CAVEOLIN-1 EXPRESSION AND MEMBRANE CHOLESTEROL CONTENT MODULATE N-TYPE CALCIUM CHANNEL ACTIVITY IN NG108-15 CELLS

¹ Universitá di Pavia
² Universitá di Milano-Bicocca

^* To whom correspondence should be addressed. E-mail: mtoselli{at}unipv.it.

Submitted on May 5, 2005
Revised on June 5, 2005
Accepted on 1 July 2005

	Abstract

Caveolins are the main structural proteins of glycolipid/cholesterol-rich plasmalemmal invaginations, termed caveolae. In addition, caveolin-1 isoform takes part in membrane remodelling as it binds and transports newly synthesized cholesterol from endoplasmic reticulum to the plasma membrane. Caveolin-1 is expressed in many cell types, including hippocampal neurons, where an abundant SNAP25-caveolin-1 complex is detected after induction of persistent synaptic potentiation. To ascertain whether caveolin-1 influences neuronal voltage-gated Ca2+ channel basal activity, we stably expressed caveolin-1 into transfected neuroblastoma x glioma NG108-15 hybrid cells [cav1(+) clone], that lack endogenous caveolins but express N-type Ca2+ channels upon cAMP-induced neuronal differentiation. Whole-cell patch-clamp recordings of cav1(+) cells evidenced that N-type current density was size reduced of about 70% without any significant change in the time courses of activation and inactivation and voltage-dependence. Moreover, the cav1(+) clone exhibited a significantly increased proportion of membrane cholesterol compared to wild type NG108-15 cells. To gain insight on the mechanism underlying caveolin-1 lowering of N-current density and more precisely to test whether this was indirectly caused by caveolin-1-induced enhancement of membrane cholesterol, we compared single N-type channel activities in cav1(+) clone and wild type NG108-15 cells, enriched with cholesterol following exposure to a methyl--cyclodextrin-cholesterol complex. A lower Ca2+ channel activity was recorded from cell-attached patches of both cell types, thus supporting the view that the increased proportion of membrane cholesterol is ultimately responsible for the effect. This is due to a reduction in the probability of channel opening caused by a significant decrease of channel mean open time and by an increase of the frequency of null sweeps.

Key Words: calcium channel, caveolae, caveolin, cholesterol, lipid rafts, methyl-beta-cyclodextrin

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