Xin-ming Shen - Academia.edu (original) (raw)
Papers by Xin-ming Shen
Neuron, 1998
et al., 1995). A functionally similar mutation, located in the N-terminal portion of the M1 domai... more et al., 1995). A functionally similar mutation, located in the N-terminal portion of the M1 domain (␣N217K), also enhances resting state ACh affinity and slows the rate of channel closing (Engel et al., 1996b; Wang et al., 1997). The physiologic converse of the SCCMS-the low
Neuromuscular disorders : NMD, 2011
Centronuclear myopathy is a pathologically diagnosed congenital myopathy. The disease genes encod... more Centronuclear myopathy is a pathologically diagnosed congenital myopathy. The disease genes encode proteins with membrane modulating properties (MTM1, DNM2, and BIN1) or alter excitation-contraction coupling (RYR1). Some patients also have myasthenic symptoms but electrodiagnostic and endplate studies in these are limited. A sporadic patient had fatigable weakness and a decremental EMG response. Analysis of centronuclear myopathy disease- and candidate-genes identified no mutations. Quantitative endplate electron microscopy studies revealed simplified postsynaptic regions, endplate remodeling with normal nerve terminal size, normal synaptic vesicle density, and mild acetylcholine receptor deficiency. The amplitude of the miniature endplate potential was decreased to 60% of normal. Quantal release by nerve impulse was reduced to 40% of normal due to a decreased number of releasable quanta. The safety margin of neuromuscular transmission is compromised by decreased quantal release by ...
Annals of Neurology, 2008
Objective-Detailed analysis of phenotypic and molecular genetic aspects of Dok-7 myasthenia in 16... more Objective-Detailed analysis of phenotypic and molecular genetic aspects of Dok-7 myasthenia in 16 patients. Methods-We assessed our patients by clinical and electromyographic studies, by intercostal muscle biopsies for in vitro microelectrode analysis of neuromuscular transmission and quantitative electron microscopy EM of 409 end plates (EPs), and by mutation analysis, and expression studies of the mutants. Results-The clinical spectrum varied from mild static limb-girdle weakness to severe generalized progressive disease. The synaptic contacts were single or multiple, and some, but not all, were small. In vitro microelectrode studies indicated variable decreases of the number of released quanta and of the synaptic response to acetylcholine; acetylcholine receptor (AChR) channel kinetics were normal. EM analysis demonstrated widespread and previously unrecognized destruction and remodeling of the EPs. Each patient carries 2 or more heteroallelic mutations: 11 in genomic DNA, 7 of which are novel; and 6 identifiable only in complementary DNA or cloned complementary DNA, 3 of which are novel. The pathogenicity of the mutations was confirmed by expression studies. Although the functions of Dok-7 include AChR β-subunit phosphorylation and maintaining AChR site density, patient EPs showed normal AChR β-subunit phosphorylation, and the AChR density on the remaining junctional folds appeared normal.
Human Molecular Genetics, 2003
A frameshifting 7 bp deletion (e553del7) in exon 7 of CHRNE encoding the acetylcholine receptor e... more A frameshifting 7 bp deletion (e553del7) in exon 7 of CHRNE encoding the acetylcholine receptor e subunit, observed in seven congenital myasthenic syndrome patients, enhances expression of an aberrantly spliced transcript that skips the preceding 101 bp exon 6. To recapitulate the aberrant splicing, we cloned the entire CHRNE spanning 12 exons and 11 introns and expressed it in COS cells. Scanning mutagenesis revealed that e553del7 does not disrupt an exonic splicing enhancer. Inhibition of protein synthesis and of nonsensemediated mRNA decay (NMD) by anisomycin shows that even wild-type CHRNE produces an exon 6-skipped transcript, and that even e553del7-CHRNE yields a normally spliced transcript. Both transcripts, however, are degraded by NMD due to a premature stop codon. In contrast, the normally spliced transcript from wild-type CHRNE and the exon 6-skipped transcript from e553del7-CHRNE carry no premature stop codon and hence are immune to NMD. Optimization of splicing signals for exon 6 prevents it being skipped even in the presence of anisomycin and/or e553del7, indicating that inherently weak splicing signals for exon 6 account for its skipping. We suggest that a similar mechanism probably operates in other genes in skipping of remote exons. The presence of weak splicing signals for exon 6 also prompted us to search for mutations in exon 6 that disrupt an exonic splicing enhancer. Indeed, we found that eEF157V and eE154X in exon 6, observed in two other patients, caused aberrant splicing of exon 6.
Annals of the New York Academy of Sciences, 1998
Annals of Clinical and Translational Neurology
Objective: To characterize the molecular and phenotypic basis of a severe slow-channel congenital... more Objective: To characterize the molecular and phenotypic basis of a severe slow-channel congenital myasthenic syndrome (SCCMS). Methods: Intracellular and single-channel recordings from patient endplates; alpha-bungarotoxin binding studies; direct sequencing of AChR genes; microsatellite analysis; kinetic analysis of AChR activation; homology modeling of adult human AChR structure. Results: Among 24 variants reported to cause SCCMS only two appear in the AChR d-subunit. We here report a 16-year-old patient harboring a novel dL273F mutation (dL294F in HGVS nomenclature) in the second transmembrane domain (M2) of the AChR d subunit. Kinetic analyses with ACh and the weak agonist choline indicate that dL273F prolongs the channel opening bursts 9.4-fold due to a 75-fold increase in channel gating efficiency, whereas a previously identified eL269F mutation (eL289F in HGVS nomenclature) at an equivalent location in the AChR e-subunit prolongs channel opening bursts 4.4-fold due to a 30-fold increase in gating efficiency. Structural modeling of AChR predicts that inter-helical hydrophobic interactions between the mutant residue in the d and e subunit and nearby M2 domain residues in neighboring a subunits contribute to structural stability of the open relative to the closed channel states. Interpretation: The greater increase in gating efficiency by dL273F than by eL269F explains why dL273F has more severe clinical effects. Both dL273F and eL269F impair channel gating by disrupting hydrophobic interactions with neighboring a-subunits. Differences in the extent of impairment of channel gating in d and e mutant receptors suggest unequal contributions of e/a and d/a subunit pairs to gating efficiency. 2067 X.-M. Shen et al. Slow-Channel Myasthenia Mutation in AChR-d Subunit
JCI insight, Jan 25, 2018
We identify 2 homozygous mutations in the ε-subunit of the muscle acetylcholine receptor (AChR) i... more We identify 2 homozygous mutations in the ε-subunit of the muscle acetylcholine receptor (AChR) in 3 patients with severe congenital myasthenia: εR218W in the pre-M1 region in 2 patients and εE184K in the β8-β9 linker in 1 patient. Arg218 is conserved in all eukaryotic members of the Cys-loop receptor superfamily, while Glu184 is conserved in the α-, δ-, and ε-subunits of AChRs from all species. εR218W reduces channel gating efficiency 338-fold and AChR expression on the cell surface 5-fold, whereas εE184K reduces channel gating efficiency 11-fold but does not alter AChR cell surface expression. Determinations of the effective channel gating rate constants, combined with mutant cycle analyses, demonstrate strong energetic coupling between εR218 and εE184, and between εR218 and εE45 from the β1-β2 linker, as also observed for equivalent residues in the principal coupling pathway of the α-subunit. Thus, efficient and rapid gating of the AChR channel is achieved not only by coupling be...
Neuromuscular disorders : NMD, Jan 28, 2017
Congenital myasthenic syndromes (CMS) are a group of hereditary disorders affecting the neuromusc... more Congenital myasthenic syndromes (CMS) are a group of hereditary disorders affecting the neuromuscular junction. Here, we present clinical, electrophysiological and genetic findings of 69 patients from 51 unrelated kinships from Turkey. Genetic tests of 60 patients were performed at Mayo Clinic. Median follow-up time was 9.8 years (range 1-22 years). The most common CMS was primary acetylcholine receptor (AChR) deficiency (31/51) and the most common mutations in AChR were c.1219 + 2T > G (12/51) and c.1327delG (6/51) in CHRNE. Four of our 5 kinships with AChE deficiency carried p.W148X that truncates the collagen domain of COLQ, and was previously reported only in patients from Turkey. These were followed by GFPT1 deficiency (4/51), DOK7 deficiency (3/51), slow channel CMS (3/51), fast channel CMS (3/51), choline acetyltransferase deficiency (1/51) and a CMS associated with desmin deficiency (1/51). Distribution of muscle weakness was sometimes useful in giving a clue to the CMS s...
Journal of the neurological sciences, May 15, 2017
Neuromuscular disorders : NMD, Jan 11, 2017
Monoclonal gammopathies due to plasma cell dyscrasias can induce diverse rare neuromuscular disor... more Monoclonal gammopathies due to plasma cell dyscrasias can induce diverse rare neuromuscular disorders. Deposition of monoclonal antibody light chains in skeletal muscle causes amyloid myopathy. Monoclonal gammopathy is occasionally associated with sporadic late-onset nemaline myopathy. Here we report a monoclonal gammopathy patient with both sporadic late-onset nemaline myopathy and amyloid myopathy. The diagnoses were based on immunofixation electrophoresis of urine, and serum for free light chain assay, Congo red staining and Thioflavin S staining of muscle biopsies, as well as immunohistochemical staining and electron-microscopic observation. Nemaline myopathy and amyloid myopathy can present in the same patient with monoclonal gammopathy.
Annals of clinical and translational neurology, 2017
To identify the molecular basis and elucidate the pathogenesis of a fatal congenital myasthenic s... more To identify the molecular basis and elucidate the pathogenesis of a fatal congenital myasthenic syndrome. We performed clinical electrophysiology studies, exome and Sanger sequencing, and analyzed functional consequences of the identified mutation. Clinical electrophysiology studies of the patient revealed several-fold potentiation of the evoked muscle action potential by high frequency nerve stimulation pointing to a presynaptic defect. Exome sequencing identified a homozygous c.340delA frameshift mutation in synaptobrevin 1 (SYB1), one of the three SNARE proteins essential for synaptic vesicle exocytosis. Analysis of both human spinal cord gray matter and normal human muscle revealed expression of the SYB1A and SYB1D isoforms, predicting expression of one or both isoforms in the motor nerve terminal. The identified mutation elongates the intravesicular C-terminus of the A isoform from 5 to 71, and of the D isoform from 4 to 31 residues. Transfection of either mutant isoform into b...
Human mutation, Jan 4, 2016
We identify two novel mutations in acetylcholine receptor (AChR) causing a slow-channel congenita... more We identify two novel mutations in acetylcholine receptor (AChR) causing a slow-channel congenital myasthenia syndrome (CMS) in three unrelated patients (Pts). Pt 1 harbors a heterozygous βV266A mutation (p.Val289Ala) in the second transmembrane domain (M2) of the AChR β subunit (CHRNB1). Pts 2 and 3 carry the same mutation at an equivalent site in the ε subunit (CHRNE), εV265A (p.Val285Ala). The mutant residues are conserved across all AChR subunits of all species and are components of a valine ring in the channel pore which is positioned four residues above the leucine ring. Both βV266A and εV265A reduce the amino acid size and lengthen the channel opening bursts by 4.0-fold by enhancing gating efficiency by approximately 30-fold. Substitution of alanine for valine at the corresponding position in the δ and α subunit prolongs the burst duration 4- and 8-fold, respectively. Replacing valine at ε codon 265 either by a still smaller glycine or by a larger leucine also lengthens the b...
The Journal of Clinical Investigation, 2008
Journal of Biological Chemistry, 2015
We identify two heteroallelic mutations in the acetylcholine receptor ␦-subunit from a patient wi... more We identify two heteroallelic mutations in the acetylcholine receptor ␦-subunit from a patient with severe myasthenic symptoms since birth: a novel ␦D140N mutation in the signature Cysloop and a mutation in intron 7 of the ␦-subunit gene that disrupts splicing of exon 8. The mutated Asp residue, which determines the disease phenotype, is conserved in all eukaryotic members of the Cys-loop receptor superfamily. Studies of the mutant acetylcholine receptor expressed in HEK 293 cells reveal that ␦D140N attenuates cell surface expression and apparent channel gating, predicting a reduced magnitude and an accelerated decay of the synaptic response, thus reducing the safety margin for neuromuscular transmission. Substituting Asn for Asp at equivalent positions in the ␣-, -, and ⑀-subunits also suppresses apparent channel gating, but the suppression is much greater in the ␣-subunit. Mutant cycle analysis applied to single and pairwise mutations reveals that ␣Asp-138 is energetically coupled to ␣Arg-209 in the neighboring pre-M1 domain. Our findings suggest that the conserved ␣Asp-138 and ␣Arg-209 contribute to a principal pathway that functionally links the ligand binding and pore domains.
Neuron, 1998
et al., 1995). A functionally similar mutation, located in the N-terminal portion of the M1 domai... more et al., 1995). A functionally similar mutation, located in the N-terminal portion of the M1 domain (␣N217K), also enhances resting state ACh affinity and slows the rate of channel closing (Engel et al., 1996b; Wang et al., 1997). The physiologic converse of the SCCMS-the low
Neuromuscular disorders : NMD, 2011
Centronuclear myopathy is a pathologically diagnosed congenital myopathy. The disease genes encod... more Centronuclear myopathy is a pathologically diagnosed congenital myopathy. The disease genes encode proteins with membrane modulating properties (MTM1, DNM2, and BIN1) or alter excitation-contraction coupling (RYR1). Some patients also have myasthenic symptoms but electrodiagnostic and endplate studies in these are limited. A sporadic patient had fatigable weakness and a decremental EMG response. Analysis of centronuclear myopathy disease- and candidate-genes identified no mutations. Quantitative endplate electron microscopy studies revealed simplified postsynaptic regions, endplate remodeling with normal nerve terminal size, normal synaptic vesicle density, and mild acetylcholine receptor deficiency. The amplitude of the miniature endplate potential was decreased to 60% of normal. Quantal release by nerve impulse was reduced to 40% of normal due to a decreased number of releasable quanta. The safety margin of neuromuscular transmission is compromised by decreased quantal release by ...
Annals of Neurology, 2008
Objective-Detailed analysis of phenotypic and molecular genetic aspects of Dok-7 myasthenia in 16... more Objective-Detailed analysis of phenotypic and molecular genetic aspects of Dok-7 myasthenia in 16 patients. Methods-We assessed our patients by clinical and electromyographic studies, by intercostal muscle biopsies for in vitro microelectrode analysis of neuromuscular transmission and quantitative electron microscopy EM of 409 end plates (EPs), and by mutation analysis, and expression studies of the mutants. Results-The clinical spectrum varied from mild static limb-girdle weakness to severe generalized progressive disease. The synaptic contacts were single or multiple, and some, but not all, were small. In vitro microelectrode studies indicated variable decreases of the number of released quanta and of the synaptic response to acetylcholine; acetylcholine receptor (AChR) channel kinetics were normal. EM analysis demonstrated widespread and previously unrecognized destruction and remodeling of the EPs. Each patient carries 2 or more heteroallelic mutations: 11 in genomic DNA, 7 of which are novel; and 6 identifiable only in complementary DNA or cloned complementary DNA, 3 of which are novel. The pathogenicity of the mutations was confirmed by expression studies. Although the functions of Dok-7 include AChR β-subunit phosphorylation and maintaining AChR site density, patient EPs showed normal AChR β-subunit phosphorylation, and the AChR density on the remaining junctional folds appeared normal.
Human Molecular Genetics, 2003
A frameshifting 7 bp deletion (e553del7) in exon 7 of CHRNE encoding the acetylcholine receptor e... more A frameshifting 7 bp deletion (e553del7) in exon 7 of CHRNE encoding the acetylcholine receptor e subunit, observed in seven congenital myasthenic syndrome patients, enhances expression of an aberrantly spliced transcript that skips the preceding 101 bp exon 6. To recapitulate the aberrant splicing, we cloned the entire CHRNE spanning 12 exons and 11 introns and expressed it in COS cells. Scanning mutagenesis revealed that e553del7 does not disrupt an exonic splicing enhancer. Inhibition of protein synthesis and of nonsensemediated mRNA decay (NMD) by anisomycin shows that even wild-type CHRNE produces an exon 6-skipped transcript, and that even e553del7-CHRNE yields a normally spliced transcript. Both transcripts, however, are degraded by NMD due to a premature stop codon. In contrast, the normally spliced transcript from wild-type CHRNE and the exon 6-skipped transcript from e553del7-CHRNE carry no premature stop codon and hence are immune to NMD. Optimization of splicing signals for exon 6 prevents it being skipped even in the presence of anisomycin and/or e553del7, indicating that inherently weak splicing signals for exon 6 account for its skipping. We suggest that a similar mechanism probably operates in other genes in skipping of remote exons. The presence of weak splicing signals for exon 6 also prompted us to search for mutations in exon 6 that disrupt an exonic splicing enhancer. Indeed, we found that eEF157V and eE154X in exon 6, observed in two other patients, caused aberrant splicing of exon 6.
Annals of the New York Academy of Sciences, 1998
Annals of Clinical and Translational Neurology
Objective: To characterize the molecular and phenotypic basis of a severe slow-channel congenital... more Objective: To characterize the molecular and phenotypic basis of a severe slow-channel congenital myasthenic syndrome (SCCMS). Methods: Intracellular and single-channel recordings from patient endplates; alpha-bungarotoxin binding studies; direct sequencing of AChR genes; microsatellite analysis; kinetic analysis of AChR activation; homology modeling of adult human AChR structure. Results: Among 24 variants reported to cause SCCMS only two appear in the AChR d-subunit. We here report a 16-year-old patient harboring a novel dL273F mutation (dL294F in HGVS nomenclature) in the second transmembrane domain (M2) of the AChR d subunit. Kinetic analyses with ACh and the weak agonist choline indicate that dL273F prolongs the channel opening bursts 9.4-fold due to a 75-fold increase in channel gating efficiency, whereas a previously identified eL269F mutation (eL289F in HGVS nomenclature) at an equivalent location in the AChR e-subunit prolongs channel opening bursts 4.4-fold due to a 30-fold increase in gating efficiency. Structural modeling of AChR predicts that inter-helical hydrophobic interactions between the mutant residue in the d and e subunit and nearby M2 domain residues in neighboring a subunits contribute to structural stability of the open relative to the closed channel states. Interpretation: The greater increase in gating efficiency by dL273F than by eL269F explains why dL273F has more severe clinical effects. Both dL273F and eL269F impair channel gating by disrupting hydrophobic interactions with neighboring a-subunits. Differences in the extent of impairment of channel gating in d and e mutant receptors suggest unequal contributions of e/a and d/a subunit pairs to gating efficiency. 2067 X.-M. Shen et al. Slow-Channel Myasthenia Mutation in AChR-d Subunit
JCI insight, Jan 25, 2018
We identify 2 homozygous mutations in the ε-subunit of the muscle acetylcholine receptor (AChR) i... more We identify 2 homozygous mutations in the ε-subunit of the muscle acetylcholine receptor (AChR) in 3 patients with severe congenital myasthenia: εR218W in the pre-M1 region in 2 patients and εE184K in the β8-β9 linker in 1 patient. Arg218 is conserved in all eukaryotic members of the Cys-loop receptor superfamily, while Glu184 is conserved in the α-, δ-, and ε-subunits of AChRs from all species. εR218W reduces channel gating efficiency 338-fold and AChR expression on the cell surface 5-fold, whereas εE184K reduces channel gating efficiency 11-fold but does not alter AChR cell surface expression. Determinations of the effective channel gating rate constants, combined with mutant cycle analyses, demonstrate strong energetic coupling between εR218 and εE184, and between εR218 and εE45 from the β1-β2 linker, as also observed for equivalent residues in the principal coupling pathway of the α-subunit. Thus, efficient and rapid gating of the AChR channel is achieved not only by coupling be...
Neuromuscular disorders : NMD, Jan 28, 2017
Congenital myasthenic syndromes (CMS) are a group of hereditary disorders affecting the neuromusc... more Congenital myasthenic syndromes (CMS) are a group of hereditary disorders affecting the neuromuscular junction. Here, we present clinical, electrophysiological and genetic findings of 69 patients from 51 unrelated kinships from Turkey. Genetic tests of 60 patients were performed at Mayo Clinic. Median follow-up time was 9.8 years (range 1-22 years). The most common CMS was primary acetylcholine receptor (AChR) deficiency (31/51) and the most common mutations in AChR were c.1219 + 2T > G (12/51) and c.1327delG (6/51) in CHRNE. Four of our 5 kinships with AChE deficiency carried p.W148X that truncates the collagen domain of COLQ, and was previously reported only in patients from Turkey. These were followed by GFPT1 deficiency (4/51), DOK7 deficiency (3/51), slow channel CMS (3/51), fast channel CMS (3/51), choline acetyltransferase deficiency (1/51) and a CMS associated with desmin deficiency (1/51). Distribution of muscle weakness was sometimes useful in giving a clue to the CMS s...
Journal of the neurological sciences, May 15, 2017
Neuromuscular disorders : NMD, Jan 11, 2017
Monoclonal gammopathies due to plasma cell dyscrasias can induce diverse rare neuromuscular disor... more Monoclonal gammopathies due to plasma cell dyscrasias can induce diverse rare neuromuscular disorders. Deposition of monoclonal antibody light chains in skeletal muscle causes amyloid myopathy. Monoclonal gammopathy is occasionally associated with sporadic late-onset nemaline myopathy. Here we report a monoclonal gammopathy patient with both sporadic late-onset nemaline myopathy and amyloid myopathy. The diagnoses were based on immunofixation electrophoresis of urine, and serum for free light chain assay, Congo red staining and Thioflavin S staining of muscle biopsies, as well as immunohistochemical staining and electron-microscopic observation. Nemaline myopathy and amyloid myopathy can present in the same patient with monoclonal gammopathy.
Annals of clinical and translational neurology, 2017
To identify the molecular basis and elucidate the pathogenesis of a fatal congenital myasthenic s... more To identify the molecular basis and elucidate the pathogenesis of a fatal congenital myasthenic syndrome. We performed clinical electrophysiology studies, exome and Sanger sequencing, and analyzed functional consequences of the identified mutation. Clinical electrophysiology studies of the patient revealed several-fold potentiation of the evoked muscle action potential by high frequency nerve stimulation pointing to a presynaptic defect. Exome sequencing identified a homozygous c.340delA frameshift mutation in synaptobrevin 1 (SYB1), one of the three SNARE proteins essential for synaptic vesicle exocytosis. Analysis of both human spinal cord gray matter and normal human muscle revealed expression of the SYB1A and SYB1D isoforms, predicting expression of one or both isoforms in the motor nerve terminal. The identified mutation elongates the intravesicular C-terminus of the A isoform from 5 to 71, and of the D isoform from 4 to 31 residues. Transfection of either mutant isoform into b...
Human mutation, Jan 4, 2016
We identify two novel mutations in acetylcholine receptor (AChR) causing a slow-channel congenita... more We identify two novel mutations in acetylcholine receptor (AChR) causing a slow-channel congenital myasthenia syndrome (CMS) in three unrelated patients (Pts). Pt 1 harbors a heterozygous βV266A mutation (p.Val289Ala) in the second transmembrane domain (M2) of the AChR β subunit (CHRNB1). Pts 2 and 3 carry the same mutation at an equivalent site in the ε subunit (CHRNE), εV265A (p.Val285Ala). The mutant residues are conserved across all AChR subunits of all species and are components of a valine ring in the channel pore which is positioned four residues above the leucine ring. Both βV266A and εV265A reduce the amino acid size and lengthen the channel opening bursts by 4.0-fold by enhancing gating efficiency by approximately 30-fold. Substitution of alanine for valine at the corresponding position in the δ and α subunit prolongs the burst duration 4- and 8-fold, respectively. Replacing valine at ε codon 265 either by a still smaller glycine or by a larger leucine also lengthens the b...
The Journal of Clinical Investigation, 2008
Journal of Biological Chemistry, 2015
We identify two heteroallelic mutations in the acetylcholine receptor ␦-subunit from a patient wi... more We identify two heteroallelic mutations in the acetylcholine receptor ␦-subunit from a patient with severe myasthenic symptoms since birth: a novel ␦D140N mutation in the signature Cysloop and a mutation in intron 7 of the ␦-subunit gene that disrupts splicing of exon 8. The mutated Asp residue, which determines the disease phenotype, is conserved in all eukaryotic members of the Cys-loop receptor superfamily. Studies of the mutant acetylcholine receptor expressed in HEK 293 cells reveal that ␦D140N attenuates cell surface expression and apparent channel gating, predicting a reduced magnitude and an accelerated decay of the synaptic response, thus reducing the safety margin for neuromuscular transmission. Substituting Asn for Asp at equivalent positions in the ␣-, -, and ⑀-subunits also suppresses apparent channel gating, but the suppression is much greater in the ␣-subunit. Mutant cycle analysis applied to single and pairwise mutations reveals that ␣Asp-138 is energetically coupled to ␣Arg-209 in the neighboring pre-M1 domain. Our findings suggest that the conserved ␣Asp-138 and ␣Arg-209 contribute to a principal pathway that functionally links the ligand binding and pore domains.