Calcium Binding To The N-lobe Or C-lobe Of CaM Plays A Opposite Role In The Activity Of Cardiac L-type Calcium Channels

ObjectiveCalcium entering through the cardiac L-type calcium channels is an essential event for the contraction of the heart muscle in response to a depolarizing stimulus.Increases in intracellular calcium concentration modulate calcium current by engaging opposing processes of enhanced inactivation(reduces ICa,CDI),and facilitation(increases ICa,CDF).Calmodulin(CaM),serving as an ubiquitous Ca²⁺sensor,is very important for CDI and CDF of L-type calcium channels.the molecular details of he processes by which CaM performs such diverse functions are not clear."Run-down" is a unique charicterition of voltage-gated L-type calcium channels.The activity of L-type calcium channels decreases when the cytoplasmic side of the channels is perfused with an artifical intracellular solution.This phenomenon is called run-down and is most pronounced in cells dialysed internally in whole-cell recording or inside-out patches.So far,it has been suggested that wash-out of cytoplasmic factors is the main cause of run-down.So,studies on run-down are important for us to understand the regulation of channel activity.Calmodulin,bound to the carboxy-terminai tail ofα1c,can affect CDI and CDF of cardiac L-type calcium channels.The molecular details of the processes by which calmodulin performs such diverse functions are not clear.So,first,we purify the mutant calmodulin,and the,use wild type-calmodulin(WT-CaM),CaM12(CaM double mutant containing E31A and E67A that abolishes calcium binding to the carboxy-terminal lobe of CaM)and CAM34 to investigate the effects of CaM on "run-down" of cardiac L-type calcium channels in inside-out mode of patch clamp in guinea pig ventricular myocytes.In order to find out which lobe of CaM(N-lobe and C-lobe)contribute to the two opposite processes(CDI and CDF).Methods1.Purification of CaM12 and CaM34CaM12 means CaM double mutant containing E31A and E67A that abolishes Ca²⁺ binding to the amino-terminal lobe of CaM.Use "QIAGEN Plasmid Purification Handbook" to purify pGEX-6P-3 plasmid that contains genes coding mutant CaM on calcium binding site-2.And than,cut the plasmid into two segments using Apa-I,add mutagenic primers(primer-1F and primer-1R that contain the desired mutation)to cycle mutant strand synthesis reaction. Digest the methylated,nonmutated parental DNA template with Dpn- I.Transform the circular,necked dsDNA into BL21(DE3)Competent Cells in 37℃overnight.Culture the clones and than check sequence.2.Preparation of Single MyocytesA guinea pig(weight 300-600g)was anaesthetized with sodium pentobarbitone(6.6mg/100g),and the aorta cannulated in situ artificial respiration. Single ventricular myocytes from guinea pig heart were dispersed by collagenase.The myocytes were stored at 4℃for using.3.Patch-clamp Record in Inside-out Mode and Data AnalysisSingle channel currents were recorded in inside-out configuration using a patch-clamp amplifier(AXOPATCH 200B).Barium currents through the calcium channels were elicited by depolarizing pulses from -70 to 0 mV with 200 ms duration at a rate of 0.5 Hz,recorded with a patch-clamp amplifier and fed to a computer at a sampling rate of 3.3 kHz.The capacity and leakage currents in the current traces were digitally subtracted.Single channel currents were recorded in cell-attatched mode for 2 minutes,than, lift up the pipette to make the channel appear run-down phenomenon for 1 minute,add drugs(CaM+ATP)immediately,wait for 20 minutes at least to see the recovery of calcium channel.The mean current during the 5-105 ms after the onset of the pulses(â… )was measured and divided by the unitary current amplitude(â…°)to yield NPo(I=N×Po×i),where N is the number of channels in the patch and Po is the time-averaged open-state probability of the channels.A mean NPo value was measured for 3 minutes as the standard of NPo.Data were presented as means±S.E.M.Student's t test was used to estimate statistical significance and a probability value(P)of less than 0.05 was considered to be significant.Results1.Results of MolecularbiologyPurify mutant CAM12 using QuikChange Site-Directed Mutagenesis Kit.Check the sequence of calmodulin after site-directed mutagenesis.The 91st site of the protein products was changed from A to C,correspondingly,the amino acid was changed from glutamic acid to alanine.Site-1 of calcium binding motif was mutant successfully. The 200th site of the protein products was changed from A to C,correspondingly,the amino acid was changed from glutamic acid to alanine.Site-2 of calcium binding motif was also the mutant site.The rest alkali bases of CaM were corresponding to the WT-CaM.So,the purification of CaM12 was successful.SDS-PAGE analysis of CAM12 was done to check the purification and concentration of protein.The results show a high purification and concentration. Moreover,check the exact concentration of CaM12.Measure the absorbance at or near 595 nm with a plate reader(Infinite-200,Tacan,i-control),both for standard and sample. Prepare a standard curve by plotting the average Blank-corrected 595 nm measurement for each BSA standard vs.its concentration inμg/ml.The exact concentration of CAM12 was 0.73 mg/ml. 2.Results of Electrophisiology(1)Calcium- and dose-dependent effects of CAM12 on Cav1.2 channelsIn this study,CaM12(with 3raM ATP)can reactivate the channel activity after "run-down" in dose-dependence,especially for high CaM concentration(7μM)can reverse "run-down" obviously.Calcium-dependent effect of CAM12 was detected by using I-O solutions with different[Ca²⁺](0,250nM)to dissolve CaM.The channel activity also increases along by the increase of[CAM]with the existence of calcium.When increasing the[Ca²⁺],the dose-effect curve shifts to the left,that is,lower[CAM]can produce the same effect with high[Ca²⁺].(2)Calcium- and dose-dependent effects of CAM34 on Cav1.2 channelsIn this study,CaM34(with 3mM ATP)can reactivate the channel activity after "run-down" in "bell-shape" dose-dependence.Calcium-dependent effect of CAM34 was detected by using I-O solutions with different[Ca²⁺](0,250,500nM)to dissolve CAM34.The channel activity has no change by the increase of[Ca²⁺]in low CaM concentration,but decreases in high CaM concentration.That is,when increasing the[Ca²⁺],the activation phase of dose-effect curve has no change,wherever,the inactivation phase of dose-effect curve shifts to the left.ConclusionApoCaM and Ca²⁺/CaM both can facilitate and inactive cardiac L-type calcium channel.Calcium binding to the sites 1,2 of CaM N-lobe may promote the inactivation of L-type calcium channels.Calcium binding to the sites 3,4 of CaM C-lobe may promote the facilitation of L-type calcium channels.