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

Sofia Fortalezas,Dorinda Marques-Da-Silva,Carlos Gutierrez-Merino

doi:10.3390/ijms19113667

Sofia Fortalezas, Dorinda Marques-Da-Silva + Show 1 more

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https://doi.org/10.3390/ijms19113667

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Abstract

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 ([Ca2+]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 [Ca2+]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.

Highlights

Cyclodextrins are used as additives to improve the aqueous solubility of poorly soluble drugs and increase their bioavailability [1], and they are being increasingly used in nanoparticle-based drug delivery [2]
It is shown that protein kinase A (PKA) and calmodulin-dependent protein kinase II (CaMK-II) associate with caveolin-1-rich lipid rafts in the plasma membrane of cerebellar granule neurons (CGNs) and that treatment with the lipid raft-disrupting agent MβCD largely decreases the phosphorylation level of the β2 subunit of L-type calcium channels (LTCCs) to levels observed in the presence of inhibitors of PKA and CaMK-II, and to those observed in the proapoptotic conditions attained by low potassium (5 mM) in the extracellular medium, an experimental condition that inactivates LTCCs [33]
The pretreatment of CGNs with MβCD and the removal of MβCD–cholesterol complexes formed results in a decrease in the steady-state cytosolic [Ca2+]i in the neuronal somas; the decrease is close to that induced by the LTCC blocker nifedipine, highlighting that MβCD treatment leads to a large functional inactivation of LTCCs, even in the partial depolarizing conditions elicited by 25 mM KCl in the extracellular medium

Summary

Introduction

Cyclodextrins are used as additives to improve the aqueous solubility of poorly soluble drugs and increase their bioavailability [1], and they are being increasingly used in nanoparticle-based drug delivery [2]. Cyclodextrins are used as anti-browning agents in different foods and foodstuffs [3]. A link between cyclodextrins and iatrogenic hearing loss has been noted in several species, including humans [4]. Methyl-β-cyclodextrin has been widely used for the selective removal of cholesterol and disruption of lipid rafts in different cell lines (see, e.g., [5,6]). The neurological risks of using methyl-β-cyclodextrin are informed by the reported toxicity of this compound to neuronal cell lines [7]. The molecular mechanism underlying selective neuronal cell death induced by methyl-β-cyclodextrin deserves to be studied

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