engleski

Ion permeabilities in rat red blood cell plasma membrane vesicles depend on vesicle orientation

Ion permeabilities in rat red blood cell plasma membrane vesicles depend on vesicle orientation Background and purpose: In the mammalian red blood cells (RBC), the intracellular pH (pHi) is mainly maintained by anion (Cl--HCO3-) and cation (Na+-H+) antiports localized in the plasma membrane. Other possible mechanisms for H+ transport in RBC plasma membrane have been poorly investigated. In this work we used rat RBC ghosts and plasma membrane vesicles to study the intrinsic conductances for protons (H+) and potassium (K+) and to characterize Na+-H+ antiport. These vesicles can be prepared in right-side-out (ROV) and inside-out (IOV) orientation. The model offers possibility to test differences in permeability for various ions and to study kinetic characteristics of the ion transport in right-side-out and inversely oriented plasma membranes. Material and methods: Sealed IOV and ROV were prepared by the established methods from the rat RBC gosts. The vesicle orientation was estimated from the activity of cholinesterase in untreated and detergent-treated vesicle preparations. H+ and K+ transport mechanisms were studied by the quench method of acridine orange fluorescence. Results: We confirmed the presence of an Na+(Li+)-H+ exchange in the RBC ghosts and membrane vesicles. The exchanger exhibited an H+ modifier site at the cytoplasmic side of the membrane and similar Vmax and Km for Na+ from both sides of the membrane. The exchanger activity was unaffected by dicyclohexylcarbodiimide and N-ethylmaleimide. Amiloride, however, inhibited it asymmetrically ; the Ki for amiloride in IOV was 2-3-fold higher than in ROV and RBC ghosts. Both RBC ghosts and membrane vesicles exhibited intrinsic H+ and K+ conductances. By using selective ionophores we found that the H+ conductance in IOV was 4-5 times higher then in ROV, whereas the K+ conductance in ROV was twice as high as that in IOV. The conductances for H+ and K+ in ROV matched those in the sealed RBC ghosts. Conclusion: The pattern of Na+-H+ exchange and ion conductances in selaed RBC ghosts and ROV is similar and may reflect the situation in vivo. Compared with the data in ROV and RBC ghosts, the IOV are more conductive for H+ and less conductive for K+. The differences in ion conductances between the IOV and ROV may result from different fluidity of the membranes due to rearrangement of membrane phospholipids generated during vesicle formation.

ion permeability; membrane vesicles; red blood cell; plasma membrane; rat

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