Molecular Physiology of Sodium-Calcium Exchange

(0609) The retinal rod Na-Ca,K exchanger

Paul Schnetkamp

Hi Paul:

I am not sure that our finding of a potassium-dependence for the NCKX1/Bvdel construct, or as for that matter the full-length dolphin NCKX1, is surprising. The various NCKX1/2 clones in Jonathan Lytton's lab or in my own lab that give functional expression are all potassium-dependent as expected for the Na-Ca+K exchanger. I think Neil Cook's work has also shown the Na-Ca+K exchanger purified from bovine ROS is clearly potassium-dependent without any apparent need for additional factors. I cannot explain the observation of Navagnioni et al. and I think both Jonathan's work and our work confirm that expression of a single polypeptide coded for by NCKX1/2 cDNA yields potassium-dependent Na-Ca exchange without need for additional factors. The only thing we have come across in our transfection experiments with HEK293 cells is the occurrence of false positives, i.e. sometimes HEK cells show some endogenous activity that looks like Na-Ca exchange.

The full-length bovine NCKX1 cDNA can give nice protein expression in a variety of expression systems (in Sf9 cells transfected with full-length bovine NCKX1 even approaching the amounts found in ROS), but we have never observed any functional activity in any cell system we tried. In contrast, expression of the full-length dolphin NCKX1 cDNA yields very strong functional activity; we were able to observe functional activity when deleting the 114 amino acids from the bovine sequence as shown in our presentation here. When we replace the cytosolic loop in the dolphin NCKX1 with the cytosolic loop of the bovine, we observe a large amount of protein, but no functional activity. In this case, there is clearly no correlation between amount of protein and functional activity. I guess it is perhaps unclear what the deeper significance of this all is. However, in practical terms we have NCKX1 cDNAs that give very good functional activity and which will be very helpful in future studies.

[ This message was edited on Wed Dec 9 by the author ]

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