Dual action of thapsigargin on calcium mobilization in sensory neurons: Inhibition of Ca²⁺ uptake by caffeine-sensitive pools and blockade of plasmalemmal Ca²⁺ channels

The action of thapsigargin on intracellular calcium homeostasis and voltage-activated calcium currents was studied on freshly isolated adult mouse dorsal root ganglia neurons. The cytoplasmic Ca²⁺ concentration ([Ca²⁺]_i) was measured using indo-1-based microfluorimetry; transmembrane Ca²⁺ currents were recorded under voltage-clamp in the whole-cell configuration of the patch-clamp technique. Extracellular applications of thapsigargin at concentrations of 20-2000 nM did not cause substantial changes of basal [Ca²⁺]_i level in the majority of neurons studied. However, 5-10 min incubation of neurons with 20 nM thapsigargin completely and almost irreversibly inhibited caffeine-mediated Ca²⁺ release from intracellular pools. This inhibition was associated with deceleration of the recovery of depolarization-induced [Ca²⁺]_i transients, presumably due to the inhibition of Ca²⁺ uptake by intracellular calcium stores. At concentrations between 200 and 2000 nM, thapsigargin markedly depressed the amplitudes of depolarization-triggered [Ca²⁺]_i transients due to the inhibition of transmembrane Ca²⁺ entry through voltage-activated Ca²⁺ channels. We found that thapsigargin discriminates between low- and high-voltage-activated Ca²⁺ channels: 2000 nM of thapsigargin decreased the amplitudes of high-voltage-activated currents by 60%, while the amplitudes of low-voltage-activated Ca²⁺ currents were reduced by only 25%. Thus, thapsigargin exerts a dual action on [Ca²⁺]_i handling mechanisms in mouse sensory neurons: at low concentrations (< 50 nM) it inhibits Ca²⁺ accumulation by endoplasmic reticulum pools, whereas at higher concentrations (200-2000 nM) thapsigargin blocks high-voltage-activated Ca²⁺ currents, reducing Ca²⁺ entry into the cell.