PROPERTIES OF CALCIUM CURRENTS AND CONTRACTION IN CULTURED RAT DIAPHRAGM MUSCLE

The characterization of calcium currents and contraction simultaneousl y measured in cultured rat diaphragm muscle cells was carried out in t he present study. Whole-cell patch-clamp experiments were designed to further elucidate the mechanism of excitation-contraction (E-C) coupli ng in diaphragm which, though generally considered a skeletal-type mus cle, has been reported to exhibit properties indicative of a cardiac-l ike E-C coupling mechanism. Normalized current/voltage (I/V) curves fo r two concentrations of external calcium (2.5 and 5 mM) were obtained from diaphragm myoballs. Both curves showed peaks corresponding to the activation of a T-type calcium current and a dihydropyridine-sensitiv e L-type calcium current. The normalized curve for the voltage depende nce of the activation of contraction in diaphragm myoballs followed a typical Boltzmann-type relationship to the peak of contraction. Therea fter, the curve declined in a manner that was more pronounced in diaph ragm compared to that measured in additional experiments using culture d rat limb muscle myoballs. This effect could be interpreted in terms of a more pronounced participation of the L-type current in E-C coupli ng in cultured diaphragm muscle. An increased likelihood of cultured d iaphragm muscle to undergo depletion of sarcoplasmic reticular calcium stores during repetitive stimulation, or a heightened propensity for the voltage sensor for E-C coupling in diaphragm to enter the inactive state could also explain this effect. Maximal contractile activity wa s only slightly affected when the L-type current was blocked by extern ally applied cadmium (2 mM) or cobalt (3 mM), suggesting that a pronou nced calcium-current-dependent component of contraction is unlikely in cultured diaphragm muscle. These results show that T- and L-type calc ium channels are expressed in cultured rat diaphragm muscle cells and that, in contrast to cardiac muscle, the entry of calcium ions via L-t ype voltage-dependent calcium channels is not a prerequisite for contr action. Differences in the voltage sensitivity of contraction, observe d at depolarized membrane potentials in cultured rat diaphragm and lim b muscle cells, suggest that the voltage sensor for E-C coupling in di aphragm might more readily enter an inactivated configuration - possib ly by a mechanism which is dependent on the concentration of external calcium.