Cation interactions within the cyclic GMP-activated channel of retinal rods from the tiger salamander.
1. The ionic dependence of current through the 3',5'-cyclic guanosine monophosphate (cyclic GMP)-activated channels of salamander rods was studied in excised inside-out membrane patches from isolated outer segments. Voltage-clamp experiments on transducing rods were performed so that the channels in intact cells could be compared with those in excised patches. 2. The reversal potential of the cyclic GMP-induced patch current was close to the Na+ equilibrium potential when the concentration of NaCl on the cytoplasmic surface of a patch was varied at constant external NaCl concentration. Fitting the Goldman-Hodgkin-Katz equation indicated that the apparent ratio of permeabilities for Na+ and Cl- was at least 50. This confirms a previous report that the channel's Na+ permeability is much larger than its Cl- permeability. 3. Na+ currents through the channel did not obey the independence principle. The outward patch current at large positive potential began to saturate with increasing concentrations of internal Na+, as if permeation required Na+ to bind to a site with an apparent dissociation constant around 180 mM. 4. In symmetrical NaCl solutions containing very low concentrations of divalent cations the current-voltage relation measured from excised patches 50 microseconds after switching the voltage showed mild outward rectification. By 1 ms the rectification was more pronounced. The rectification at 50 microseconds is attributed to voltage dependence of Na+ permeation. The additional rectification at later times is attributed to voltage dependence of the channel's probability of being open, depolarization favouring the open state. 5. In symmetrical Mg2+ solutions the cyclic GMP-induced patch currents were smaller and the outward rectification was more pronounced. 6. Addition of Mg2+ or Ca2+ to an internal Na+ solution blocked the cyclic GMP-induced Na+ current through the channels, as if by occupying a single binding site with an affinity in the 0.1-2 mM range. Block by Mg2+ was voltage dependent, suggesting that the binding site was within the channel's transmembrane electric field. Raising the Mg2+ concentration on the external surface of the patch increased the apparent dissociation constant of block by internal Mg2+, as expected if external and internal Mg2+ compete for the same binding site. 7. Block by internal Ca2+ had an opposite and weaker voltage dependence than block by internal Mg2+. 8. In symmetrical solutions containing both Na+ and Mg2+ the outward rectification was more pronounced than in solutions containing Na+ alone. In solutions thought to be close to physiological the outward patch current increased e-fold for a depolarization of 24-30 mV.(ABSTRACT TRUNCATED AT 400 WORDS)
Zimmerman
A L
AL
Department of Neurobiology, Stanford University School of Medicine, CA 94305.
Baylor
D A
DA
D000818:Animals; D001665:Binding Sites / Q000502:physiology; D002118:Calcium / Q000378:metabolism; D006152:Cyclic GMP / Q000378:metabolism*; D007473:Ion Channels / Q000378:metabolism*; D008274:Magnesium / Q000378:metabolism; D008564:Membrane Potentials / Q000502:physiology; D010786:Photoreceptor Cells / Q000378:metabolism*; D012964:Sodium / Q000378:metabolism; D014562:Caudata
D007473:Ion Channels; D012964:Sodium; D006152:Cyclic GMP; D008274:Magnesium; D002118:Calcium