Neurons And Into The Muscle Cell Membrane Na + Channel Regulation And Its Mechanism Of Ceramide

Sphingolipid is one of the major components of eukaryote membrane,and also a major ingredient of lipid raft.Ceramide is a multi-fuctional second messager as a motabolin of sphingolipid,it can alter the lipid environments of cell membrane. Ceramide can induce cell apoptosis or affect cell cycle through modulating the intracellular signal pathway.Besides,ceramide and its analogs modulate K⁺ and Ca²⁺ channels directly in many cell types.While it's modulation on Na⁺ channels is seldom reported.Our research use two cell types,the rat cerebellar granule neuron and myoblast. With the method of whole-cell patch clamp recording,we study the modulation mechanism of C₆-ceramide on voltage-dependent inward Na⁺ channels in these two cell types in vitro.NaV_1.2,which is the most prevalent subtype of Na⁺ channels in cerebellar granule cell,locates in membrane lipid raft.We find this modulation is associated with membrane lipid raft.Disruption of lipid raft results in loss of modulation of C₆-ceramide significantly.Our results suggest that C₆-ceramide inhibits I_Na of cerebellar granule neuron rapidly and reversibly in a dose-dependent manner between 0.01-1μM,dose-independent manner when the concentration is higher, without altering its steady-state activation and inactivation.C₂-ceramide,an active analog of C₆-ceramide,shows a similar inhibitory effect on I_Na of granule neuron, while its inactive analog,dihydro-C₆-ceramide,fails to mimik the effect on I_Na. Application of caffeine(1 mM) to the bath solution could stimulate release of Ca²⁺ from the endoplasmic reticulum and this caused a rapid and marked reduction of I_Na, while intracellular infusion of 1,2-bis(2-aminophenoxy) ethane-N,N,N9, N9-tetraacetic acid(BAPTA),a fast Ca²⁺-chelating agent,vanishes the effect of C₆-ceramide on I_Na.infusion of ruthenium red,a ryonodine-sensitive Ca²⁺ receptor antagonist,into cerebellar granule cells results in gradual increase of I_Na amplitude and abolishes the inhibitory effect of C₆-ceramide on I_Na.Xestospongin C and heparin, potent IP₃ receptor antagonists,fail to affect I_Na.Intracellular infusion into rat myoblast with xestospongin C or heparin to block the release of Ca²⁺ from sarcoplasmic reticulum and/or endoplasmic reticulum also results in gradual increase of I_Na and abolishment of C₆-ceramide on I_Na,while rethenium red fails to mimik this effect.Further investigation with Ca²⁺ imaging suggests C₆-ceramide can increase intracellular Ca²⁺ concentration significantly.Intracellular infusion of GTPγS,a potent agonist of G protein,can reduce I_Na amplitude significantly in both cell types,the activation and inactivation curves are significantly shifted.GDPβS,a potent G protein inhibitor,abolished the effects of C₆-ceramide on I_Na.These results indicated that the inhibitory effects of C₆-ceramide on I_Na was a downstream reaction of the activation of G protein and phospholipase C (PLC) pathway and intracellular Ca²⁺ release.Ca²⁺ releases from ryanodine-sensitive receptor in cerebellar granule neurons and IP₃ receptor in myoblasts.Extracellular Ca²⁺ and membrane Ca²⁺ channels are not involved in this progress.And the apoptosis of cerebellar granule cells induced by C₆-ceramide is not associated with Na⁺ channels.Our research suggests the downstream reactions of C₆-ceramide on cerebellar granule cell and myoblast,but how they interact with each other needs further investigation.