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 from the external and internal face. We studied ion selectivity and blockage by Ca2+of wild-type and mutant channels in a heterologous expression system. External Ca2+blocks the inward current at micromolar concentrations in a highly voltage-dependent manner. The blockage at negative membrane voltages shows a steep concentration dependence with a Hill coefficient of ≈2. The blockage from the internal face requires ≈1000-fold higher Ca2+concentrations. Neutralization 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 permeabilities nearly unaffected. The current blockage at -80 mV requires ≈2000-fold higher external Ca2+concentrations and the voltage dependence is almost abolished. These results demonstrate that E363 represents a binding site for monovalent and divalent cations and resides in the pore lumen.