Abstract
In fast-spiking neurons such as those in the medial nucleus of the trapezoid body (MNTB) in the auditory brainstem, Kv3.1 potassium channels are required for high frequency firing. The Kv3.1b splice variant of this channel predominates in the mature nervous system and is a substrate for phosphorylation by protein kinase C (PKC) at Ser-503. In resting neurons, basal phosphorylation at this site decreases Kv3.1 current, reducing neuronal ability to follow high frequency stimulation. We used a phospho-specific antibody to determine which PKC isozymes control serine 503 phosphorylation in Kv3.1b-tranfected cells and in auditory neurons in brainstem slices. By using isozyme-specific inhibitors, we found that the novel PKC-delta isozyme, together with the novel PKC-epsilon and conventional PKCs, contributed to the basal phosphorylation of Kv3.1b in MNTB neurons. In contrast, only PKC-epsilon and conventional PKCs mediate increases in phosphorylation produced by pharmacological activation of PKC in MNTB neurons or by metabotropic glutamate receptor activation in Kv3.1/mGluR1-cotransfected cells. We also measured the time course of dephosphorylation and recovery of basal phosphorylation of Kv3.1b following brief high frequency electrical stimulation of the trapezoid body, and we determined that the recovery process is mediated by both novel PKC-delta and PKC-epsilon isozymes and by conventional PKCs. The association between Kv3.1b and PKC isozymes was confirmed by reciprocal coimmunoprecipitation of Kv3.1b with multiple PKC isozymes. Our results suggest that the Kv3.1b channel is regulated by both conventional and novel PKC isozymes and that novel PKC-delta contributes specifically to the maintenance of basal phosphorylation in auditory neurons.
Highlights
Able to fire at frequencies of hundreds of Hz [9, 10] and to lock their action potentials precisely to the phase of auditory stimuli at frequencies of up to 2– 4 kHz or to rapid fluctuations in the amplitude of high frequency sounds [11]
Of the novel protein kinase C (PKC), only nPKC-⑀ contributes to Phorbol 12-myristate 13-acetate (PMA)-induced increases in the phosphorylation over basal levels. By using both immunocytochemistry and coimmunoprecipitation approaches, we have shown that the Kv3.1b potassium channel is basally phosphorylated at serine 503 in resting medial nucleus of the trapezoid body (MNTB) neurons but not in Kv3.1b-transfected CHO cells
Synaptic input pathway to MNTB neurons for 5 s or more results in the dephosphorylation of Kv3.1b channels, and the recovery of basal phosphorylation occurs over 1–5 min
Summary
Able to fire at frequencies of hundreds of Hz [9, 10] and to lock their action potentials precisely to the phase of auditory stimuli at frequencies of up to 2– 4 kHz or to rapid fluctuations in the amplitude of high frequency sounds [11]. In contrast to the Kv3.1b-transfected CHO cells, profuse immunostaining was seen on the membrane of MNTB principal neurons in adult rats using the phospho-specific Kv3.1b antibody, even in the absence of pharmacological activation of PKC. We found that preincubation of Kv3.1b-transfected CHO cells with group I inhibitors, i.e. 100 nM Go6976, 1 M Go6976, 50 nM Ro 31-7549, or 50 nM GF109203X, diminished but did not abolish PMA-induced phosphorylation of Kv3.1b channels (Fig. 2A).
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