| IntroductionTwo opposing forms of autoregulatory feedback, Ca2 * - dependent facilitation and inactivation are displayed in L - type Ca + channel in response to increased intracellular Ca + concentration( [Ca + ];). Despite extensive biophysical analysis, the molecular basis of autoregulation remains unclear. Calmodulin (CaM) as a crucial Ca 2+ sensor,may mediate the two autoregulatory feedback. To confirm this hypothesis, we observed Ca + channel activity which was first potentiated and then suppressed after constantly superfusion of high - Ca solution. And study the effects of CaM and calmodulin kinase E ( CaMK H ) in the regulation of calcium - dependent facilitation and inactivation.Materials and Methods1. Preparation of single myocytesA guinea -pig (weight 400 -600g) was anaesthetized with sodium pento-barbitone (30 mg kg-1 i. p. ) , and the aorta cannulated in situ under artificial respiration. Single ventricular myocytes from guinea - pig hearts were dispersed by collagenase. The myocytes were kept at 4 C.2. Patch - clamp and data analysisSingle - channel currents were recorded in the cell - attached configuration using a patch - clamp amplifier (EPC -7, List, Darmstadt, Germany). Barium currents through the Ca 2+ 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. The mean current during the period 5 -105 ms after the onset of the test pulses ( I )was measured and divided by the unitary current amplitude ( i ) to yield NP0 ( since I = N x P0 x i ) , where N is the number of channels in the patch and P0 is the time - averaged open - state probability of the channels. A mean NP0 value was measured for 3 minutes just before changing to the high - Ca bath solution.3. [ Ca2 + ]; measurementCells on a glass coverslip were loaded with Fura - 2 by exposure to 2mM acetoxymethyl ester ( Fura -2/AM) in Ca + - free bath solution for Ih at 37 ^C. The coverslips were then mounted in a superfiision chamber and placed on the stage of an inverted microscope equipped with epifluorescence optics ( TE 2000 -U, Nikon, Japan). Cells were continuously superfused at a rate of 1 ml/min with Ca2+ - free bath solution for 3 min before changing to high - Ca2 + bath solution at 20 C through a polyethylene tube, the tip of which was placed 1-2 mm away from the cells.Cells were viewed with a 40 x Fluor objective lens ( Nikon) , the changes in fluorscence ratios at 340 and 380nm excitation wavelengths ( R340/380) were measured using Aquacosmos/Ratio (hamamatsu, Japan). The absolute value of [ Ca2 + ] i was calculated using the formula.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.ResultsSingle - channel currents were recorded in the cell - attached configuration. After seal formation, stability of channel was confirmed by recording unitary current 3 min in the absence of intervention. When control high - K + , Ca2 +- free bath solution was switched to high - K + , high - Ca2 + solution containing 2mM EGTA and 2.5 mM Ca2+ (calculated [ Ca2+ ] 0 about 500 M), NP0 was first increased ( Ca2+ - dependent facilitation). After raising [ Ca2 + ] 0 for 4. 89 1. 27 min (n = 14) , the effect was reversed to suppression ( Ca2+ - dependent inactivation). After using CPZ (1,10,100 M) (n =5 -9) , a CaM inhibitor,both facilitation and inactivation were inhibited dose - dependendy represented by highest and lowest normalized NP0, which was compared by dividing average NP0 each minute after changing to high - Ca solution by 3 - min average NP0 in Ca2 + -free solution. However, KN -62 (0. 1,1,3 M) (n =6) , a specific CaMK II inhibitor, could only delay the time to highest NP0 dose - dependendy without affecting the amplitude of highest an...
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