Calcium-dependent inactivation of neuronal calcium channel currents is independent of calcineurin.
Dephosphorylation by the Ca2+/calmodulin-dependent phosphatase calcineurin has been suggested as an important mechanism of Ca2+-dependent inactivation of voltage-gated Ca2+ channels. We have tested whether calcineurin plays a role in the inactivation process of two types of high-voltage-activated Ca2+ channels (L and N type) widely expressed in the central nervous system, using the immunosuppressive drug FK506 (tacrolimus), which inhibits calcineurin after binding to intracellular FK506 binding proteins. Inactivation of L- and N-type Ca2+ channels was studied in a rat pituitary tumor cell line (GH3) and chicken dorsal root ganglion neurons, respectively. With the use of antisera directed against the calcineurin subunit B and the 12,000 mol. wt binding protein, we show that both proteins are present in the cytoplasm of GH3 cells and chicken dorsal root ganglion neurons. Ionic currents through voltage-gated Ca2+ channels were investigated in the perforated-patch and whole-cell configurations of the patch-clamp technique. The inactivation of L- as well as N-type Ca2+ currents could be well fitted with a bi-exponential function. Inactivation was largely reduced when Ba2+ substituted for extracellular Ca2+ or when the Ca2+ chelator EGTA was present intracellularly, indicating that both types of Ca2+ currents exhibited Ca2+-dependent inactivation. Extracellular (perforated-patch configuration) or intracellular (whole-cell configuration) application of FK506 to inactivate calcineurin had no effect on the amplitude and time-course of Ca2+ channel current inactivation of either L- or N-type Ca2+ channels. In addition, we found that recovery from inactivation and rundown of N-type Ca2+ channel currents were not affected by FK506. Our results provide direct evidence that the calcium-dependent enzyme calcineurin is not involved in the inactivation process of the two Ca2+ channel types which are important for neuronal functioning, such as gene expression and transmitter release.
Zeilhofer
H U
HU
Institut für Experimentelle und Klinische Pharmakologie und Toxikologie, Erlangen, Germany.
Blank
N M
NM
Neuhuber
W L
WL
Swandulla
D
D
D000818:Animals; D019703:Calcineurin / Q000378:metabolism / Q000502:physiology*; D002118:Calcium / Q000502:physiology*; D015220:Calcium Channels / Q000502:physiology*; D020746:Calcium Channels, L-Type / Q000502:physiology; D020864:Calcium Channels, N-Type / Q000502:physiology; D002642:Chick Embryo; D004553:Electric Conductivity; D020104:Immunophilins / Q000378:metabolism; D009474:Neurons / Q000378:metabolism*; D051381:Rats; D022021:Tacrolimus Binding Proteins; D014018:Tissue Distribution; D014407:Tumor Cells, Cultured
D015220:Calcium Channels; D020746:Calcium Channels, L-Type; D020864:Calcium Channels, N-Type; D019703:Calcineurin; D022021:Tacrolimus Binding Proteins; D020104:Immunophilins; D002118:Calcium