Voltage-clamp analysis of membrane currents and excitation–contraction coupling in a crustacean muscle

A single fibre preparation from the extensor muscle of a marine isopod crustacean is described which allows the analysis of membrane currents and simultaneously recorded contractions under two-electrode voltage-clamp conditions. We show that there are three main depolarisation-gated currents, two are outward and carried by K+, the third is an inward Ca2+ current, ICa. Normally, the K+ currents which can be isolated by using K+ channel blockers, mask ICa. ICa activates at potentials more positive than −40 mV, is maximal around 0 mV, and shows strong inactivation at higher depolarisation. Inactivation depends on current rather than voltage. Ba2+, Sr2+ and Mg2+ can substitute for Ca2+. Ba2+ currents are about 80% larger than Ca2+ currents and inactivate little. The properties of ICa characterise it as a high threshold L-type current. The outward current consists primarily of a fast, transient A current, IK(A), and a maintained, delayed rectifier current, IK(V). In some fibres, a small Ca2+-dependent K+ current is also present. IK(A) activates fast at depolarisation above −45 mV, shows pronounced inactivation and is almost completely inactivated at holding potentials more positive than −40 mV. IK(A) is half-maximally blocked by 70 μM 4-aminopyridine (4-AP), and 70 mM tetraethylammonium (TEA). IK(V) activates more slowly, at about −30 mV, and shows no inactivation. It is half-maximally blocked by 2 mM TEA but rather insensitive to 4-AP. Physiologically, the two K+ currents prevent all-or-nothing action potentials and determine the graded amplitude of active electrical responses and associated contractions. Tension development depends on and is correlated with depolarisation-induced Ca2+ influx mediated by ICa. The voltage dependence of peak tension corresponds directly to the voltage dependence of the integrated ICa. The threshold potential for contraction is at about −38 mV. Peak tension increases with increasing voltage steps, reaches maximum at around 0 mV, and declines with further depolarisation.