How 2C a channel that's (almost) not there

The development of patch clamp techniques for recording single ion channels has greatly facilitated our understanding of cell physiology. But how do you really know that you're recording just one channel? Although single channel patchers don't like to mention it, membrane patches usually contain more than one active channel. In this issue of The Journal of Physiology, Dravid et al. (2008) report measurements of one-channel patches containing one of the least active of all ligand gated channels, the NMDA NR1–NR2C subtype of glutamate receptor. Obtaining such recordings is an exacting task, but one which the authors have convincingly completed. NMDA receptors underlie the slow component of excitatory synaptic currents, and those containing NR2C subunits have a unique expression pattern in the brain, being found mainly in the cerebellum, cortex and in oligodendrocytes. Dravid et al. measured the fraction of the time spent in the open state by these channels (also called Popen, the open probability), being careful to check that they were watching only one channel. Amazingly, they found that NR1–NR2C receptors are open for only 1% of the time, even when saturated by their agonists, glutamate and glycine. The low Popen of NR2C-containing receptors is in stark contrast to the widely studied NR2A and NR2B subtypes, and is more reminiscent of the long, meandering activations of NR2D-containing receptors. When channel openings are long or frequent – that is they occupy an appreciable proportion of the time – simultaneous openings (‘doubles’) are easily detected, and one can usually select parts of the record where only one channel is active, without knowing how many channels are in the patch. But when Popen is small, because the probability of observing a double opening scales approximately with the square of the open probability, doubles could be so rare that the recording could end before one was seen. How, then, to tell the difference between one channel and two identical channels with about half the activity? In their search for perfect one-channel patches, Dravid et al. screened 60 patch records, and found eight that exceeded the criteria (with > 99.9% confidence) for the number of openings expected before a ‘double’ occurs, in a patch containing two channels (Colquhoun & Hawkes, 1990). The authors also provide strong evidence that their analysis was correct by examining simulations of plausible channel activity. The big advantage of using a one-channel patch is that the entire record, including the gaps between openings, becomes available for analysis. And for NR2C-containing receptors, these gaps are a big part of the story, because the fully bound receptor spends 99% of the time in the resting state, and in other states that bind transmitter tightly, without being open. Direct fitting of mechanisms suggests that these other states do not relate to desensitization, which has slower kinetics, and is barely seen in the NR2C-containing channels anyway. Instead, these recordings clearly reveal intermediate states in the activation pathway, between resting and open states, that are becoming increasingly obvious in other voltage- and ligand-gated channels. Such states are seen in the nicotinic superfamily (the ‘flip’ state; Lape et al. 2008) in other glutamate receptor subtypes (Zhang et al. 2008), and in the BK-type voltage-gated potassium channels. The existence of these states has profound consequences for understanding the action of drugs and agonists that do not fully activate receptors. It is surprising just how bad glutamate is at opening the channel! To pass on the excitatory signal reliably, one might expect a channel to open for a healthier fraction of the time. Such inefficiency seems profligate, but in fact it makes a great deal of sense. The authors' results predict synaptic activations of NR1–NR2C receptors with a decay constant approaching 500 ms, in keeping with observations in neurons (Cathala et al. 2000; and other references in Dravid et al. 2008). The NR2C subunit reduces the channel block by magnesium that keeps other NMDA receptors silent at the resting membrane potential. Taking into account these properties, and the fatal consequences for neurons of excessive calcium influx through NMDA receptors, it isn't surprising that the opening of NR2C-containing receptors is heavily damped. Cerebellar granule neurons, in which the NR2C subunit is expressed in mature animals (Cathala et al. 2000), are depolarized more easily than most neurons because of their small size. Thus, the role of the NR2C subunit may be analogous to turning down the volume on the stereo in a small room. Using a large population of sparsely active receptors could provide the same depolarization (on average) as a few strongly active channels (such as those containing the NR2A subunit), but with much less variability between responses. In this way the low Popen of NR2C-containing receptors might confer reliability, especially important for coincidence detection. The single channel work in this study was done at saturating concentrations. Unfortunately, working at lower glutamate concentrations (and hence lower open probabilities) is probably impossible because the problem of detecting one-channel patches would be greatly amplified. Dravid et al. used macroscopic currents in this study to determine the rates of glutamate binding, and this approach could be extended to the coagonist glycine using recently developed methods to fit activation mechanisms directly to the excess noise in macroscopic records. Previous studies from the senior author's lab have gone one better and measured the non-equilibrium response of other NMDA receptor subtypes in one channel patches (Banke & Traynelis, 2003), so this is another route, notwithstanding potential problems with assessing the number of channels. Future directions aside, the elegant study by Dravid et al. shows that when it comes to understanding neurotransmitter receptors, you can't beat a truly single channel.