Rationale: The paper reports the cloning and functional characterization of NCX2 as a Ca2+/Na+ exchanger. The authors use 'inside out' patches from Xenopus oocytes expressing NCX2.
The patch pipette, facing the extracellular side, contains 8 mM Ca2+ and no Na+.
The external solution, facing the cytoplasmic side of the membrane patch, contains 100 mM cesium. Figure 5a shows that a current can be elicited when the cesium is replaced by Na+, in the presence of 1, or 3 microMol Ca2+ in the external solution. When no Ca2+ is present at the external solution, no current is elicited, indicating a requirement of Ca2+ at the cytoplasmic domain of the exchanger.
Figure 5b shows that when the Ca2+ is removed from the external solution, the current decays.
Figure 5d shows the IV curve for the exchanger.
The data presented indicates a function for NCX2 as a Na+/Ca2+ exchanger that is dependent on the ion gradients.. Experimental description: 'A rat genomic library (Stratagene) was then screened with the 0.6-kb probe. Four genomic clones were isolated and subcloned into pBluescript SK’. Sequence analysis showed that one genomic clone, G23-2, overlapped with the 5’ end of pRll and also had an apparent codon for the initiating methionine'.
* 'We were initially unable to express NCX2 by injection of cRNA into Xenopus oocytes. We thus used the same strategy which improved expression of NCXl by replacing the 3’-untranslated region of NCXl with that of the Na+-glucose cotransporter clone which includes a poly(A)+tail'.