A SINGLE NEGATIVE CHARGE WITHIN THE PORE REGION OF A CGMP-GATED CHANNEL CONTROLS RECTIFICATION, CA2+ BLOCKAGE, AND IONIC SELECTIVITY

Ca2+ ions control the cGMP-gated channel of rod photoreceptor cells fr om the external and internal face. We studied ion selectivity and bloc kage by Ca2+ of wild-type and mutant channels in a heterologous expres sion system. External Ca2+ blocks the inward current at micromolar con centrations in a highly voltage-dependent manner. The blockage at nega tive membrane voltages shows a steep concentration dependence with a H ill coefficient of almost-equal-to 2. The blockage from the internal f ace requires almost-equal-to 1000-fold higher Ca2+ concentrations. Neu tralization of a glutamate residue (E363) in the putative pore region between transmembrane segments H4 and H5 induces outward rectification and changes relative ion conductances but leaves relative ion permeab ilities nearly unaffected. The current blockage at -80 mV requires alm ost-equal-to 2000-fold higher external Ca2+ concentrations and the vol tage dependence is almost abolished. These results demonstrate that E3 63 represents a binding site for monovalent and divalent cations and r esides in the pore lumen.