INTERACTIONS BETWEEN DIVALENT-CATIONS AND THE GATING MACHINERY OF CYCLIC GMP-ACTIVATED CHANNELS IN SALAMANDER RETINAL RODS
INTERACTIONS BETWEEN DIVALENT-CATIONS AND THE GATING MACHINERY OF CYCLIC GMP-ACTIVATED CHANNELS IN SALAMANDER RETINAL RODS
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DOI：
10.1085/jgp.101.1.1
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发表时间：
1993-01-01
影响因子：
3.8
通讯作者：
BAYLOR, DA
中科院分区：
文献类型：
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作者：
KARPEN, JW;BROWN, RL;BAYLOR, DA
通过记录从蝾螈视网膜杆切下的膜斑中的宏观电流，研究了二价阳离子对cGMP激活通道的门控的影响，以及门控对二价阳离子进出通道孔的运动的影响。内、外Mg ~（2+）和内Ca ~（2+）对cGMP激活的Na ~+电流的阻断作用与cGMP浓度无关。这表明Mg 2+和Ca 2+以相似的亲和力结合到通道的打开和关闭状态。与此相反，由内部Cd 2+或Zn 2+的块的效率成比例增加的开放通道的分数，表明这些离子优先占据开放通道。内部镍2+，与镁2+竞争，但块更慢，块的动力学被发现是不受影响的通道开放的分数。然而，外部Ni 2+，封锁和解除封锁更迅速时，通道大多是开放的。这表明在孔内，门位于离子的结合位点和通道的细胞外口之间。微摩尔浓度的过渡金属二价阳离子Ni 2+，Cd 2+，Zn 2+，Mn 2+施加到细胞质表面的补丁增强响应亚饱和浓度的cGMP，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。在用不含二价离子的溶液洗涤实验室并施加新鲜cGMP后，增强持续存在，表明其不是由溶液中二价阳离子与cGMP之间的相互作用引起的; ImM EDTA或等渗MgCl 2逆转增强。电压跳跃实验表明，增强的结果从cGMP结合率的增加。降低浴溶液的离子强度增强增强，这表明它涉及静电相互作用。对cGMP结合的强静电效应和对通过开放通道的离子渗透的不存在效应意味着通道上的cGMP结合位点与渗透途径很好地分离。
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.