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Neuronal Ca2+ channels are rapidly inactivated by a mechanism that is termed Ca2+-dependent inactivation (CDI). In this study we investigated the influence of intracellular Ca2+ release on CDI of high-voltage-activated Ca2+ channels in rat thalamocortical relay neurons by combining voltage-clamp, Ca2+ imaging and immunological techniques. Double-pulse protocols revealed CDI, which depended on the length of the conditioning pulses. Caffeine caused a concentration-dependent increase in CDI that was accompanied by an increase in the duration of Ca2+ transients. Inhibition of ryanodine receptors and endoplasmic Ca2+ pumps (by thapsigargin or cyclopiazonic acid) resulted in a reduction of CDI. In contrast, inhibition of inositol 1,4,5-tris-phosphate receptors by intracellular application of 2-aminoethoxy diphenyl borate or heparin did not influence CDI. The block of transient receptor potential channels by extracellular application of 2-aminoethoxy diphenyl borate, however, resulted in a significant reduction of CDI. The central role of L-type Ca2+ channels was emphasized by the near-complete block of CDI by nifedipine, an effect only surpassed when Ca2+ was replaced by Ba2+ and chelated by 1,2-bis(o-aminophenoxy)ethane-N,N,N′,N′,-tetraacetic acid (BAPTA). Trains of action potential-like stimuli induced a strong reduction in high-voltage-activated Ca2+ current amplitude, which was significantly reduced when intracellular Ca2+ stores were made inoperative by thapsigargin or Ba2+/BAPTA. Western blotting revealed expression of L-type Ca2+ channels in thalamic and hippocampal tissue but not liver tissue. In summary, these results suggest a cross-signalling between L-type Ca2+ channels and ryanodine receptors that controls the amount of Ca2+ influx during neuronal activity. [ABSTRACT FROM AUTHOR]

Abstract Rhythmic low and high frequency activity in thalamocortical networks depend critically on activation of low- and high-voltage-activated (LVA, HVA) Ca2+ currents. In order to test whether Ca2+ currents are modified during repetitive activation, acutely isolated thalamocortical relay neurons of rats, at postnatal days 12 (P12) to P20, were investigated using patch-clamp, Ca2+ imaging and Western blot techniques. High-voltage-activated, but not LVA Ca2+ currents were reduced significantly during 2 Hz stimulation. Ca2+ imaging experiments demonstrated a close correlation between the increase in intracellular Ca2+ levels and the decrease in HVA Ca2+ current amplitudes. Further examination of HVA Ca2+ currents revealed a ‘U-shaped’ inactivation curve and a time-dependent inactivation process that could be described by a two-exponential function. The ‘U-shape’ was significantly reduced, current amplitude was increased significantly and time-dependent inactivation revealed a one-exponential decline with Ba2+ as the charge carrier, following activation of the cAMP/PKA pathway, and following application of phosphatase inhibitors (ascomycin, calyculin A). Western blot analysis and the effect of ascomycin indicated an involvement of calcineurin in the inactivation process. Isolation of HVA Ca2+ current components by subtype-specific blockers revealed that changes in time-dependent inactivation, inactivation curve and current amplitude were carried mainly by L-type and N-type Ca2+ currents. Furthermore, Ca2+ -dependent inactivation was operative during stimulation protocols mimicking tonic action potential firing. These data indicate a modulation of L- and N-type Ca2+ channels by phosphorylation, resulting jointly in an increased intracellular Ca2+ influx during activity of the ascending... [ABSTRACT FROM AUTHOR]