Na(+)-Ca2+,K+ exchange in bovine retinal rod outer segments: quantitative characterization of normal and reversed...

Ca2+ homeostasis of bovine retinal rod outer segments is maintained through Na(+)-Ca2+,K+ exchangers and cGMP-gated channels in the plasma membrane. It has recently been demonstrated that both proteins are associated. This novel finding allowed us to investigate quantitatively normal and reversed mode Na(+)-Ca2+,K+ exchange in rod outer segment membrane vesicles and reconstituted proteoliposomes both containing exchangers in rightside-out and inside-out orientations. Addition of Na+ activated both normal and reversed mode exchange; if, however, initially Ca2+ from vesicles containing inside-out oriented exchangers has been released by activation of the associated channels, only normal mode exchange was observed upon addition of Na+. Using this approach, the fractions of vesicles containing rightside-out and inside-out oriented exchangers were about similar in these vesicle preparations. Normal and reversed mode exchange had similar Na+ concentrations of about 70 mM for half maximal activation (in the presence of 115 mM K+) and cooperativity parameters, nHill, of about 2.0. Furthermore, both modes were electrogenic, and showed only little Na(+)-Ca2+,K+ exchange in the absence of K+. The two modes of exchange differed, however, in the maximal exchange rate, the normal mode being about twice as fast as the reversed mode.