The mechanisms that underlie activation of nicotinic receptors are investigated using human recombinant receptors, both wild type and receptors that contain the slow channel myasthenic syndrome mutation, epsilonL221F. The method uses the program HJCFIT, which fits the rate constants in a specified mechanism directly to a sequence of observed open and shut times by maximising the likelihood of the sequence with exact correction for missed events. A mechanism with two different binding sites was used. The rate constants that apply to the diliganded receptor (opening, shutting and total dissociation rates) were estimated robustly, being insensitive to the exact assumptions made during fitting, as expected from simulation studies. They are sufficient to predict the main physiological properties of the receptors. The epsilonL221F mutation causes an approximately 4-fold reduction in dissociation rate from diliganded receptors, and a smaller increase in opening rate and mean open time. These are sufficient to explain the approximately 6-fold slowing of decay of miniature synaptic currents seen in patients. The distinction between the two binding sites was less robust, the estimates of rate constants being dependent to some extent on assumptions, e.g. whether an extra short-lived shut state was included or whether the EC50 was constrained. The results suggest that the two binding sites differ by roughly 10-fold in the affinity of the shut receptor for ACh in the wild type, and that in the epsilonL221F mutation the lower affinity is increased so the sites become more similar.
SCCMS mutations may show a recessive inheritance pattern and variable penetrance. A diagnosis of SCCMS should not be ruled out in cases of CMS with an apparent recessive inheritance pattern.
The discovery of the genetic basis for many of the human congenital myasthenic syndromes Beeson & Newsom-Davis, 2000) has added to the inherent interest in investigation of the relationship between structure and function in the nicotinic acetylcholine receptor (AChR) of the muscle endplate. The slow channel congenital myasthenic syndromes result from single amino acid 'gain-of-function' mutations in the receptor protein that give rise to prolonged endplate currents. Muscle weakness is thought to result from endplate damage caused by excess calcium entry. In addition, at physiological rates of stimulation the prolonged endplate potentials summate, leading to persistent depolarisation at the endplate and the consequent inactivation of the voltage-gated sodium channels.The mechanisms that underlie activation of nicotinic receptors are investigated using human recombinant receptors, both wild type and receptors that contain the slow channel myasthenic syndrome mutation, eL221F. The method uses the program HJCFIT, which fits the rate constants in a specified mechanism directly to a sequence of observed open and shut times by maximising the likelihood of the sequence with exact correction for missed events. A mechanism with two different binding sites was used. The rate constants that apply to the diliganded receptor (opening, shutting and total dissociation rates) were estimated robustly, being insensitive to the exact assumptions made during fitting, as expected from simulation studies. They are sufficient to predict the main physiological properties of the receptors. The eL221F mutation causes an approximately 4-fold reduction in dissociation rate from diliganded receptors, and a smaller increase in opening rate and mean open time. These are sufficient to explain the approximately 6-fold slowing of decay of miniature synaptic currents seen in patients. The distinction between the two binding sites was less robust, the estimates of rate constants being dependent to some extent on assumptions, e.g. whether an extra short-lived shut state was included or whether the EC 50 was constrained. The results suggest that the two binding sites differ by roughly 10-fold in the affinity of the shut receptor for ACh in the wild type, and that in the eL221F mutation the lower affinity is increased so the sites become more similar. Journal of Physiologyinvolved in gating. At the other extreme, the individual openings in the mutant might have exactly the same mean duration as for the wild type (or even be shorter), but there would be more of them -the activation would look much like that for the wild type but go on for longer. This would, for example, be expected if the mutation caused the acetylcholine to dissociate more slowly from the resting (shut) state of the receptor, an effect on the binding reaction. The longer occupancy of the shut state would give more time for the channel to re-open, before dissociation prevented it from doing so. However a similar outcome would be seen if the mutation had a quite different effect, to spe...
cDNA sequences encompassing the full coding region for the human muscle acetylcholine receptor (AChR) E and y subunits have been isolated. The deduced amino-acid sequences indicate that the mature E subunit contains 473 amino acids and is preceded by a 20-amino-acid signal peptide. As predicted from genomic clones, the y subunit contains 495 amino acids preceded by a 22-aminoacid signal peptide. In common with the human a, /?, y and 6 subunits the E subunit is highly conserved between mammalian species. The E subunit gene is not closely linked to the y and 6 subunits on chromosome 2 but rather is located with the / 3 subunit on chromosome 17. Expression of the a-, p-, y-, 6-and E-subunit cRNAs in rabbit-reticulocyte lysates followed by analysis on SDS/ PAGE show glycosylated proteins with apparent molecular masses of 44-60 kDa.The muscle nicotinic acetylcholine receptor (AChR) mediates synaptic transmission at the vertebrate neuromuscular junction. The binding of acetylcholine to its receptor gives rise to the opening of cation-selective channels, a vital step in the process which leads to muscle contraction. The AChR is implicated in at least two human disorders. In the autoimmune disease myasthenia gravis, characterised by fatiguable muscle weakness, the autoantibodies are directed against muscle AChRs [l]. The sera from patients with myasthenia gravis show limited cross-reactivity with AChRs from other species despite substantial sequence similarity [ 2 ] . In congenital myasthenia there may be inherited abnormalities of the AChRs [3, 41. Detailed study of the role of the AChR in these disorders is restricted by the low level of AChR (<2 pmol/g) found in human muscle. One means of overcoming this difficulty is to isolate and express the cDNAs which encode the human AChR.AChRs from the electric ray Torpedo have been well characterised at both the biochemical and molecular level p, y and 6 subunits, whereas in adult muscle the y subunit is replaced by the E subunit [8, 91.Genomic clones encoding the human AChR a and y sub- . Although the mRNA encoding the P3A isoform of the human AChR is present in all human skeletal muscle tissue we have examined, the functional significance of the P3A exon has yet to be determined. In this study we report the isolation of cDNAs for the human E and y subunits, and thus the availability of all five human muscle AChR cDNAs for the study of the functional expression of the P3A isoform. We also localise the gene for the E subunit to chromosome 17. MATERIALS AND METHODS Radiochemicals [32P]dCTP (5000 Cilmmol) and [a- 35S]dATP[S](600 Cilmmol) and 1 packaging extracts were obtained from Amersham International. T7 DNA sequencing kits were obtained from United States Biochemicals or Pharmacia. Restriction endodeoxyribonucleases, and enzymes used in the construction of cDNA libraries and subcloning were from Promega, Gibco BRL and Pharmacia; reactions were performed according to the conditions recommended by the manufacturer. EcoRI linkers were from New England Biolabs. RNAseI, DNAseI, ...
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