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The activation of L-type calcium channels (LTCCs) prevents cerebellar granule neurons (CGNs) from entering low-K⁺-induced apoptosis. In previous works, we showed that LTCCs are largely associated with caveolin-1-rich lipid rafts in the CGN plasma membrane. In this work, we show that protein kinase A (PKA) and calmodulin-dependent protein kinase II (CaMK-II) are associated with caveolin-1-rich lipid rafts of mature CGNs, and we further show that treatment with the cholesterol-trapping and lipid raft-disrupting agent methyl-β-cyclodextrin decreases the phosphorylation level of the LTCC β₂ subunit and the steady-state calcium concentration in neuronal somas ([Ca 2+ ] i ) to values close to those measured in 5 mM KCl proapoptotic conditions. These effects correlate with the effects produced by a short (15 min) treatment of CGNs with H-89 and KN-93-inhibitors of PKA and CaMK-II, respectively-in 25 mM KCl medium. Moreover, only a 15 min incubation of CGNs with H-89 produces about a 90% inhibition of the calcium entry that would normally occur through LTCCs to increase [Ca 2+ ] i upon raising the extracellular K⁺ from 5 to 25 mM, i.e., from proapoptotic to survival conditions. In conclusion, the results of this work suggest that caveolin-1-rich lipid rafts play a major role in the control of the PKA- and CaMK-II-induced phosphorylation level of the LTCC β₂ subunit, thus preventing CGNs from entering apoptosis.

international journal of molecular sciences

Methyl-β-Cyclodextrin Impairs the Phosphorylation of the β2 Subunit of L-Type Calcium Channels and Cytosolic Calcium Homeostasis in Mature Cerebellar Granule Neurons