Karpen J W, Brown R L, Stryer L, Baylor D A
Department of Neurobiology, Sherman Fairchild Center, Stanford University School of Medicine, California 94305.
J Gen Physiol. 1993 Jan;101(1):1-25. doi: 10.1085/jgp.101.1.1.
The effects of divalent cations on the gating of the cGMP-activated channel, and the effects of gating on the movement of divalent cations in and out of the channel's pore were studied by recording macroscopic currents in excised membrane patches from salamander retinal rods. The fractional block of cGMP-activated Na+ currents by internal and external Mg2+ as well as internal Ca2+ was nearly independent of cGMP concentration. This indicates that Mg2+ and Ca2+ bind with similar affinity to open and closed states of the channel. In contrast, the efficiency of block by internal Cd2+ or Zn2+ increased in proportion to the fraction of open channels, indicating that these ions preferentially occupy open channels. The kinetics of block by internal Ni2+, which competes with Mg2+ but blocks more slowly, were found to be unaffected by the fraction of channels open. External Ni2+, however, blocked and unblocked much more rapidly when channels were mostly open. This suggests that within the pore a gate is located between the binding site(s) for ions and the extracellular mouth of the channel. Micromolar concentrations of the transition metal divalent cations Ni2+, Cd2+, Zn2+, and Mn2+ applied to the cytoplasmic surface of a patch potentiated the response to subsaturating concentrations of cGMP without affecting the maximum current induced by saturating cGMP. The concentration of cGMP that opened half the channels was often lowered by a factor of three or more. Potentiation persisted after the experimental chamber was washed with divalent-free solution and fresh cGMP was applied, indicating that it does not result from an interaction between divalent cations and cGMP in solution; 1 mM EDTA or isotonic MgCl2 reversed potentiation. Voltage-jump experiments suggest that potentiation results from an increase in the rate of cGMP binding. Lowering the ionic strength of the bathing solution enhanced potentiation, suggesting that it involves electrostatic interactions. The strong electrostatic effect on cGMP binding and absence of effect on ion permeation through open channels implies that the cGMP binding sites on the channel are well separated from the permeation pathway.
通过记录蝾螈视网膜杆细胞分离膜片中的宏观电流，研究了二价阳离子对cGMP激活通道门控的影响，以及门控对二价阳离子进出通道孔运动的影响。内部和外部的Mg2+以及内部的Ca2+对cGMP激活的Na+电流的分数阻断几乎与cGMP浓度无关。这表明Mg2+和Ca2+以相似的亲和力结合到通道的开放和关闭状态。相比之下，内部Cd2+或Zn2+的阻断效率与开放通道的分数成比例增加，表明这些离子优先占据开放通道。内部Ni2+的阻断动力学与Mg2+竞争但阻断较慢，发现不受开放通道分数的影响。然而，当通道大多开放时，外部Ni2+的阻断和解除阻断要快得多。这表明在孔内，一个门位于离子结合位点和通道细胞外口之间。将微摩尔浓度的过渡金属二价阳离子Ni2+、Cd2+、Zn2+和Mn2+施加到膜片的细胞质表面，增强了对亚饱和浓度cGMP的反应，而不影响饱和cGMP诱导的最大电流。打开一半通道的cGMP浓度通常降低三倍或更多。在用无二价溶液冲洗实验腔并施加新鲜cGMP后，增强作用仍然存在，表明它不是由溶液中二价阳离子与cGMP之间的相互作用引起的；1 mM EDTA或等渗MgCl2可逆转增强作用。电压跳跃实验表明，增强作用是由于cGMP结合速率的增加。降低浴液的离子强度增强了增强作用，表明它涉及静电相互作用。对cGMP结合的强烈静电效应以及对通过开放通道的离子渗透没有影响，这意味着通道上的cGMP结合位点与渗透途径相距很远。
