The Regulation And Mechanism Of Intracellular Mg2+ On Cav1.2 Channels

Purpose:Magnesium ions as natural calcium antagonists,ischemic stroke,a large number of L-type calcium channel open,calcium overload and triggered a series of harmful metabolic reactions are the key factors that lead to neuronal injury and death,magnesium or calcium channel blockers can have very good protection effect on neurons.We can hypothesize that in ischemic stroke,magnesium may play its protective role in neurons by blocking the opening of l-type calcium channels.Intracellular Mg2+has been reported to regulate cardiac Cav1.2 channel.Come so far,the mechanism of the regulation of Mg2+is not clear.Mg2+,a divalent ion,competing with Ca2+for EF hand either in CaM or Cav1.2 channel may modulate dynamic CaM interaction with Cav1.2channel which are previously suggested to be the determinant of channel activity.The purpose of this paper is to study the regulation and mechanism of intracellular Mg2+on Cav1.2 channels,and to explore the possible regulatory mechanisms of magnesium in the treatment of ischemic cerebral apoplexy.Methods:Respectively using transgenic technology can express CaM and Cav1.2channel near the c-terminal fragment of e.coli（CT1）,CaM is obtained by cell culture,protein extraction and curing of Cav1.2 channel near the c-terminal fragment（CT1）,GST-CT1.In this paper,the binding of CaM and Cav1.2 channel near C end（CT1）was set up to study the influence of Mg2+on the dynamic combination of CaM and Cav1.2channels.Then the calcium ion binding sites on CaM or CT1 were respectively mutated,and CaM₁₂₃₄234 and CT1B of the mutant protein were obtained.Respectively set up CaM and CT1,CT1B contrast test and CaM₁₂₃₄234 and CT1,CT1B contrast test,the mutation off on CaM or CT1 EF hand calcium binding site,the regulating role of magnesium 2+is affected.The fluorescence intensity changes of Ca2+or Mg2+with CaM or CaM₁₂₃₄234 were compared under different concentration gradients.The fluorescence intensity represents the complexity of the conformational changes that can cause CaM or CaM₁₂₃₄,and the experimental results are in accordance with the pull-down experiment。Result:We found,in the low Ca2+condition（100nM）,Mg2+accelerated CaM binding at 1?M?100?M,and inhibited binding at 100?M?100mM,while in the high Ca2+condition（500?M）,Mg2+had no effect until 100mM of Mg2+which inhibited CaM binding.To our expectation,the result of CaM binding to CT1B（a short CT1 with EF hand truncated）showed a similar effect of Mg2+as that of CT1 binding,suggesting that the EF hand in the channel was not involved in Mg2+effect.Finally,mutation of all Ca2+-binding sites in CaM（CaM1234）abolished Mg2+effect on CaM binding.Discussion:We found,in the low Ca2+condition（100nM）,the regulation of Mg2+has a bidirectional effect on the activity of Cav1.2 channel.Mg²⁺accelerated CaM binding at1?M?100?M,and inhibited binding at 100?M?100mM,while in the high Ca²⁺condition（500?M）,Mg²⁺had no effect until 100mM of Mg²⁺which inhibited CaM binding.These results suggest that internal Mg2+regulates Cav1.2 channel might through modulating dynamic CaM interaction with the channel at physiological and pathophysiological condition,the EF hand in the channel was not involved in Mg²⁺effect.