Myosin light chain phosphorylation in vertebrate striated muscle: regulation and function.

The regulatory light chain of myosin (RLC) is phosphorylated in striated muscles by Ca2+/calmodulin-dependent myosin light chain kinase. Unique biochemical and cellular properties of this phosphorylation system in fast-twitch skeletal muscle maintain RLC in the phosphorylated form for a prolonged period after a brief tetanus or during low-frequency repetitive stimulation. This phosphorylation correlates with potentiation of the rate of development and maximal extent of isometric twitch tension. In skinned fibers, RLC phosphorylation increases force production at low levels of Ca2+ activation, via a leftward shift of the force-pCa relationship, and increases the rate of force development over a wide range of activation levels. In heart and slow-twitch skeletal muscle, the functional consequences of RLC phosphorylation are probably similar, and the primary physiological determinants are phosphorylation and dephosphorylation properties unique to each muscle. The mechanism for these physiological responses probably involves movement of the phosphorylated myosin cross bridges away from the thick-filament backbone. The movement of cross bridges may also contribute to the regulation of myosin interactions with actin in vertebrate smooth and invertebrate striated muscles.

Pre-steady-state kinetics of the activation of rabbit skeletal muscle myosin light chain kinase by Ca2+/calmodulin.

Myosin light chain kinase is activated by Ca2+/calmodulin. Insights into the kinetic mechanism of this activation by Ca2+/calmodulin have now been obtained using extrinsically labeled fluorescent calmodulin, a fluorescent peptide substrate, and a stopped-flow spectrophotofluorimeter. We employed spinach calmodulin labeled with the sulfhydryl-selective probe, 2-(4-maleimidoanilino)naphthalene-6-sulfonic acid, to measure changes in the fluorescence intensity of the 2-(4-maleimidoanilino)naphthalene-6-sulfonic acid-calmodulin upon binding to rabbit skeletal muscle myosin light chain kinase. The fluorescent peptide substrate KKRAARAC(sulfobenzo-furazan)SNVFS-amide was used to measure kinase activity. Our results showed that the binding interaction could be modeled as a two-step process: a bimolecular reaction with an association rate of 4.6 x 10(7) M-1 s-1 followed by an isomerization with a rate of 2.2 s-1. Phosphorylation of the peptide during stopped-flow experiments could be modeled by a two-step process with a catalytic association rate of 6.5 x 10(6) M-1 s-1 and a turnover rate of 10-20 s-1. Our results also indicated that kinase activity occurred too rapidly for the slower isomerization rate of 2.2 s-1 to be linked specifically to the activation process.

Cytoplasmic Ca2+ is a primary determinant for myosin phosphorylation in smooth muscle cells.

Initiation of smooth muscle contraction is associated with Ca2+/calmodulin activation of myosin light chain kinase which catalyzes the phosphorylation of the 20-kDa light chain of myosin. In tracheal smooth muscle cells in culture, the extent of myosin light chain phosphorylation is less than 10% at basal cytosolic free Ca2+ concentrations of 150 nM. Stimulation of these cells with serotonin, histamine, carbachol, or the Ca2+ ionophore, ionomycin, increases free cytosolic Ca2+ concentrations and the extent of myosin light chain phosphorylation. Light chain phosphorylation reaches a maximal value of 67% at Ca2+ concentrations below 1 microM. The relationship between the extent of light chain phosphorylation and cytosolic free Ca2+ concentration is apparently independent of the source of free intracellular Ca2+ or the agent used to stimulate the cells and is not altered by pre-exposure of the contractile apparatus to high concentrations of free Ca2+. Pretreatment of cells with 8-bromo-cyclic GMP or forskolin decreases free cytosolic Ca2+ concentrations and the extent of myosin light chain phosphorylation in response to histamine or ionomycin. Pretreatment with 8-bromo-cyclic GMP also decreases the maximal extent of light chain phosphorylation. These results indicate that cytosolic free Ca2+ concentration, per se, is a primary determinant for myosin light chain phosphorylation in tracheal smooth muscle cells.

Theory of the kinetic analysis of patch-clamp data.

This paper describes a theory of the kinetic analysis of patch-clamp data. We assume that channel gating is a Markov process that can be described by a model consisting of n kinetic states and n(n - 1) rate constants at each voltage, and that patch-clamp data describe the occupancy of x different conductance levels over time. In general, all the kinetic information in a set of patch-clamp data is found in either two-dimensional dwell time histograms describing the frequency of observation of sequential dwell times of durations tau 1 and tau 2 (Fredkin, D. R., M. Montal, and J. A. Rice, 1985, Proceedings of the Berkeley Conference in Honor of Jerzy Neyman and Jack Kiefer, vol. 1, 269-289) or in three-point joint probability functions describing the probability that a channel is in a given conductance at time t, and at time t + tau 1, and at time t + tau 1 + tau 2. For the special case of channels with a single open state plus multiple closed states, one-dimensional analyses provide all of the kinetic information. Stationary patch-clamp data have information that can be used to determine H rate constants, where H = n(n - 1) - G and G is the number of intraconductance rate constants. Thus, to calculate H rate constants, G rate constants must be fixed. In general there are multiple sets of G rate constants that can be fixed to allow the calculation of H rate constants although not every set of G rate constants will work. Arbitrary assignment of the G intraconductance rate constants equal to zero always provides a solution and the calculation of H rate constants. Nonstationary patch-clamp data have information for the determination of H rate constants at a reference voltage plus n(n - 1) rate constants at all test voltages. Thus, nonstationary data have extra information about the voltage dependencies of rate constants that can be used to rule out kinetic models that cannot be disqualified on the basis of stationary data.