Abstract
Purpose: :
Light–dependent changes in free Ca2+ in the outer segment cytoplasm critically determine the time course and sensitivity of the photocurrent in rods and cones. To understand the molecular mechanism of Ca2+ homeostasis in cones we investigated the functional and structural features of the K+–dependent Na+ /Ca2+ exchanger in intact single cone photoreceptors isolated from the striped bass retina. Further, we cloned several exchanger molecule (NCKX) isoforms from the bass retina and recognized them as specifically rod or cone in origin through single cell RT–PCR.
Methods: :
Membrane currents and cytoplasmic free Ca2+ concentration were measured in individual cells, either intact single bass cones or tsA–201 cultured mammalian cells. Currents were measured under voltage clamp using methods of whole–cell recording while Ca2+ was measured from the emission intensity of the fluorescent dye bis–Fura2 excited at 380 nm. The function of the Na+/Ca2+ exchanger was assessed by measuring current and Ca2+ under ionic conditions designed to drive the "reverse mode" of exchanger transport (Ca2+ inward flux/Na+ outward flux). A bass retinal cDNA library was prepared and various isoforms of the NCKX cloned using known rod and cone exchanger sequences. The NCKX isoforms expressed in cones were specifically identified using single–cell RT–PCR.
Results: :
Single and twin cones in the bass retina express a K+–dependent, Na+/ Ca2+ exchanger. When all other ionic currents are blocked in the cell, the exchanger drives a Ca2+ and K+–dependent outward current causing a large change in cytoplasmic Ca2+. The exchanger current amplitude depends on K+ as described by binding to a single site (I/Imax=[K+]/(Km+[K+]) (Ka=3.6 mM and Imax=57 pA). Exchanger Imax is 50 times larger than the exchanger current under normal conditions consistent with the notion that only about 2% of the exchangers are active in darkness. There are 5 NCKX isoforms in bass retina, one putative rod: bassNCKX1, and 4 putative cones: NCKX2.1, 2.2, 2.3 and 2.6. Single cell RT–PCR suggests the single cones express NCKX2.2 and 2.3. The clones expressed in tsA201 yield currents and changes in cytoplasmic Ca smaller in amplitude than in the intact cells, but of undistinguishable K+ and Ca2+ –dependence.
Conclusions: :
We cloned the rod and cone photoreceptor K+–dependent Na+ / Ca2+ exchangers from the striped bass retina and assessed their functional characteristics in intact single bass cones and heterologous expression system.
Keywords: ion transporters • electrophysiology: non-clinical • photoreceptors