CALCIUM TRANSIENTS ASSOCIATED WITH THE T-TYPE CALCIUM CURRENT IN MYOTUBES

Immature skeletal muscle cells, both in vivo and in vitro, express a high density of T type calcium current and a relatively low density of the dihydropyridine receptor, the protein thought to function as the I-show calcium channel and as the voltage sensor for excitation-contraction coupling. Although the role of the voltage sensor in eliciting elevations of myoplasmic, free calcium (calcium transients) has been examined, the role of the T type current has not. In this study we examined calcium transients associated with the T type current in cultured myotubes from normal and dysgenic mice, using the whole cell configuration of the patch clamp technique in conjunction with the calcium indicator dye Fluo-3. In both normal and dysgenic myotubes, the T type current was activated by weak depolarizations and was maximal for test pulses to similar to -20 mV. In normal myotubes that displayed T type calcium current, the calcium transient followed the amplitude and the integral of the current at low membrane potentials (-40 to -20 mV) but not at high potentials, where the calcium transient is caused by SR calcium release. The amplitude of the calcium transient for a pulse to -20 mV measured at 15 ms after depolarization represented, on average, 4.26 +/- 0.68% (n = 19) of the maximum amplitude of the calcium transient elicited by strong, 15-ms test depolarizations. In dysgenic myotubes, the calcium transient followed the integral of the calcium current at all test potentials, in cells expressing only T type current as well as in cells possessing both T type current and the L type current I-dys. Moreover, the calcium transient also followed the amplitude and time course of current in dysgenic myotubes expressing the cardiac, DHP-sensitive calcium channel. Thus, in those cases where the transient appears to be a consequence of calcium entry, it has the same time course as the integral of the calcium current. Inactivation of the T type calcium current with 1-s prepulses, or block of the current by the addition of amiloride (0.3-1.0 mM) caused a reduction in the calcium transient which was similar in normal and dysgenic myotubes. To allow calculation of expected changes of intracellular calcium in response to influx, myotubes were converted to a roughly spherical shape (myoballs) by adding 0.5 mu M colchicine to culture dishes of normal cells. Calcium currents and calcium transients recorded from myoballs were similar to those in normal myotubes. The calculated change in calcium due to influx through the T type channel disagreed with the measured calcium concentration, suggesting that additional calcium might be released from the SR or that calcium buffers in the pipette had not equilibrated with the interior of the cell.