Park C S, MacKinnon R
Department of Neurobiology, Harvard Medical School, Boston, Massachusetts 02115, USA.
Biochemistry. 1995 Oct 17;34(41):13328-33. doi: 10.1021/bi00041a008.
Divalent metal cation selectivity was studied in guanosine 3',5'-cyclic monophosphate-gated ion channels. Channels from bovine retina were expressed in Xenopus laevis oocytes, and currents were measured using tight-seal patch recording methods. The ability of divalent cations to block Na+ currents was used to determine the occupancy of divalent cations in the ion conduction pore. At positive membrane voltages, where extracellular divalent cations are near equilibrium with their binding site, the occupancy reflects the affinity of the blocking ion. The selectivity sequence based on relative affinity was Ca2+ > Mg2+ = Sr2+ = Ba2+. In addition to its higher affinity, Ca2+ was more permeant and blocked with a weaker voltage dependence. Ca2+ was the only ion that blocked with a high Hill coefficient (n = 2.7), suggesting the presence of multiple binding sites. When Glu 363, located in the pore-forming region, was mutated to Asp, the affinity of all four ions increased and the selectivity sequence became Ca2+ > Sr2+ > Ba2+ > Mg2+. These results show that the channel is highly selective for Ca2+ and that Glu 363 mediates divalent cation selectivity of the channel.
在鸟苷3',5'-环一磷酸门控离子通道中研究了二价金属阳离子选择性。将来自牛视网膜的通道在非洲爪蟾卵母细胞中表达，并使用紧密封接膜片钳记录方法测量电流。利用二价阳离子阻断Na+电流的能力来确定二价阳离子在离子传导孔中的占据情况。在正膜电位下，细胞外二价阳离子与其结合位点接近平衡，此时占据情况反映了阻断离子的亲和力。基于相对亲和力的选择性序列为Ca2+ > Mg2+ = Sr2+ = Ba2+。除了具有更高的亲和力外，Ca2+的通透性更高，且其阻断具有较弱的电压依赖性。Ca2+是唯一以高希尔系数（n = 2.7）阻断的离子，表明存在多个结合位点。当位于孔形成区域的Glu 363突变为Asp时，所有四种离子的亲和力均增加，选择性序列变为Ca2+ > Sr2+ > Ba2+ > Mg2+。这些结果表明该通道对Ca2+具有高度选择性，且Glu 363介导了该通道的二价阳离子选择性。
