Calcium hotspots caused by L-channel clustering promote morphological changes in neuronal growth cones

Abstract

THE increase in intracellular Ca²⁺concentration ([Ca²⁺]_i) that follows electrical activity in excitable cells influences various cellular functions. But the measured increase in average cytosolic [Ca²⁺]_i(typically 10 nM per action potential^1–3) is often less than the micromolar [Ca²⁺]_i required to activate proteins controlling cell function^4–5. We now report that clustering of L-type Ca²⁺ channels causes [Ca²⁺]_i hotspots of average diameter 7 μm at the neuronal growth cone. At the hotspot, [Ca²⁺]_i changes of the order of 1 μM were recorded during 1-s voltage-clamp depolarizations, whereas a single action potential raised [Ca²⁺]_i by 89 nM. Depolarization will therefore activate enzymes with a micromolar requirement for Ca²⁺ at the hotspot. Local morphological changes near the site of the hotspot were induced by action potentials. We observed hotspots in all regions of the growth cone, usually at the base of processes extending from the growth-cone palm, but never in the neurite. The role of voltage-dependent Ca²⁺ influx in controlling nerve cell outgrowth has been a puzzle: although raised [Ca²⁺]_i triggers outgrowth of the growth cone margin, neurite elongation requires low [Ca²⁺]_i(refs 6–8). Our results resolve this paradox: electrical activity can selectively raise [Ca²⁺]_i in the growth cone, leaving neurite [Ca²⁺]_ilow. Gross [Ca²⁺]_i gradients and localized hotspots have been previously reported in depolarized neurons^3,9–12. Patch-clamp, toxin-binding and freeze-fracture studies have demonstrated that calcium channels are grouped in clusters^13–17. However, this is the first report that calcium channel clusters can cause [Ca²⁺]_i hotspots, and that channel clustering has a physiological role.