Na+-Ca2+-K+ currents measured in insect cells transfected with the retinalcone or rod Na+-Ca2+-K+ exchanger cDNA

The recently cloned retinal cone Na+-Ca2+-K+ exchanger (NCKX) was expressed in cultured insect cells, and whole-cell patch clamp was used to measure t ransmembrane currents generated by this transcript and compare them with cu rrents generated by retinal rod NCKX or by a deletion mutant rod NCKX from which the two large hydrophilic loops were removed. We have characterized t he ionic currents generated by both the forward (Ca2+ extrusion) and revers e (Ca2+ influx) modes of all three NCKX proteins. Reverse NCKX exchange gen erated outward current that required the simultaneous presence of both exte rnal Ca2+ and external K+. Forward NCKX exchange carried inward current wit h Na+, but not with Li+ in the bath solution. The cation dependencies of th e three NCKX tested (external K+, external Na+, internal Ca2+) were very si milar to each other and to those reported previously for the in situ rod NC KX. These findings provide the first electrophysiological characterization of cone NCKX and the first electrophysiological characterization of potassi um-dependent Na+-Ca+ exchangers in heterologous systems. Our results demons trate the feasibility of combining heterologous expression and biophysical measurements for detailed NCKX structure/function studies.