Direct evidence of Na+/Ca2+exchange in squid rhabdomeric membranes

Abstract

Na+/Ca2+exchange has been investigated in squid ( Loligo pealei) rhabdomeric membranes. Ca2+-containing vesicles have been prepared from purified rhabdomeric membranes by extrusion through polycarbonate filters of 1-μm pore size. After removal of external Ca2+, up to 90% of the entrapped Ca2+ could be specifically released by the addition of Na+; this finding indicates that most of the vesicles contained Na+/Ca2+exchanger. The Na+-induced Ca2+ efflux had a half-maximum value ( K 1/2) of ∼44 mM and a Hill coefficient of ∼1.7. The maximal Na+-induced Ca2+ efflux was ∼0.6 nmol Ca2+ ⋅ s−1 ⋅ mg protein−1. Similar Na+-induced Ca2+ effluxes were measured if K+ was replaced with Li+ or Cs+. Vesicles loaded with Ca2+ by Na+/Ca2+exchange also released this Ca2+by Na+/Ca2+exchange, suggesting that Na+/Ca2+exchange operated in both forward and reverse modes. Limited proteolysis by trypsin resulted in a rate of Ca2+ efflux enhanced by approximately fivefold when efflux was activated with 95 mM NaCl. For vesicles subjected to limited proteolysis by trypsin, Na+/Ca2+exchange was characterized by a K 1/2 of ∼25 mM and a Hill coefficient of 1.6. For these vesicles, the maximal Na+-induced Ca2+ efflux was about twice as great as in control vesicles. We conclude that Na+/Ca2+exchange proteins localized in rhabdomeric membranes mediate Ca2+ extrusion in squid photoreceptors.