphatase (●) to 0.79 ± 0.04 after alkaline phosphatase (○); n = 6; P = 0.05], suggesting that either little active CAMKII is bound to these channels or that there already is strong phosphatase activity in the inside-out patches.

Fig. 5.

Inhibition of phosphatase activity decreases the open probability of BK channels. A, left: example traces show that inhibition of endogenous phosphatases by the phosphatase blocker microcystin LR (MC-LR; 1 μM) induced a decrease in the open probability of BK channels recorded at −50 mV. A, right: within patch comparisons showed a highly significant effect of MC-LR on BK channel open probability [from 0.48 ± 0.07 before MC-LR (●) to 0.35 ± 0.08 after MC-LR (○); n = 7; P = 0.01]. B: example of the voltage-dependence of a BK channel before (●) and after application of MC-LR (○). MC-LR induced a mean shift of 51 ± 11 mV (n = 5; P = 0.009; B) towards more depolarized potentials, with no significant change on the slope factor (−0.9 ± 0.5 mV; n = 5; P = 0.12). C: control recordings of active BK channels in time showed a slight, but nonsignificant, run-up in BK channel open probability [from 0.79 ± 0.04 before 0.2% DMSO (●) to 0.83 ± 0.04 after 0.2% DMSO (○); n = 4; P = 0.09]. **P < 0.01.

Fig. 6.

Activated PKC activity decreases BK channel open probability A: blockade of phosphatase activity by microcystin LR in the presence of a peptide-inhibitor of PKA (0.45 μM) did not prevent the decrease in open probability in 5 out of 5 patches tested [from 0.68 ± 0.09 before MC-LR + PKA inhibitor (●) to 0.22 ± 0.10 after MC-LR + PKA inhibitor (○); n = 5; P = 0.004; left], while blockade of phosphatases in the presence of a peptide-inhibitor of PKC (1 μM) did prevent the decrease in open probability [from 0.69 ± 0.05 before MC-LR + PKC inhibitor (●) to 0.67 ± 0.06 after MC-LR + PKC inhibitor (○); n = 10; P = 0.55; right], thus identifying PKC as the active kinase that is unmasked by inhibition of endogenous phosphatase activity. B, left: example traces show that application of the PKC agonist PMA (10 μM) to patches with intact phosphatase activity reduced BK channel open probability. On the right, within patch comparisons showed a significant effect of PMA on BK channel open probability [from 0.71 ± 0.06 before PMA (●) to 0.55 ± 0.09 after PMA (○); n = 7; P = 0.02]. *P < 0.05; **P < 0.01.

Fig. 7.

Schematic of bidirectional phosphorylation of BK channels by CAMKII and PKC. The effect of phosphorylation of MVN BK channels by CAMKII and PKC is bidirectional: active CAMKII increases open probability, while active PKC decreases open probability. As a decrease in active CAMKII increases gain, an effect that is occluded by blockade of BK channels (Nelson et al. 2005) and blockade of BK channels increases the input-output gain of MVN neurons (Smith et al. 2002), it can be concluded that in MVN neurons CAMKII phosphorylation of BK channels decreases firing rates while PKC phosphorylation of BK channels increases firing rates. This bidirectional BK channel modulation by CAMKII and PKC thus constitutes a powerful and dynamic mechanism to regulate the excitability of MVN neurons.