Kinetics and Energetics of Mg2+,ATP-Dependent Ca2+Transport in the Plasma Membrane of Smooth Muscle Cells

Abstract

Calcium ions play a crucial role in the excitation/contraction coupling in smooth muscles. I would like to interpret the biochemical mechanisms underlying Ca2+ exchange and dynamics of such an exchange in the smooth muscles. Particular emphasis is laid on the examination of kinetic, energetic, and catalytic properties of the membrane-linked energy-dependent Ca2+-transporting systems involved in regulation of the intracellular Ca2+ concentration in smooth muscle cells (SMC). It was suggested that the Mg2+,ATP-dependent plasma membrane calcium pump (Ca2+,Mg2+-ATPase) plays a key role in regulation of the Ca2+ concentration in SMC. The purpose of this review is to analyze some of our own results concerning kinetic, energetic, and catalytic properties of the calcium pump of the SMC plasma membrane. In our experiments, we used different biochemical models (namely, fractions of the membrane subcellular structures, highly purified Ca2+,Mg2+-ATPase of the SMC plasma membrane solubilized and reconstituted in the lyposomes, and suspension of digitonin-treated SMC) and a number of methods (including preparative biochemistry, enzymology, membranology, tracer 45Ca2+ flux analysis, and chemical and enzymological kinetics). We have shown that sodium azide-insensitive Mg2+,ATP-dependent Ca2+ accumulation in ureter smooth muscle microsomes is determined by two components. One component represents the Mg2+,ATP-dependent calcium pump of the sarcoplasmic reticulum functionally potentiated by Ca2+-precipitating permeating anions, oxalate or phosphate and inhibited by thapsigargin or cyclopiazonic acid, the highly selective inhibitors of the calcium pump of sarco(endo)plasmic rerticulum. Another component represents the Mg2+,ATP-dependent calcium pump of the plasma membrane functionally potentiated by phosphate. This pump is not inhibited by thapsigargin and cyclopiazonic acid. The effects of temperature, dielectric permeability (D), and ionic strength on the activity of purified Ca2+,Mg2+-ATPase solubilized from the myometrial sarcolemma were studied. The results suggest that changes in the polarity of the incubation medium markedly affect the activity of transport Ca2+,Mg2+-ATPase, and electrostatic interactions between the enzyme activity center and specific ligands (Mg·ADP-, in particular) significantly contribute to the energetics of ATP hydrolysis. Therefore, our data show that changes in the incubation medium polarity significantly affects the ATP-hydrolase activity of Ca2+,Mg2+-ATPase solubilized from the SMC plasma membranes, and electrostatic interactions between the enzyme active sites and reactants (in particular, Mg·ADP-) contribute to a significant extent to the energetics of ATP hydrolysis. We cannot rule out that under physiological conditions the local D values of the myoplasm may differ from that of water, and, moreover, may change (especially near the membrane surface) depending on the metabolic level of SMC. We suppose that local changes in the cytoplasmic D value will affect the plasma membrane calcium pump and, consequently, the efficiency of control of intracellular Ca2+ homeostasis in smooth muscle. So, our biochemical models are suitable experimental objects for studying the kinetic, energetic, and catalytic properties of the Mg2+,ATP-dependent calcium pump of the SMC plasma membrane. In addition, our data might be useful for screening of the mechanisms underlying the action of different physico-chemical factors involved in modulation of the contraction/relaxation cycle.