Na(+)-Ca(2+)-K(+) currents measured in insect cells transfected with the retinal cone or rod Na(+)-Ca(2+)-K(+) exchanger cDNA.
The recently cloned retinal cone Na(+)-Ca(2+)-K(+) exchanger (NCKX) was expressed in cultured insect cells, and whole-cell patch clamp was used to measure transmembrane currents generated by this transcript and compare them with currents generated by retinal rod NCKX or by a deletion mutant rod NCKX from which the two large hydrophilic loops were removed. We have characterized the ionic currents generated by both the forward (Ca(2+) extrusion) and reverse (Ca(2+) influx) modes of all three NCKX proteins. Reverse NCKX exchange generated outward current that required the simultaneous presence of both external Ca(2+) and external K(+). Forward NCKX exchange carried inward current with Na(+), but not with Li(+) in the bath solution. The cation dependencies of the three NCKX tested (external K(+), external Na(+), internal Ca(2+)) were very similar to each other and to those reported previously for the in situ rod NCKX. These findings provide the first electrophysiological characterization of cone NCKX and the first electrophysiological characterization of potassium-dependent Na(+)-Ca(+) exchangers in heterologous systems. Our results demonstrate the feasibility of combining heterologous expression and biophysical measurements for detailed NCKX structure/function studies.
Sheng
J Z
JZ
Department of Physiology and Biophysics and the MRC Group on Ion Channels/Transporters, Faculty of Medicine, University of Calgary, Calgary, Alberta T2N 4N1, Canada.
Prinsen
C F
CF
Clark
R B
RB
Giles
W R
WR
Schnetkamp
P P
PP
D000595:Amino Acid Sequence; D000818:Animals; D055592:Biophysical Phenomena; D001703:Biophysics; D002352:Carrier Proteins / Q000235:genetics* / Q000378:metabolism*; D002417:Cattle; D002460:Cell Line; D002645:Chickens; D018076:DNA, Complementary / Q000235:genetics; D004289:Dolphins; D006801:Humans; D007313:Insecta; D017136:Ion Transport; D008969:Molecular Sequence Data; D018408:Patch-Clamp Techniques; D017949:Retinal Cone Photoreceptor Cells / Q000378:metabolism; D017948:Retinal Rod Photoreceptor Cells / Q000378:metabolism; D019831:Sodium-Calcium Exchanger; D014162:Transfection
D002352:Carrier Proteins; D018076:DNA, Complementary; D019831:Sodium-Calcium Exchanger; C074530:potassium-dependent sodium-calcium exchanger