Neurotransmitter-gated ion channels (LGIC) transduce a presynaptic event (release of transmitter) into a postsynaptic event (opening of channels). Given the vagaries of life for a synapsethe quantal content, the number of transmitter molecules in a vesicle, the exact release site, the statistics of transmitter binding, noise in the postsynaptic cell, and so forthit would seem to be advantageous for fidelity in signal transmission for some aspects of the process to be tightly controlled. Indeed, the idea that the functional properties of the ion channels are well defined and reasonably homogeneous is necessary for biophysicists to analyze the kinetics from population measurements. All in all, relatively few data have to be swept under the rug in our effort to define "the" characteristic behavior of a given type of LGIC. However, there are recurring observations of variability in the kinetic properties of LGIC. Some reports have noted relatively subtle effects (for example, that the mean open times differ between individual muscle nicotinic receptors;
The paper by subunit identified in patients showing a congenital myasthenic syndrome, which harbors a point mutation in the "amphipathic helix" (HA), a region of the receptor near the carboxy terminus. The study demonstrates that this particular mutation,
A411P (alanine mutated to proline in the
subunit), produces a receptor with increased heterogeneity in gating kinetics.
The HA region was first identified by analysis of amino acid sequences as a likely helix that had one polar and one nonpolar face, and was first suggested as a candidate for the channel-lining regions of the subunits. It is located at the COOH-terminal end of the major intracellular loop of each subunit, just before the fourth membrane-spanning helix (Fig 1). After it was shown that receptors could function even when major portions of the HA region were deleted in the subunit (
, ß,
, and
subunits, whereas the adult form contains
, ß,
, and
subunits. Fetal receptors have a (two- to fourfold) longer burst duration, resulting from a smaller channel closing rate. Work by
subunit found in other patients with congenital myasthenic syndrome identified a six amino acid repeat, again located in the HA region (
subunit can affect the gating behavior of the AChR. In general, the results are consistent with the idea that the HA region, in some fashion, can affect the modal kinetic behavior of the receptor.
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subunit, which does not produce clear changes in modal behavior, but increases the variability in channel gating kinetics. The gating properties of the AChR containing the mutated subunit are, in some ways, very frustrating to anyone who wants to quantitatively analyze the kinetics. The experiments therefore were conducted using a relatively high concentration of ACh so that, most of the time, all the receptors in a patch were desensitized and inactive. Occasionally, a single receptor recovered from desensitization and underwent a paroxysm of activityopening and closing repeatedlyuntil it finally desensitized again and the patch became silent. The advantage of studying the activity in such a "cluster" is that it results from the activation of a single receptor. Even then, frustration arose because different clusters from AChR containing mutated
subunits showed obviously different kinetics. Even a straightforward parameter, the probability that the channel is open during the cluster, showed more variabilityfor the wild-type AChR, the mode under the experimental conditions was
0.65, with a range from 0.45 to 0.9, while the AChR with the mutated receptor showed a mode at
0.5, with a range from 0.1 to 0.95.
subunits. However, the variability of the estimates for the channel opening and closing rate constants was greater for the receptors with the mutated subunit. The increase in the heterogeneity of the rates was relatively large in terms of channel behavior, about twofold. However, this reflects a relatively small difference in terms of the energetics of gating. As a rough indication, suppose the limits of the variability in the rate constants are set by ±3 SD from the mean values. In this case, the spread in the case of wild-type receptors would correspond to
1.2 RT units (0.7 kcal/mol), while the spread in the mutated receptors would be
1.9 RT units (1.1 kcal/mol). Since both the opening and closing rates became more variable, it might be that the energy of the transition state was affected by the mutation (applying the simplest two well, one barrier model to the open/closed transition). However, this does not seem to be the case, since there was not a significant correlation between the estimated open and closing rate constants across clusters (S.M. Sine, personal communication). Therefore, it seems that the depth of the energy wells for the liganded-closed and -open states became more variable. The variability in kinetics was not manifest by the appearance of additional kinetic modes.
subunit, the mode transitions appeared to occur at a rate similar to that seen in the wild type.
The present and previous structure studies have in common that the single-channel conductance is not altered (suggesting that the open channel is similar), that the changes in rates are relatively modest (<10-fold), that the opening and closing rates change independently, and that there seems to be more change in channel gating than in ACh binding. It is intriguing that in some cases the occurrence of distinct modes is enhanced, while in others the heterogeneity of kinetic behavior (in the absence of clear modes) in increased. Many other mutations have been identified that alter channel kinetics or ACh binding. In general, the kinetics change, but have not been reported to be markedly more variable than for wild type AChR, nor have the mutations been reported to accentuate multiple kinetic modes.
What does this mean in terms of understanding AChR function (and, by extrapolation, learning more about the properties of other LGIC)? It is reassuring that a recent analysis of AChR gating in terms of linear free energy relationships supports the idea, at least for the wild-type receptor, that channel gating proceeds along a single reaction path ( subunit HA deletions or either of the
HA mutations, there are significant reductions in the number of receptors on the surface of the cell. It is not known whether subunit assembly or interactions with transport processes are affected.
Unfortunately, the actual location of the HA region with respect to the membrane or the rest of the cytoplasmic loop is not known. After a possible role in forming the channel was dismissed, it was thought that it might lie parallel to the membrane with one face in lipid and the other exposed to the cytoplasm. Most recently, a projection from the cytoplasmic side of each subunit has been identified that has helical content, so it may be that the HA region projects into the cytoplasm and interacts with the intracellular loops from other subunits as well (
Two additional points are of interest. First, the present study reports that homologous mutations in the HA regions of the ß and subunits do not produce these effects on kinetics (no currents were recorded from AChR-containing
subunits with the homologous mutation, and the
subunit was not examined). Does this mean that there is a unique capacity of the HA region in the
subunit, or that there are minor structural differences in other subunits and that a mutation nearby would show effects? Finally, these results yet again emphasize the novelty of the insights that are provided from studies of subunits identified in patients with congenital myasthenia. There was no reason a priori to examine the HA region for a role in controlling kinetic variability in the AChR. It is still not clear whether the kinetic alterations contribute directly to development of symptoms, since both of the mutations studied by Sine's group have shown up as recessivepatients present with symptoms when the other allele of the
subunit contains a null mutation. Accordingly, it is possible that the clinical symptoms reflect the lower expression levels for receptors in the patients.
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Acknowledgements |
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I thank Gustav Akk and Chris Lingle for their comments on this Commentary.
Submitted: 26 July 2000
Revised: 27 July 2000
Accepted: 27 July 2000
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References |
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