Alterations Of L-type Calcium Channel Binding Proteins Of Rat Hippocampus After Transient Forebrain Ischemia

Stroke is a leading cause of death and disability. The loss of neurologicalfunctions following stroke is caused by massive loss of neurons resulting fromhypoxic-ischemic insults. The mechanism underling the brain ischemic injury isunclear and there is no effective clinical treatment yet. L-type Ca²⁺ channels areprevalent in dendrites and cell bodies of hippocampal pyramidal neurons,contributing 30-50% of total calcium current. In cultured hippocampal neurons, BayK8644, the agonist of L-type calcium channel protects neurons from ischemic celldeath. Specially, L-type Ca²⁺ channel can transduce calcium signal into nucleus andregulate the expression of a group of Ca²⁺-regulated genes, including those encodingc-Fos, brain-derived neurotrophic factor (BDNF), and Bcl-2, that have been shown tobe required and important for neuronal survival. Therefore, L-type calcium channelplays an important role in hippocampal neuronal death following brain ischemia, yetthe mechanism is still to be elucidated.L-type calcium channel mediates the calcium influx, thus the changes ofintracellular calcium signal regulate the cell function and activity. As the integralprotein of electrical and chemical signals, the direct protein-protein interaction canalso regulate the channel activities and cell functions, however, the mechanisms of which are still not known, especially in the downstream target and signaling pathways.It was reported that many protein kinases and phosphotases can interact with L-typecalcium channel, such as protein kinase A (PKA), protein kinase C (PKC), proteinphosphatase 1 (PP1) and src. Calmodulin (CAM) and calcium binding protein 1(CABP1), can competitively bind to C terminus ofα_1C subunit of L-type calciumchannel. Shank, neuronal interleukin-16 (NIL-16), rab interacting molecular (RIM),sorcin, and endophillin were also found to associate with L-type calcium channel,which can regulate synaptic transduction and communication between channels.However, there is no report on the L-type calcium channel binding proteins involvedin neuronal cell death until now.In the present study, we use C terminus ofα_1C subunit of L-type calcium channelas a bait to screen the L-type calcium channel binding proteins involved in neuronalcell death by GST-pull down, and attempt to find out the downstream signalingpathways underlying the ischemic neuronal cell death.Firstly, we design and synthesize a pair of specific primers of C terminus(1808-2139) based on theα_1C nucleotide sequence in GenBank, PCR productsamplified from pcDNA3/α_1C plasmid were inserted into pGEX-KG vector, aglutathione S-transferase (GST) fusion protein expression vector. The construction ofC terminus ofα_1C subunit and GST recombinant protein expression vector wasidentified by BamHâ… and Hindâ…¢double digestion and sequencing.The recombinant protein was expressed in E.coli BL21 induced byisopropyl-β-D-thiogalactoside (IPTG). A 70,000 KD protein was obtained andconfirmed by GST or C terminus ofα_1C subunit antibodies. The recombinant proteinwas purified by Sepharose 4B and three bands, with the molecular weight of 70 KD,40 KD, 30 KD respectively, were obtained in SDS-PAGE. All the three bands wererecognized by C terminus ofα_1C subunit antibody in Western blot, which is probably due to the presence of protein degradation.Finally, transient forebrain ischemia (15 min) was induced by the use of themodified 4-vessel occlusion method. After 24 hr reperfusion, hippocampus lysate wasincubated with either GST-α_1C or GST protein. The binding proteins were eluted andanalyzed by SDS-PAGE. Compare with sham group, there are three differential bandsin the ischemia group, which means 1) there are several proteins binding to Cterminus ofα_1C subunit in vitro; 2) the component of the L-type calcium channelbinding proteins changed after ischemia, and the differential proteins were presumedto participate in the ischemic neuronal cell death.All together, we successfully constructed the pGEX-KG/α_1C plasmid andpurified the GST-α_1C fusion protein. Moreover, we found the alterations of L-typecalcium binding proteins after transient forebrain ischemia. That means theinteraction of these proteins with L-type calcium channels may participate in theischemic neuronal cell death, which help to identify and study the role of L-typecalcium channel in the ischemic neuronal cell death.