The structure of sarcoplasmic reticulum membranes was studied in the presence of modeled transmembrane Ca2+ gradient corresponding to the status of Ca2+ depot at different stages of the muscle contraction-relaxation cycle in health and disease. Various sites of the membrane were characterized using spectral analysis of tryptophan, pyrene, and merocyanine-540 fluorescence without evaluating specific changes in the molecules of membrane components (Ca2+-ATPase, ryanodine receptor, and lipids). The transmembrane Ca2+ gradient modulates the protein-lipid interactions and structural characteristics of the membrane. The proposed model can be used for studies of the effects of pharmacologically active substances and endogenous regulators.