Cryosection-Elemental Analysis Studies on the Regulatory Mechanism of Contraction by Calcium bound to Inner Surface of Smooth Muscle Plasma Membrane

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To make clear the regulatory mechanism of contraction byCa ions which are nound to the inner surface of smooth muscle plasma membrane in the resting state, intracellular Ca localization and its translocation during the contraction-relaxation cycle in the anterior byssal retractor muscle (ABRM) of Mytilus edulis were studied by the electron probe X-ray microanalysis of cryosections. By the spot analyzes of inner surface of plasma membrane in the resting fibers, significant amounts of Ca (-8mmol/kg dry wt) were detected frequently, although in some cases the Ca concentration was negligible. The averaged Ca concentration at the inner surface of plasma membrane was approximately 3.0mmol/kg dry wt (n=25). On the other hand, the Ca concentration detected by the spot analyzes of myoplasm was low (-1.5mmol/kg dry wt). This result indicates that the Ca accumulation occurs actually at the inner surface of plasma membrane. In the spot analysis along the inner surface of plasma membrane, a siginificant amount of Ca is not always detected. This fact may be related to the distribution of Ca-binding protein (MCBP-450) isolated recently from the plasma membrane fraction of ABRM.Then, we further investigated the ultrastructural features of this Ca-binding protein. The low-angle rotary shadowing replicas of purified MCBP-450 showed that the protein is roughly ellipsoidal in shape. The dimensions measured by digital image processor were approximately 22 nm and 18 nm in long and short diameters respectively. Electron microscope observations of freeze-fracture and freeze-etching replicas showed that a part of some intramembranous particles, possibly the Ca-binding prtein, protrudes into the myoplasm at the inner surface of plasma membrane. Such a protein of molecules may play a role as Ca-binding sites, and release Ca during the membrane excitation. Further experiments to reveal the Ca translocation during contraction is now in progress.