Davis B A, Hogan E M, Cooper G J, Bashi E, Zhao J, Boron W F
Department of Cellular and Molecular Physiology, Yale University School of Medicine, New Haven, CT 06520, USA.
J Membr Biol. 2001 Sep 1;183(1):25-32. doi: 10.1007/s00232-001-0050-0.
Previous squid-axon studies identified a novel K/HCO3 cotransporter that is insensitive to disulfonic stilbene derivatives. This cotransporter presumably responds to intracellular alkali loads by moving K(+) and HCO(3)(-) out of the cell, tending to lower intracellular pH (pH(i)). With an inwardly directed K/HCO(3) gradient, the cotransporter mediates a net uptake of alkali (i.e., K(+) and HCO(3)(-) influx). Here we test the hypothesis that intracellular quaternary ammonium ions (QA(+)) inhibit the inwardly directed cotransporter by interacting at the intracellular K(+) site. We computed the equivalent HCO(3)(-) influx (J(HCO3)) mediated by the cotransporter from the rate of pH(i) increase, as measured with pH-sensitive microelectrodes. We dialyzed axons to pH(i) 8.0, using a dialysis fluid (DF) free of K(+), Na(+) and Cl(-). Our standard artificial seawater (ASW) also lacked Na(+), K(+) and Cl(-). After halting dialysis, we introduced an ASW containing 437 mm K(+) and 0.5% CO(2)/12 mm HCO(3)(-), which (i) caused membrane potential to become transiently very positive, and (ii) caused a rapid pHi decrease, due to CO(2) influx, followed by a slower plateau-phase pH(i) increase, due to inward cotransport of K(+) and HCO(3)(-). With no QA(+) in the DF, J(HCO3) was approximately 58 pmole cm(-2) sec(-1). With 400 mm tetraethylammonium (TEA(+)) in the DF, J(HCO3) was virtually zero. The apparent K(i) for intracellular TEA(+) was approximately 78 mm, more than two orders of magnitude greater than that obtained by others for inhibition of K(+) channels. Introducing 100 mm inhibitor into the DF reduced J(HCO3) to approximately 20 pmole cm(-2) sec(-1) for tetramethylammonium (TMA(+)), approximately 24 for TEA(+), approximately 10 for tetrapropylammonium (TPA(+)), and virtually zero for tetrabutylammonium (TBA(+)). The apparent K(i) value for TBA(+) is approximately 0.86 mm. The most potent inhibitor was phenyl-propyltetraethylammonium (PPTEA(+)), with an apparent K(i) of approximately 91 microm. Thus, trans-side quaternary ammonium ions inhibit K/HCO(3) influx in the potency sequence PPTEA(+) > TBA(+) > TPA(+) > TEA(+) congruent with TMA(+). The identification of inhibitors of the K/HCO(3) cotransporter, for which no inhibitors previously existed, will facilitate the study of this transporter.
先前对鱿鱼轴突的研究发现了一种新型钾/碳酸氢根共转运体，它对二磺酸芪衍生物不敏感。该共转运体可能通过将钾离子（K⁺）和碳酸氢根离子（HCO₃⁻）转运出细胞来响应细胞内碱负荷，从而倾向于降低细胞内pH值（pH(i)）。在存在内向性钾/碳酸氢根梯度的情况下，该共转运体介导碱的净摄取（即钾离子和碳酸氢根离子内流）。在这里，我们测试了一个假设，即细胞内季铵离子（QA⁺）通过在细胞内钾离子位点相互作用来抑制内向性共转运体。我们根据用pH敏感微电极测量的pH(i)升高速率计算出共转运体介导的等效碳酸氢根离子内流（J(HCO₃)）。我们使用不含钾离子、钠离子和氯离子的透析液（DF）将轴突透析至pH(i) 8.0。我们的标准人工海水（ASW）也不含钠离子、钾离子和氯离子。停止透析后，我们引入含有437 mM钾离子和0.5%二氧化碳/12 mM碳酸氢根离子的ASW，这（i）导致膜电位瞬间变得非常正，并且（ii）由于二氧化碳内流导致pH(i)迅速下降，随后由于钾离子和碳酸氢根离子的内向共转运导致pH(i)在平台期缓慢升高。当DF中没有QA⁺时，J(HCO₃)约为58 pmol cm⁻² s⁻¹。当DF中含有400 mM四乙铵（TEA⁺）时，J(HCO₃)几乎为零。细胞内TEA⁺的表观解离常数（K(i)）约为78 mM，比其他人获得的抑制钾离子通道的K(i)值大两个数量级以上。将100 mM抑制剂引入DF中，对于四甲铵（TMA⁺），J(HCO₃)降至约20 pmol cm⁻² s⁻¹，对于TEA⁺约为24，对于四丙铵（TPA⁺）约为10，对于四丁铵（TBA⁺）几乎为零。TBA⁺的表观K(i)值约为0.86 mM。最有效的抑制剂是苯丙基四乙铵（PPTEA⁺），表观K(i)约为91 μM。因此，跨侧季铵离子以PPTEA⁺ > TBA⁺ > TPA⁺ > TEA⁺ ≈ TMA⁺的效力顺序抑制钾/碳酸氢根离子内流。钾/碳酸氢根共转运体抑制剂的鉴定（此前不存在此类抑制剂）将有助于对该转运体的研究。
