Activators of protein kinase C selectively enhance inactivation of a calcium current component of cultured sensory neurons in a pertussis toxin-sensitive manner.
1. Three calcium current components were recorded in mouse dorsal root ganglion (DRG) neurons using the single-electrode voltage-clamp technique, the transient low-threshold (T), the slowly inactivating high-threshold (L), and the transient high-threshold (N) calcium current components. 2. Two activators of protein kinase C, the phorbol ester, phorbol 12,13-dibutyrate (PDBu) and the diacylglycerol analogue, 1,2-oleoylacetylglycerol (OAG), were tested to determine their effects on the three calcium current components. Neither compound affected the isolated T current, but both had similar effects on calcium currents containing both the N and L current components. PDBu and OAG reduced the peak but had little effect on the late (greater than or equal to 300 ms) calcium current evoked at clamp potentials (Vc) positive to -20 mV from holding potentials (Vh) near the resting membrane potential (-70 to -55 mV). The reduction in peak calcium current ranged from 15 to 40% and was present in all neurons tested. In contrast, there was little or no effect of these compounds when calcium currents were evoked from Vh at or negative to -80 mV. A phorbol derivative, 4-alpha-phorbol, which does not activate protein kinase C, had no effect on calcium currents at any potential. 3. Pretreatment of cultures with pertussis toxin [(PTX); 100-200 ng/ml] for 4-24 h abolished the PDBu-induced reduction of calcium current. 4. Multiexponential curve-fitting of current traces was used to determine the amplitudes and inactivation time constants (tau i) of the T, N, and L current components. PDBu selectively reduced the amplitude of the N current component but had no effect on any of the tau i. 5. PDBu had no effect on the voltage dependence of the current-voltage relation or on the time dependence of recovery from inactivation at time intervals up to 500 ms. In contrast, PDBu produced a -7.5-mV shift in the steady-state inactivation curve, sufficient to account for its effect near the resting membrane potential. 6. PDBu and OAG had selective, voltage-dependent effects on the calcium current components of mouse DRG neurons in culture. We conclude that activation of protein kinase C caused a selective reduction of the N current component; this effect was due to increased steady-state inactivation at membrane potentials positive to -80 mV and was mediated by a G protein.
Gross
R A
RA
Department of Neurology, University of Michigan Medical Center, Ann Arbor 48104.
MacDonald
R L
RL
D000200:Action Potentials / Q000187:drug effects; D000818:Animals; D002118:Calcium / Q000502:physiology*; D002478:Cells, Cultured; D004075:Diglycerides / Q000494:pharmacology*; D004566:Electrodes; D004789:Enzyme Activation; D005989:Glycerides / Q000494:pharmacology*; D008564:Membrane Potentials / Q000187:drug effects; D051379:Mice; D009474:Neurons / Q000187:drug effects* / Q000502:physiology; D037342:Pertussis Toxin; D015240:Phorbol 12,13-Dibutyrate / Q000494:pharmacology*; D010703:Phorbol Esters / Q000494:pharmacology; D011493:Protein Kinase C / Q000378:metabolism*; D010566:Virulence Factors, Bordetella / Q000494:pharmacology
D004075:Diglycerides; D005989:Glycerides; D010703:Phorbol Esters; D010566:Virulence Factors, Bordetella; D015240:Phorbol 12,13-Dibutyrate; C038237:1-oleoyl-2-acetylglycerol; D037342:Pertussis Toxin; D011493:Protein Kinase C; D002118:Calcium