Caveolin-1 expression and membrane cholesterol content modulate N-type calcium channel activity in NG108-15 cells.
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 demonstrated that N-type current density was reduced in size by approximately 70% without any significant change in the time course 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 into 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 after exposure to a methyl-beta-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.
Toselli
M
M
Department of Cellular and Molecular Physiological and Pharmacological Sciences, and INFM (National Institute of Matter Physics), University of Pavia, Pavia, Italy.
Biella
G
G
Taglietti
V
V
Cazzaniga
E
E
Parenti
M
M
D000818:Animals; D020864:Calcium Channels, N-Type / Q000502:physiology*; D002460:Cell Line; D002784:Cholesterol / Q000235:genetics / Q000378:metabolism*; D015640:Ion Channel Gating / Q000502:physiology*; D007700:Kinetics; D008564:Membrane Potentials / Q000502:physiology*; D051379:Mice; D009474:Neurons / Q000378:metabolism*; D011994:Recombinant Proteins / Q000378:metabolism
D020864:Calcium Channels, N-Type; D011994:Recombinant Proteins; D002784:Cholesterol