Navanglone A, Rispoli G, Gabellini N, Carafoli E
Instituto Nazionale per la Fisica della Materia (INFM), Dipartimento di Biologia dell'Università, Ferrara, Italy.
Biophys J. 1997 Jul;73(1):45-51. doi: 10.1016/S0006-3495(97)78046-9.
The retinal Na+:Ca2+, K+exchanger cDNA was transiently expressed in human embryonic kidney (HEK 293) cells by transfection with plasmid DNA. The correct targeting of the expressed protein to the plasma membrane was confirmed by immunocytochemistry. The reverse exchange offrent (Ca2+ imported per Na+ extruded) was measured in whole-cell voltage-clamp experiments after intracellular perfusion with Na+ (Na+i, 128 mM) and extracellular perfusion with Ca2+ (Ca2o+, 1 mM) and Ko+ (20 mM). As expected, the exchange current was suppressed by removing Ca2o+. Surprisingly, however, it was also abolished by increasing Na+o to almost abolish the Na+ gradient, and it was almost unaffected by the removal of Ko+. Apparently, then, at variance with the exchanger in the rod outer segment, the retinal exchanger expressed in 293 cells acts essentially as a Na+:Ca2+ exchanger and does not require K+ for its electrogenic activity.
通过用质粒DNA转染，视网膜Na⁺:Ca²⁺、K⁺交换体cDNA在人胚肾（HEK 293）细胞中瞬时表达。通过免疫细胞化学证实了表达的蛋白质正确靶向到质膜。在用Na⁺（Na⁺i，128 mM）进行细胞内灌注以及用Ca²⁺（Ca²⁺o，1 mM）和K⁺（20 mM）进行细胞外灌注后，在全细胞电压钳实验中测量了反向交换比率（每排出一个Na⁺所摄入的Ca²⁺）。正如预期的那样，通过去除Ca²⁺o抑制了交换电流。然而，令人惊讶的是，通过增加Na⁺o几乎消除Na⁺梯度也使其消失，并且去除K⁺对其几乎没有影响。显然，与视杆外段中的交换体不同，在293细胞中表达的视网膜交换体基本上作为Na⁺:Ca²⁺交换体起作用，并且其电生活动不需要K⁺。
