Ca2+ influx in resting rat sensory neurones that regulates and is regulated by ryanodine-sensitive Ca2+ stores

摘要

class="enumerated" style="list-style-type:decimal">Store-operated, voltage-independent Ca²⁺ channels are activated by depletion of intracellular Ca²⁺ stores and mediate Ca²⁺ influx into non-excitable cells at resting membrane potential. We used microfluorimetry, patch-clamp and Mn²⁺-quench techniques to explore the possibility that a similar mechanism exists in rat dorsal root ganglion (DRG) neurones in primary culture.Following caffeine-induced depletion, ryanodine-sensitive Ca²⁺ stores refilled with Ca²⁺ at resting membrane potential. The refilling process required extracellular Ca²⁺, was blocked by 2 mM Ni²⁺, and was facilitated by membrane hyperpolarization from −55 to −80 mV, indicating a key role for Ca²⁺ influx. This influx of Ca²⁺ was not affected by the voltage-operated Ca²⁺ channel (VOCC) antagonists nicardipine (10 μM), nimodipine (10 μm) or ω-grammotoxin SIA (1 μm).When ryanodine-sensitive Ca²⁺ stores were depleted in Ca²⁺-free media, a return to 2 mM external Ca²⁺ resulted in a pronounced [Ca²⁺]i overshoot, indicating an increased permeability to Ca²⁺. Depletion of Ca²⁺ stores also produced a 2-fold increase in the rate of Mn²⁺ influx. The [Ca²⁺]i overshoot and Mn²⁺ entry were both inhibited by Ni²⁺, but not by VOCC antagonists.Caffeine induced periodic Ca²⁺ release from, and reuptake into, ryanodine-sensitive stores. The [Ca²⁺]i oscillations were arrested by removal of extracellular Ca²⁺ or by addition of Ni²⁺, but they were not affected by VOCC antagonists. Hyperpolarization increased the frequency of this rhythmic activity.These data suggest the presence of a Ca²⁺ entry pathway in mammalian sensory neurones that is distinct from VOCCs and is regulated by ryanodine-sensitive Ca²⁺ stores. This pathway participates in refilling intracellular Ca²⁺ stores and maintaining [Ca²⁺]i oscillations and thus controls the balance between intra- and extracellular Ca²⁺ reservoirs in resting DRG neurones.