Mechanism Regulating Kv2.1. Lipid Raft Composition And Role In Apoptosis

Kv2.1 is a major subunit contributing to the regulation of cell apoptosis in cerebellar granule neurons (CGNs), according to our previous work. Kv2.1 is reported to be located in the lipid raft, and the distribution of this channel, as well as the kinetic properties and current density, will be significantly changed by adding or depleting cholesterol in the lipid raft. Cholesterol is most abundant in the brain than other tissues. It is essential for maintaining brain function and a major component of lipid raft. In the first part of the paper, we study the effect of cholesterol on the apoptosis and Ik channels. We report here that cholesterol enrichment of the neuronal plasma membrane did not induce apoptosis of rat cerebellar granule neurons (CGNs) incubated with 25 mM K+/serum (HK+S) medium but significantly enhanced CGN susceptibility to apoptosis when incubated with 5 mM K+/serum free (LK-S) medium. Electrophysiological recordings, gel electrophoresis analysis and western blot analysis indicated that LK-S-induced Ik currents increased and DNA fragmentation appeared 4 hours earlier in the presence of cholesterol than without cholesterol; cholesterol-treated CGNs had upregulated Kv2.1 expression. Increased cAMP levels which co-localized with lipid rafts marker flotillin-1 were observed after treating CGNs with cholesterol-containing LK-S medium. Moreover, western blots showed that the activated form of PKA and phosphorylated CREB significantly increased after treating CGNs with cholesterol-containing LK-S medium for 4 hours. The PKA inhibitor H89 blocked this cholesterol-induced Kv2.1 upregulation. In addition, inhibiting PKA or Gs with a specific inhibitor eliminated the increase in Ik and removed the CGN susceptibility induced by LK-S and cholesterol enrichment. Our results demonstrate that elevation of membrane cholesterol enhances CGN susceptibility to apoptotic stimuli via cAMP/PKA/CREB-dependent upregulation of Kv2.1. This work provides new evidence as to how increased cholesterol elicits neuronal cell death or apoptosis.In the mammal cells, there are two kinds of lipid rafts:caveolae and non-caveolar rafts. Caveolin and flotillin are usually labeled as mark protein respectively. In the neurons, because of lack of caveolin and caveolae, signal molecules and ion channels are mainly located in the lipid raft with plenty of flotilins. Flotillin-1 is the major scaffolding protein in the non-caveolar rafts, and plays a key role in the forming of lipid rafts and signal transduction in the lipid rafts. In the second part of the paper, we study the interaction between the lipid raft protein-flotillin-1 and Kv2.1 channels. In this part, we report flotillin-1 has direct interaction with Kv2.1 either in HEK293 cells or CGNs by combination of immunofluorescence, Co-immunoprecipitation and bimolecular fluorescence complementation (BIFC) technique. In additional, flotillin-1 can modulate the kinetic properties of Kv2.1 slightly but change its distribution significantly in HEK 293 cells. Overexpressed flotillin-1 down-regulated the Kv2.1 level in HEK 293 cells and significantly decrease the Ik currents in CGNs with no changes of the kinetic properties of Ik currents. The proteasome inhibitor MG132 can significantly increases the protein level of Kv2.1 in HEK293 cells. These results suggest flotillin-1 modulates Kv2.1 in many aspects and the down-regulation of Kv2.1 via proteasome-mediated degradation maybe a major part in the modifications. In CGNs the interaction of flotillin-1 and Kv2.1 can be significant enhanced by LK-S treatment. It reveals the interaction between flotillin-1 and Kv2.1 may play an important role in neural apoptotic processes. More experiments should be performed to further clarify the mechanism of flotillin-1 modulating Kv2.1 functions, not only in physiology conditions but also in pathology conditions.