Turnover rate and number of Na+-Ca2+, K+ exchange sites in retinal photoreceptors

TThe vertebrate photoreceptor exchanger plays an important role in phototransduction, since the sustained exchanger activity in the light induces [Ca2+]i fall, which in turn triggers several mechanisms taking part in dark state recovery and light adaptation of the photoreceptor. It has been found that the exchanger imports 4 Na+ ions for every Ca2+ and K+ ion extruded (forward mode of exchange), thereby accounting for one net positive charge imported per exchange cycle. The Ca2+ extrusion in vertebrate photoreceptors relies entirely on the density of exchange sites and on the number of Ca2+ ions transported per molecule per second. Several studies have attempted to quantify the density and the turnover number T of the photoreceptor exchanger, but large discrepancies were found in these estimates: the former ranged from 200-450 molecules/um2 to 3.5104 molecules/um2 while the latter ranged from 10 sec-1 to 115 sec-1. T can be estimated reliably by measuring the effect of a jump in the concentration of one or more of the transported ion(s) on the exchange current (recorded in situ). This approach would be particularly feasible if the ion transport was as slow as suggested by the above studies, and in particular if the entire reaction cycle was rate limited by one or more steps that can be forced to occur by manipulating the extracellular solution. This would allow to measure the step(s) kinetics (or T), no matter if the step is electrogenic or not, and to calculate the number of the exchanger molecules N by the equation: N=IE/(E0*T) where IE is the saturating exchange current and E0 the elementary charge. If the ions are not translocated simultaneously, then one would suspect that the reaction(s) implicated in the Ca2+ transport are slower than the ones implicated in the transport of Na+ and K+. In fact, the exchanger affinity for Ca2+ must change of more than 3 orders of magnitude in order to bind Ca2+ intracellularly (where its concentration is 500 nM at the most) and release it extracellularly (where its concentration is about 1 mM). It is reasonable to expect that this affinity change would require some time to be accomplished. Alternatively, the affinity for extracellular Ca2+ could remain elevated, but then the Ca2+ release at the extracellular side would take time to be completed. In the present work, the Ca2+ dependence of the exchanger reaction cycle was studied by recording the outer segment (OS) membrane current under whole-cell or excised-patch voltage-clamp conditions, while performing fast solution changes of the ions transported by the exchanger. The peculiar architecture of the OS, in which the entire transduction machinery, the cGMP channels and the exchanger are segregated, makes the patch clamp recording from mechanically isolated OS a powerful technique to study photoreceptor physiology. It was found that the turnover of photoreceptor exchanger is indeed rate limited by the Ca2+ transport but not by the Na+ or K+ transport and resulted in the extrusion of about 2-3 Ca2+ ions/s, which yields a density of about 104 molecules/um2.