THE MULTIION NATURE OF THE CGMP-GATED CHANNEL FROM VERTEBRATE RODS

1. Native cGMP-gated channels were studied in rod outer segments of th e larval tiger salamander, Ambystoma tigrinum. The alpha-subunit of th e cGMP-gated channel, here referred to as the wild type (WT), and muta nt channels were heterologously expressed in Xenopus laevis oocytes. T hese channels were studied in excised membrane patches in the inside-o ut configuration and were activated by the addition of 100 or 500 mu M cGMP. The current carried by monovalent cations was measured under vo ltage-clamp conditions. 2. In the presence of 110 mM Na+ in the extrac ellular medium and different amounts of Na+ in the intracellular mediu m, the I-V relations of the native channel could be described by a sin gle-site model with a profile of Gibbs free energy with two barriers a nd a well. A similar result was obtained in the presence of 110 mM Li in the extracellular medium and different amounts of Lif in the intra cellular medium. The well depth was 1.4RT (where R is the gas constant and T is the absolute temperature) for both Li+ and Na+. 3. The I-V r elations of the native channel in the presence of 110 mM Na+ on one si de of the membrane and 110 mM Li+ on the other side could not be descr ibed by the same single-site model with identical values of barriers a nd well obtained in the presence of Li+ or Na+ alone: the well for Li had to be at least 4RT. 4. In the presence of mixtures of 110 mM Liand Cs+ on the cytoplasmic side of the membrane, an anomalous mole fra ction effect was observed both in the native and the WT channel. No an omalous behaviour was seen in the presence of Li+-Na+ and Li+-NH4+ mix tures. 5. The anomalous mole fraction effect with mixtures of Li+ and Cs+ was not observed in the channel where glutamate 363 was mutated to a glutamine (E363Q) or an asparagine (E363N). When glutamate 363 was mutated to an aspartate (E363D), the anomalous mole fraction effect wi th mixtures of Li+ and Cs+ was still observed, although significantly reduced. 6. When lysine 346, arginine 369, aspartate 370 and glutamate 372 were neutralized by mutation to glutamine, the ion permeation thr ough the mutant channels and the WT channel had largely similar proper ties. 7. The results here reported indicate that: (i) the native and t he WT cGMP-gated channels are both multi-ion pores; (ii) the mutant ch annels E363Q and E363N behave as a single-ion pore; (iii) the multi-io n nature of the WT channel is primarily controlled by glutamate 363 an d not by other charged residues in the pore region.