title: Modulation of calcium efflux from cultured rat dorsal root ganglion neurons.
abstract: The free intracellular Ca2+ concentration ([Ca2+]i) is governed by the balance between the activation of Ca2+ channels and buffering and efflux processes. We tested the hypothesis that Ca2+ efflux pathways are susceptible to modulation. The whole-cell patch-clamp technique was used in combination with Indo-1-based microfluorometry to record Ca2+ current and [Ca2+]i simultaneously from single rat dorsal root ganglion (DRG) neurons grown in culture. Depolarizing test pulses (-80 to 0 mV, 100-300 msec) elicited [Ca2+]i transients that recovered to basal levels by a process best-fit with a single exponential (tau = 5.1 +/- 0.4 sec; n = 14) and were independent of Ca2+ load (40-500 pC) over this range of test pulses. [Ca2+]i transients recorded in whole-cell configuration were similar to those elicited by a brief train of action potentials in unclamped neurons. Inhibition of Ca2+ sequestration into intracellular stores with thapsigargin had no effect on the kinetics of recovery. Inhibition of plasma membrane Ca2+ ATPase (PMCA) function by including a peptide inhibitor (C28R2) in the patch pipette significantly slowed recovery to basal [Ca2+]i (tau = 9.9 +/- 0.8 sec; n = 4). Preincubation with calmidazolium, a calmodulin antagonist, produced modest slowing of Ca2+ efflux. Phorbol dibutyrate, an activator of protein kinase C (PKC), accelerated Ca2+ efflux only when the PMCA had been inhibited by C28R2. We conclude that in DRG neurons PMCAs are responsible for lowering [Ca2+]i after small Ca2+ loads and that PMCA-mediated Ca2+ efflux is modulated by calmodulin- and PKC-signaling pathways.
authors.affiliation: Department of Pharmacology, University of Minnesota Medical School, Minneapolis 55455, USA.
authors.forename: J L
authors.initials: JL
authors.lastname: Werth
authors.forename: Y M
authors.initials: YM
authors.lastname: Usachev
authors.forename: S A
authors.initials: SA
authors.lastname: Thayer
mesh_terms: D000200:Action Potentials; D000595:Amino Acid Sequence; D000818:Animals; D001693:Biological Transport, Active / Q000187:drug effects; D002118:Calcium / Q000378:metabolism*; D015220:Calcium Channels / Q000187:drug effects / Q000378:metabolism*; D000252:Calcium-Transporting ATPases / Q000037:antagonists & inhibitors / Q000502:physiology*; D002147:Calmodulin / Q000037:antagonists & inhibitors / Q000502:physiology*; D002451:Cell Compartmentation; D002478:Cells, Cultured; D004558:Electric Stimulation; D005470:Fluorometry; D005727:Ganglia, Spinal / Q000166:cytology*; D007093:Imidazoles / Q000494:pharmacology; D007424:Intracellular Fluid / Q000378:metabolism; D008969:Molecular Sequence Data; D009419:Nerve Tissue Proteins / Q000502:physiology*; D009474:Neurons / Q000187:drug effects / Q000378:metabolism*; D018408:Patch-Clamp Techniques; D010455:Peptides / Q000494:pharmacology; D015240:Phorbol 12,13-Dibutyrate / Q000494:pharmacology; D011493:Protein Kinase C / Q000378:metabolism / Q000502:physiology*; D051381:Rats; D017207:Rats, Sprague-Dawley; D015398:Signal Transduction; D013729:Terpenes / Q000494:pharmacology; D019284:Thapsigargin
chemical_list: C097865:C28R2 peptide; D015220:Calcium Channels; D002147:Calmodulin; C097866:G25 peptide; D007093:Imidazoles; D009419:Nerve Tissue Proteins; D010455:Peptides; D013729:Terpenes; D015240:Phorbol 12,13-Dibutyrate; C031938:calmidazolium; D019284:Thapsigargin; D011493:Protein Kinase C; D000252:Calcium-Transporting ATPases; D002118:Calcium