The plasma membrane of retinal rod outer segments contains a cyclic GMP-activated conductance which appears to be the light-sensitive conductance involved in phototransduction. Recently, it has been found that this conductance is partially blocked by Mg2+ and Ca2+ at physiological concentrations, thus possibly accounting for the absence of observable single-channel activity in excised membrane patches and for the unusually small apparent unit conductance deduced from noise measurements on intact cells. We now report that, as expected from this idea, single cGMP-activated channel activity can be detected from an excised rod membrane patch in the absence of divalent cations. The most prominent unitary current had a mean conductance of approximately 25 pS. Both individual channel openings (mean open time approximately 1 ms) and short bursts of openings (mean burst duration of about a few milliseconds) were observed. In addition, there were smaller events which probably represented other states of the conductance. The mean current increased with the third power of cGMP concentration, suggesting that there are at least three cGMP-binding sites on the channel molecule. With 0.2 mM Mg2+ in the cGMP-containing solution, a flickering block of the open channel was observed; the effect of Ca2+ was similar. The results resolve a puzzle about the light-sensitive conductance by demonstrating that it is an aqueous pore rather than a carrier.

Receptors for excitatory amino-acid transmitters on nerve cells fall into two main categories associated with non-selective cationic channels, the NMDA (N-methyl-D-aspartate) and non-NMDA (kainate and quisqualate) receptors. Special properties of NMDA receptors such as their voltage-dependent blockade by Mg2+ (refs 3, 4) and their permeability to Na+, K+ as well as to Ca2+ (refs 5, 6), have led to the suggestion that these receptors are important in plasticity during development and learning. They have been implicated in long-term potentiation (LTP), a model for the study of the cellular mechanisms of learning. We report here that glutamate and NMDA, acting at typical NMDA receptors, stimulate the release of arachidonic acid (as well as 11- and 12-hydroxyeicosatetraenoic acids from striatal neurons probably by stimulation of a Ca2+-dependent phospholipase A2. Kainate and quisqualate, as well as K+-induced depolarization were ineffective. Our results provide direct evidence in favour of the hypothesis, that arachidonic acid derivatives, produced by activation of the postsynaptic cell, could be messengers that cross the synaptic cleft to modify the presynaptic functions known to be altered during LTP. In addition, we suggest that NMDA receptors are the postsynaptic receptors which trigger the synthesis of these putative transynaptic messengers.

A cyclic GMP-sensitive conductance has recently been observed with patch-clamp recording in excised inside-out patches of plasma membrane from frog and toad rod outer segments. This conductance has properties suggesting that it is probably the light-sensitive conductance involved in visual transduction. We now report a similar conductance in the outer segment membrane of catfish cones. Cyclic GMP showed positive cooperativity in opening this conductance, with a Hill coefficient of 1.6-3.0 and a half-saturating cGMP concentration of 35-70 microM. Cyclic AMP at 1 mM, or changing Ca concentration (in the presence of Mg), had little effect on the conductance. In physiological solutions the cGMP-induced current had a reversal potential near +10 mV; the current amplitude increased roughly exponentially with membrane potential in both depolarizing and hyperpolarizing directions. Our results suggest that cGMP is also the internal transmitter for phototransduction in cones.

Inside-out patches were excised from catfish rod or cone outer segments. Single channel and macroscopic currents were recorded from GMP-gated channels activated by 1 mM cGMP in low divalent buffered saline. Currents were blocked by the application of micromolar concentrations of/-c/s-diltiazem to the cytoplasmic side of the patch. The concentration dependence of block indicated that a single molecule was sufficient to block a channel and that all channels were susceptible to block. The dissociation constant for the rod channel was an order of magnitude smaller than for the cone channel, but the voltage dependence of block was nearly identical. The macroscopic current-voltage relation in the presence of blocker was inwardly rectifying and superficially resembled voltage-dependent block by an impermeant blocker occluding the ion-conducting pore of the channel. Block by diltiazem acting from the extracellular side of the channel was investigated by including 5 ~.M diltiazem in the recording pipette solution. The macroscopic current-voltage relation again showed inward rectification, inconsistent with the idea that diltiazem acts by occluding the pore at the external side. The kinetics of block by diltiazem applied to the intra-and extracellular side were measured in cone patches containing only a single channel. The unbinding rates were similar in both cases, suggesting a single binding site. Differences in the binding rate were consistent with greater accessibility to the binding site from the cytoplasmic side. Block from the cytoplasmic side was independent of pH, suggesting that the state of ionization of diltiazem was not related to its ability to block the channel in a voltage-dependent fashion. These observations are inconsistent with a pore-occluding blocker, but could be explained if the hydrophobic portion of diltiazem partitioned into the hydrophobic core of the channel protein, perhaps altering the gating of the channel.