Influence of Ca2+-binding proteins and the cytoskeleton on Ca2+-dependent inactivation of high-voltage activated Ca2+ currents in thalamocortical relay neurons

Ca²⁺-dependent inactivation (CDI) of high-voltage activated (HVA) Ca²⁺ channels was investigated in acutely isolated and identified thalamocortical relay neurons of the dorsal lateral geniculate nucleus (dLGN) by combining electrophysiological and immunological techniques. The influence of Ca²⁺-binding proteins, calmodulin and the cytoskeleton on CDI was monitored using double-pulse protocols (a constant post-pulse applied shortly after the end of conditioning pre-pulses of increasing magnitude). Under control conditions the degree of inactivation (34±9%) revealed a U-shaped and a sigmoid dependency of the post-pulse current amplitude on pre-pulse voltage and charge influx, respectively. In contrast to a high concentration (5.5 mM) of EGTA (31±3%), a low concentration (3 µM) of parvalbumin (20±2%) and calbindin_D28K (24±4%) significantly reduced CDI. Subtype-specific Ca²⁺ channel blockers indicated that L-type, but not N-type Ca²⁺ channels are governed by CDI and modulated by Ca²⁺-binding proteins. These results point to the possibility that activity-dependent changes in the intracellular Ca²⁺-binding capacity can influence CDI substantially. Furthermore, calmodulin antagonists (phenoxybenzamine, 22±2%; calmodulin binding domain, 17±1%) and cytoskeleton stabilizers (taxol, 23±5%; phalloidin, 15±3%) reduced CDI. Taken together, these findings indicate the concurrent occurrence of different CDI mechanisms in a specific neuronal cell type, thereby supporting an integrated model of this feedback mechanism and adding further to the elucidation of the role of HVA Ca²⁺ channels in thalamic physiology.