Phorbol ester-induced inhibition of potassium currents in rat sensory neurons requires voltage-dependent entry of calcium

The whole cell patch-clamp technique was used to examine the effects of protein kinase C (PKC) activation (via the phorbol ester, phorbol 12,13 dibutyrate, PDBu) on the modulation of potassium currents (I-K) in cultured capsaicin-sensitive neurons isolated from dorsal root ganglia from embryonic rat pups and grown in culture. PDBu, in a concentration- and time-dependent manner, reduced I-K measured at +60 mV by similar to 30% if the holding potential (V-h) was -20 or -47 mV but had no effect if V-h was -80 mV. The PDBu-induced inhibition of I-K was blocked by pretreatment with the PKC inhibitor bisindolylmaleimide I and I-K was unaffected by 4-alpha phorbol, indicating that the suppression of I-K was mediated by PKC. The inhibition of I-K by 100 nM PDBu at a V-h of -50 mV was reversed over several minutes if V-h was changed to -80 mV. In addition, intracellular perfusion with 5 mM bis-(o-aminophenoxy)-N,N,N',N'-tetraacetic acid (BAPTA) or pretreatment with omega -conotoxin GVIA or Cd2+-Ringer, but not nifedipine, prevented the PDBu-induced suppression of I-K at -50 mV, suggesting that a voltage-dependent influx of calcium through N-type calcium channels was necessary for the activation of PKC. The potassium channel blockers tetraethylammonium (TEA, 10 mM) and 4-aminopyridine (4-AP, 3 mM and 30 muM) reduced I-K, but only TEA attenuated the ability of PDBu to further inhibit the current, suggesting that the I-K modified by PDBu was sensitive to TEA. Interestingly, in the presence of 3 mM or 30 muM 4-AP, 100 nM PDBu inhibited I-K when V-h was -80 mV. Thus 4-AP promotes the capacity of PDBu to reduce I-K at -80 mV. We find that activation of PKC inhibits I-K in rat sensory neurons and that voltage-dependent calcium entry is necessary for the development and maintenance of this inhibition.