Activation of protein kinase C in sensory neurons accelerates Ca ²⁺ uptake into the endoplasmic reticulum

The rate of Ca²⁺ clearance from the neuronal cytoplasm affects the amplitude, duration, and localization of Ca²⁺ signals and influences a variety of Ca²⁺-dependent functions. We reported previously that activation of protein kinase C (PKC) accelerates Ca⁺ efflux in rat sensory neurons mediated by the plasma membrane Ca ⁺-ATPase isoform 4 (PMCA4). Here we show that sarco-endoplasmic reticulum Ca⁺-ATPase (SERCA)-mediated Ca⁺ uptake into intracellular stores is also accelerated by PKC activation. The rate of intracellular Ca⁺ concentration ([Ca⁺]_i) clearance was studied after small (<350 nM) action potential-induced Ca ⁺ loads in rat dorsal root ganglion neurons. Under these conditions, mitochondrial Ca⁺ uptake and Na⁺/Ca⁺ exchange do not significantly influence [Ca⁺]_i recovery. Phorbol dibutyrate (PDBu) increased the rate of [Ca⁺]_i clearance by 71% in a manner sensitive to the selective PKC inhibitors GF109203x (2-[1-(3-dimethylaminopropyl)-1H-indol-3-yl]-3-(1H-indol-3-yl)maleimide) and calphostin. PKC-dependent acceleration was still observed (∼39%) when the PKC-sensitive PMCA isoform was knocked down by expression of an antisense PMCA4 cDNA (AS4). Direct measurement of Ca⁺ in the endoplasmic reticulum (ER) lumen revealed that PKC increased the rate of store refilling more than twofold after depletion by treatment with cyclopiazonic acid. ER refilling was less complete in PDBu-treated cells, although, in AS4-expressing cells, PDBu accelerated the rate without reducing the ER capacity, suggesting that PMCA and SERCA compete for Ca⁺. Thus, activation of PKC accelerates the clearance of Ca⁺ from the cytoplasm by the concerted stimulation of Ca⁺ sequestration and Ca⁺ efflux.