髓袢升支粗段细胞中的钠/钾/2氯共转运：动力学与布美他尼结合

Renal Section, Veterans Affairs Medical Center, Bronx, New York.

Biochim Biophys Acta. 1993 Nov 7;1152(2):289-99. doi: 10.1016/0005-2736(93)90260-7.

We examined the properties of Na+/K+/2Cl- cotransport in cultured mouse mTAL cells with respect to its kinetics, the contribution of K/K exchange to K fluxes mediated by the cotransporter, and [3H]bumetanide binding and turnover numbers in media with varying osmolality. The addition of bumetanide, the replacement of external Na+ or the replacement of external Cl- resulted in an almost identical (approx. 50%) decrease in K+ influx, suggesting that Na(+)-dependent, Cl(-)-dependent, BS K+ influx was a measure of Na+/K+/2Cl- cotransport. The kinetics of the BS K+ influx revealed a high affinity for external Na+ (apparent Km 7 mM) and external K+ (apparent Km 1.3 mM), but a very low affinity for external Cl- (apparent Km 67 mM with a two-site model). Of interest was the finding that none of the K+ (86Rb+) efflux was sensitive to bumetanide, suggesting the absence of cotransport mediated K/K exchange in this cell type. Specific [3H]bumetanide binding was a saturable function of free bumetanide concentration with a Kd of 0.20 microM and maximum binding (Bmax) of 0.63 pmol/mg, or about 53,000 sites per cell. Simultaneous transport and bumetanide binding assays yielded a turnover number of 255 min-1. The omission of external Na+, K+ or Cl- reduced specific [3H]bumetanide binding to values indistinguishable from zero. Changing medium osmolarity resulted in a co-ordinate change in BS K+ influx and bumetanide binding, with a monotonic increase in both transport and bumetanide binding with increase in osmolality from 200 to 400 mosmol/kg. About 85% of the cotransporter sites were located on the apical side, as in the intact mTAL tubule. The simultaneous measurement of BS ion transport and [3H]bumetanide binding in the mTAL cell may provide valuable insights into the regulation of Na+/K+/2Cl- cotransport in this nephron segment.

我们研究了培养的小鼠髓袢升支粗段（mTAL）细胞中Na⁺/K⁺/2Cl⁻共转运体的特性，包括其动力学、K⁺/K⁺交换对由该共转运体介导的K⁺通量的贡献，以及在不同渗透压的培养基中[³H]布美他尼结合和周转数。添加布美他尼、替换细胞外Na⁺或替换细胞外Cl⁻均导致K⁺内流几乎相同（约50%）的降低，这表明依赖Na⁺、依赖Cl⁻的基底侧K⁺内流是Na⁺/K⁺/2Cl⁻共转运的一种度量。基底侧K⁺内流的动力学显示对细胞外Na⁺（表观Km为7 mM）和细胞外K⁺（表观Km为1.3 mM）具有高亲和力，但对细胞外Cl⁻具有非常低的亲和力（双位点模型的表观Km为67 mM）。有趣的是，发现没有K⁺（⁸⁶Rb⁺）外流对布美他尼敏感，这表明在这种细胞类型中不存在共转运介导的K⁺/K⁺交换。特异性[³H]布美他尼结合是游离布美他尼浓度的饱和函数，Kd为0.20 μM，最大结合量（Bmax）为0.63 pmol/mg，即每个细胞约53,000个位点。同时进行转运和布美他尼结合测定得出周转数为255 min⁻¹。省略细胞外Na⁺、K⁺或Cl⁻会使特异性[³H]布美他尼结合降低到与零无显著差异的值。改变培养基渗透压会导致基底侧K⁺内流和布美他尼结合发生协同变化，随着渗透压从200 mosmol/kg增加到400 mosmol/kg，转运和布美他尼结合均单调增加。与完整的髓袢升支粗段小管一样，约85%的共转运体位点位于顶端侧。在mTAL细胞中同时测量基底侧离子转运和[³H]布美他尼结合可能为该肾单位节段中Na⁺/K⁺/2Cl⁻共转运的调节提供有价值的见解。