Intramembrane charge movements and excitation– contraction coupling expressed by two-domain fragments of the Ca2+ channel

To investigate the molecular basis of the voltage sensor that triggers excitation–contraction (EC) coupling, the four-domain pore subunit of the dihydropyridine receptor (DHPR) was cut in the cytoplasmic linker between domains II and III. cDNAs for the I-II domain (α1S 1–670) and the III-IV domain (α1S 701-1873) were expressed in dysgenic α1S-null myotubes. Coexpression of the two fragments resulted in complete recovery of DHPR intramembrane charge movement and voltage-evoked Ca2+ transients. When fragments were expressed separately, EC coupling was not recovered. However, charge movement was detected in the I-II domain expressed alone. Compared with I-II and III-IV together, the charge movement in the I-II domain accounted for about half of the total charge (Qmax = 3 ± 0.23 vs. 5.4 ± 0.76 fC/pF, respectively), and the half-activation potential for charge movement was significantly more negative (V1/2 = 0.2 ± 3.5 vs. 22 ± 3.4 mV, respectively). Thus, interactions between the four internal domains of the pore subunit in the assembled DHPR profoundly affect the voltage dependence of intramembrane charge movement. We also tested a two-domain I-II construct of the neuronal α1A Ca2+ channel. The neuronal I-II domain recovered charge movements like those of the skeletal I-II domain but could not assist the skeletal III-IV domain in the recovery of EC coupling. The results demonstrate that a functional voltage sensor capable of triggering EC coupling in skeletal myotubes can be recovered by the expression of complementary fragments of the DHPR pore subunit. Furthermore, the intrinsic voltage-sensing properties of the α1A I-II domain suggest that this hemi-Ca2+ channel could be relevant to neuronal function.

Indirect coupling of phosphate release to de novo tension generation during muscle contraction.

A key question in muscle contraction is how tension generation is coupled to the chemistry of the actomyosin ATPase. Biochemical and mechanochemical experiments link tension generation to a change in structure associated with phosphate release. Length-jump and temperature-jump experiments, on the other hand, implicate phase 2slow, a significantly faster, markedly strain-sensitive kinetic process in tension generation. We use a laser temperature jump to probe the kinetics and mechanism of tension generation in skinned rabbit psoas fibers--an appropriate method since both phosphate release and phase 2slow are readily perturbed by temperature. Kinetics characteristic of the structural change associated with phosphate release are observed only when phosphate is added to fibers. When present, it causes a reduction in fiber tension; otherwise, no force is generated when it is perturbed. We therefore exclude this step from tension generation. The kinetics of de novo tension generation by the temperature-jump equivalent of phase 2slow appear unaffected by phosphate binding. We therefore propose that phosphate release is indirectly coupled to de novo tension generation via a steady-state flux through an irreversible step. We conclude that tension generation occurs in the absence of chemical change as the result of an entropy-driven transition between strongly bound crossbridges in the actomyosin-ADP state. The mechanism resembles the operation of a clock, with phosphate release providing the energy to tension the spring, and the irreversible step functions as the escapement mechanism, which is followed in turn by tension generation as the movement of the hands.