The effects of the divalent cations Ca2+, Mg2+ and Ni2+ on unitary Na+ currents through receptor-regulated non-selective cation channels were studied in inside-out and cell-attached patches from rat adrenal zona glomerulosa cells. 2. External Ca2+ caused a concentration-dependent and voltage-independent inhibition of inward Na+ current, exhibiting an IC50 of 1.4 mM. The channel was also Ca2+ permeant and external Ca2+ shifted the reversal potential as expected for a channel exhibiting a constant Ca2+ : Na+ permeability ratio near to 4. 3. External and internal 2 mM Mg2+ caused voltage-dependent inhibition of inward and outward Na+ current, respectively. Modelling Mg2+ as an impermeant fast open channel blocker indicated that external Mg2+ blocked the pore at a single site exhibiting a zero voltage Kd of 5.1 mM for Mg2+ and located 19 % of the distance through the transmembrane electric field from the external surface. Internal Mg2+ blocked the pore at a second site exhibiting a Kd of 1.7 mM for Mg2+ and located 36% of the distance through the transmembrane electric field from the cytosolic surface. 4. External Ni2+ caused a voltage- and concentration-dependent slow blockade of inward Na+ current. Modelling Ni2+ as an impermeant slow open channel blocker indicated that Ni2+ blocked the pore at a single site exhibiting a Kd of 1.09 mM for Ni2+ and located 13.7% of the distance through the transmembrane electric field from the external surface. 5. External 2 mM Mg2+ increased the Kd for external Ni2+ binding to 1.27 mM, consistent with competition for a single binding site. Changing ionic strength did not substantially affect Ni2+ blockade indicating the absence of surface potential under physiological ionic conditions. 6. It is concluded that at least two divalent cation binding sites, separated by a high free energy barrier (the selectivity filter), are located in the pore and contribute to Ca2+ selectivity and permeability of the channel.
摘要
在大鼠肾上腺球状带细胞的内面向外和细胞贴附膜片中，研究了二价阳离子Ca2+、Mg2+和Ni2+对通过受体调节的非选择性阳离子通道的单位Na+电流的影响。2. 细胞外Ca2+引起内向Na+电流的浓度依赖性和电压非依赖性抑制，IC50为1.4 mM。该通道也对Ca2+通透，细胞外Ca2+使反转电位发生移动，这与表现出接近4的恒定Ca2+ : Na+通透率的通道预期一致。3. 细胞外和细胞内2 mM Mg2+分别引起内向和外向Na+电流的电压依赖性抑制。将Mg2+模拟为非通透的快速开放通道阻滞剂表明，细胞外Mg2+在单个位点阻断孔道，该位点对Mg2+的零电压Kd为5.1 mM，位于跨膜电场从外表面算起距离的19%处。细胞内Mg2+在第二个位点阻断孔道，该位点对Mg2+的Kd为1.7 mM，位于跨膜电场从胞质表面算起距离的36%处。4. 细胞外Ni2+引起内向Na+电流的电压和浓度依赖性缓慢阻断。将Ni2+模拟为非通透的缓慢开放通道阻滞剂表明，Ni2+在单个位点阻断孔道，该位点对Ni2+的Kd为1.09 mM，位于跨膜电场从外表面算起距离的13.7%处。5. 细胞外2 mM Mg2+使细胞外Ni2+结合的Kd增加到1.27 mM，这与对单个结合位点的竞争一致。改变离子强度对Ni2+阻断没有实质性影响，表明在生理离子条件下不存在表面电位。6. 得出的结论是，至少有两个被高自由能屏障（选择性过滤器）隔开的二价阳离子结合位点位于孔道中，有助于通道对Ca2+的选择性和通透性。
