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

Abstract

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 microM) 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.

Impaired Regulation of Thalamic Pacemaker Channels through an Imbalance of Subunit Expression in Absence Epilepsy

“…Indeed, I h in TC neurons, which mostly express HCN2 and HCN4, possesses slower kinetics and higher cAMP sensitivity than, for instance, I h in CA1 pyramidal neurons, which mostly express HCN1 and HCN2 (Franz et al, 2000;Santoro et al, 2000). The following line of evidence indicates that the reduced cAMP sensitivity and faster kinetics of I h activation observed in individual TC neurons from WAG/Rij rats compared with ACI seem is correlated with the increase in HCN1: (1) HCN1 expression mostly localizes to parvalbumin-immunopositive cells in thalamic relay nuclei, representing the population of TC neurons (Jones and Hendry, 1989;Sieg et al, 1998;Meuth et al, 2005). (2) The selective alteration in HCN1 expression, with unaltered HCN2-4 expression, in thalamic relay nuclei from epileptic versus nonepileptic animals, and the unchanged passive membrane properties and I h current densities in the respective TC neurons, vote against more general influences, such as neuronal morphologies.…”

“…8 A, left). To confirm that HCN1 isoforms are expressed in TC neurons, in situ hybridization for HCN1 was combined with immunohistochemical labeling of parvalbumin, a TC neuron-specific cell marker in the dLGN of several species (Jones and Hendry, 1989;Sieg et al, 1998;Meuth et al, 2005). The large majority of parvalbuminpositive neurons in the dLGN of WAG/Rij were positive for HCN1, whereas parvalbumin-immunonegative HCN1-positive neurons were rarely detected (Fig.…”

Endogenous and Exogenous Ca2+ Buffers Differentially Modulate Ca2+-dependent Inactivation of CaV2.1 Ca2+ Channels

“…Infusion of PV and CB in thalamic relay neurons caused an inhibition of CDI of high voltage activated Ca 2ϩ current, although this effect was primarily on L-type channels (26). In addition, loss of CB from surviving granule cells isolated from the hippocampus of patients with mesial temporal lobe epilepsy correlated with increased CDI of voltage-gated Ca 2ϩ currents, which was restored by inclusion of CB in the recording electrode (25).…”

“…Similarly, PV and CB might physiologically regulate Ca 2ϩ -dependent modulation of Ca v 2.1, as PV and CB are concentrated in subsets of neurons, such as cerebellar Purkinje neurons, where Ca v 2.1 channels are also highly expressed (21)(22)(23)(24). Previous studies implicate a role for PV and CB in modulating CDI of L-type voltage-gated Ca 2ϩ channels in neurons (25,26), although whether these proteins also affect Ca v 2.1 (P/Q-type) channels is not known.…”

Differential Regulation of Endogenous N- and P/Q-Type Ca2+Channel Inactivation by Ca2+/Calmodulin Impacts on Their Ability to Support Exocytosis in Chromaffin Cells

“…Here we examined CDI of endogenous Ca V 2.1 and Ca V 2.2 channels in chromaffin cells. Because CaM-dependent CDI of Ca V 2 channels is sensitive to intracellular Ca 2ϩ chelation (Liang et al, 2003;Meuth et al, 2005;Kreiner and Lee, 2006), we used the perforated-patch configuration to maintain physiological buffering conditions. We show that endogenous Ca V 2.2 channels undergo prominent CDI through a CaM-dependent mechanism; Ca V 2.1 channels lack such modulation.…”