Mutation in the M1 Domain of the Acetylcholine Receptor α Subunit Decreases the Rate of Agonist Dissociation

We describe the kinetic consequences of the mutation N217K in the M1 domain of the acetylcholine receptor (AChR) α subunit that causes a slow channel congenital myasthenic syndrome (SCCMS). We previously showed that receptors containing αN217K expressed in 293 HEK cells open in prolonged activation episodes strikingly similar to those observed at the SCCMS end plates. Here we use single channel kinetic analysis to show that the prolonged activation episodes result primarily from slowing of the rate of acetylcholine (ACh) dissociation from the binding site. Rate constants for channel opening and closing are also slowed but to much smaller extents. The rate constants derived from kinetic analysis also describe the concentration dependence of receptor activation, revealing a 20-fold shift in the EC₅₀ to lower agonist concentrations for αN217K. The apparent affinity of ACh binding, measured by competition against the rate of ¹²⁵I-α-bungarotoxin binding, is also enhanced 20-fold by αN217K. Both the slowing of ACh dissociation and enhanced apparent affinity are specific to the lysine substitution, as the glutamine and glutamate substitutions have no effect. Substituting lysine for the equivalent asparagine in the β, ε, or δ subunits does not affect the kinetics of receptor activation or apparent agonist affinity. The results show that a mutation in the amino-terminal portion of the M1 domain produces a localized perturbation that stabilizes agonist bound to the resting state of the AChR.     

Reactivity of Cysteines in the Transmembrane Region of the Na,K-ATPase α Subunit Probed with Hg2+

To gain insight into the structure and conformational coupling in the Na,K-ATPase, this study characterized the reaction of the α1 subunit transmembrane cysteines with a small probe. Intact HeLa cells expressing heterologous Na,K-ATPase were treated with (μm) HgCl₂ after placing the enzyme predominantly in either of two conformations, phosphorylated E2P.Na/E2P or dephosphorylated ATP.E1.K/ATP.E1. Under both conditions the treatment led to enzyme inactivation following a double exponential kinetic as determined by ouabain-sensitive K⁺ uptake measurements. However, the rate constant of the slow reacting component was ten times larger when the protein was probed in a medium that would favor enzyme phosphorylation. Enzymes carrying mutations of cysteines located in the α1 subunit transmembrane region were used to identify the reacting–SH groups. Replacement Cys104Ser reduced enzyme inactivation by removing the slow reacting component under both treatment conditions. Replacement of Cys964 reduced the inactivation rate constant of the fast reacting component (79%) and removed the slow reacting component when the dephosphorylated enzyme was treated with Hg²⁺. Moreover, Cys964Ser substituted enzyme was insensitive to Hg²⁺ when treated under phosphorylation conditions. These results indicate that Cys964 is involved in the fast inactivation by Hg²⁺. Although the double mutant Cys964, 104Ser was still partially inactivated by treatment under nonphosphorylating conditions, an enzyme devoid of transmembrane cysteines was insensitive to Hg²⁺ under all treatment conditions. Thus, this enzyme provides a background where accessibility of engineered transmembrane cysteines can be tested. Received: 13 March 2000/Revised: 23 June 2000     

Species- and Subtype-Specific Recognition by Antibody WF6 of a Sequence Segment Forming an α-Bungarotoxin Binding Site on the Nicotinic Acetylcholine Receptor α Subunit

Abstract

The monoclonal antibody WF6 competes with acetylcholine and α-bungarotoxin (α-BGT) for binding to the Torpedo nicotinic acetylcholine receptor (nAChR) α1 subunit. Using synthetic peptides corresponding to the complete Torpedo nAChR α1 subunit, we previously mapped a continuous epitope recognized by WF6, and the prototope for α-BGT, to the sequence segment α1(181–200). Single amino acid substitution analogs have been used as an initial approach to determine the critical amino acids for WF6 and α-BGT binding. In the present study, we continue our analysis of the structural features of the WF6 epitope by comparing its cross-reactivity with synthetic peptides corresponding to the α1 subunits from the muscle nAChRs of different species, the rat brain α2, α3, α4 and α5 nAChR subtypes, and the chick brain α-BGT binding protein subunits, αBGTBP α1 and αBGTBP α2. Our results indicate that WF6 is able to cross-react with the muscle α1 subunits of different species by virtue of conservation of several critical amino acid residues between positions 190–198 of the α1 subunit. These studies further define the essential structural features of the sequence segment α1(181–200) required to form the epitope for WF6.     

Determinants in the β and δ Subunit Cytoplasmic Loop Regulate Golgi Trafficking and Surface Expression of the Muscle Acetylcholine Receptor

The molecular determinants that govern nicotinic acetylcholine receptor (AChR) assembly and trafficking are poorly defined, and those identified operate largely during initial receptor biogenesis in the endoplasmic reticulum. To identify determinants that regulate later trafficking steps, we performed an unbiased screen using chimeric proteins consisting of CD4 fused to the muscle AChR subunit cytoplasmic loops. In C2 mouse muscle cells, we found that CD4-β and δ subunit loops were expressed at very low levels on the cell surface, whereas the other subunit loops were robustly expressed on the plasma membrane. The low surface expression of CD4-β and δ loops was due to their pronounced retention in the Golgi apparatus and also to their rapid internalization from the plasma membrane. Both retention and recovery were mediated by the proximal 25–28 amino acids in each loop and were dependent on an ordered sequence of charged and hydrophobic residues. Indeed, βK353L and δK351L mutations increased surface trafficking of the CD4-subunit loops by >6-fold and also decreased their internalization from the plasma membrane. Similarly, combined βK353L and δK351L mutations increased the surface levels of assembled AChR expressed in HEK cells to 138% of wild-type levels. This was due to increased trafficking to the plasma membrane and not decreased AChR turnover. These findings identify novel Golgi retention signals in the β and δ subunit loops that regulate surface trafficking of assembled AChR and may help prevent surface expression of unassembled subunits. Together, these results define molecular determinants that govern a Golgi-based regulatory step in nicotinic AChR trafficking.     

Crystal Structure of the Human Pol α B Subunit in Complex with the C-terminal Domain of the Catalytic Subunit

In eukaryotic DNA replication, short RNA-DNA hybrid primers synthesized by primase-DNA polymerase α (Prim-Pol α) are needed to start DNA replication by the replicative DNA polymerases, Pol δ and Pol ϵ. The C terminus of the Pol α catalytic subunit (p180_C) in complex with the B subunit (p70) regulates the RNA priming and DNA polymerizing activities of Prim-Pol α. It tethers Pol α and primase, facilitating RNA primer handover from primase to Pol α. To understand these regulatory mechanisms and to reveal the details of human Pol α organization, we determined the crystal structure of p70 in complex with p180_C. The structured portion of p70 includes a phosphodiesterase (PDE) domain and an oligonucleotide/oligosaccharide binding (OB) domain. The N-terminal domain and the linker connecting it to the PDE domain are disordered in the reported crystal structure. The p180_C adopts an elongated asymmetric saddle shape, with a three-helix bundle in the middle and zinc-binding modules (Zn1 and Zn2) on each side. The extensive p180_C-p70 interactions involve 20 hydrogen bonds and a number of hydrophobic interactions resulting in an extended buried surface of 4080 Ų. Importantly, in the structure of the p180_C-p70 complex with full-length p70, the residues from the N-terminal to the OB domain contribute to interactions with p180_C. The comparative structural analysis revealed both the conserved features and the differences between the human and yeast Pol α complexes.     

Cloning of the Fatty Acid Synthetase β Subunit from Fission Yeast, Coexpression with the α Subunit, and Purification of the Intact Multifunctional Enzyme Complex

We have cloned and sequenced the fission yeast (Schizosaccharomyces pombe)fas1⁺gene, which encodes the fatty acid synthetase (FAS) β subunit, by applying a PCR technique to conserved regions in the β subunit of the α₆β₆types of FAS among different organisms. The deduced amino acid sequence of the Fas1 polypeptide, consisting of 2073 amino acids (M_r= 230,616), exhibits the 48.1% identity with the β subunit from the budding yeast (Saccharomyces cerevisiae). This subunit, with five different catalytic activities, bears four distinct domains, while the α subunit, the sequence of which was previously reported by Saitohet al.(S. Saitohet al.,1996,J. Cell Biol.134, 949–961), carries three domains. We have developed a co-expression system of the FAS α and β subunits by cotransformation of two expression vectors, containing thelsd1⁺/fas2⁺gene and thefas1⁺gene, into fission yeast cells. The isolated FAS complex showed quite high specific activity, of more than 4000 mU/mg, suggesting complete purification. Its molecular weight was determined by dynamic light scattering and ultracentrifugation analysis to be 2.1–2.4 × 10⁶, and one molecule of the FAS complex was found to contain approximately six FMN molecules. These results indicate that the FAS complex fromS. pombeforms a heterododecameric α₆β₆structure. Electron micrographs of the negatively stained molecule suggest that the complex adopts a unique barrel-shaped cage architecture.     

A Novel α2/α4 Subtype-selective Positive Allosteric Modulator of Nicotinic Acetylcholine Receptors Acting from the C-tail of an α Subunit

Positive allosteric modulators (PAMs) of nicotinic acetylcholine receptors (nAChR) are important therapeutic candidates as well as valuable research tools. We identified a novel type II PAM, (R)-7-bromo-N-(piperidin-3-yl)benzo[b]thiophene-2-carboxamide (Br-PBTC), which both increases activation and reactivates desensitized nAChRs. This compound increases acetylcholine-evoked responses of α2* and α4* nAChRs but is without effect on α3* or α6* nAChRs (* indicates the presence of other nAChR subunits). Br-BPTC acts from the C-terminal extracellular sequences of α4 subunits, which is also a PAM site for steroid hormone estrogens such as 17β-estradiol. Br-PBTC is much more potent than estrogens. Like 17β-estradiol, the non-steroid Br-PBTC only requires one α4 subunit to potentiate nAChR function, and its potentiation is stronger with more α4 subunits. This feature enables Br-BPTC to potentiate activation of (α4β2)(α6β2)β3 but not (α6β2)₂β3 nAChRs. Therefore, this compound is potentially useful in vivo for determining functions of different α6* nAChR subtypes. Besides activation, Br-BPTC affects desensitization of nAChRs induced by sustained exposure to agonists. After minutes of exposure to agonists, Br-PBTC reactivated short term desensitized nAChRs that have at least two α4 subunits but not those with only one. Three α4 subunits were required for Br-BPTC to reactivate long term desensitized nAChRs. These data suggest that higher PAM occupancy promotes channel opening more efficiently and overcomes short and long term desensitization. This C-terminal extracellular domain could be a target for developing subtype or state-selective drugs for nAChRs.     

Three-Dimensional Localization of the α and β Subunits and of the II-III Loop in the Skeletal Muscle L-type Ca2+ Channel

The L-type Ca²⁺ channel (dihydropyridine receptor (DHPR) in skeletal muscle acts as the voltage sensor for excitation-contraction coupling. To better resolve the spatial organization of the DHPR subunits (α_1s or Ca_V1.1, α₂, β_1a, δ1, and γ), we created transgenic mice expressing a recombinant β_1a subunit with YFP and a biotin acceptor domain attached to its N- and C- termini, respectively. DHPR complexes were purified from skeletal muscle, negatively stained, imaged by electron microscopy, and subjected to single-particle image analysis. The resulting 19.1-Å resolution, three-dimensional reconstruction shows a main body of 17 × 11 × 8 nm with five corners along its perimeter. Two protrusions emerge from either face of the main body: the larger one attributed to the α₂-δ1 subunit that forms a flexible hook-shaped feature and a smaller protrusion on the opposite side that corresponds to the II-III loop of Ca_V1.1 as revealed by antibody labeling. Novel features discernible in the electron density accommodate the atomic coordinates of a voltage-gated sodium channel and of the β subunit in a single docking possibility that defines the α1-β interaction. The β subunit appears more closely associated to the membrane than expected, which may better account for both its role in localizing the α_1s subunit to the membrane and its suggested role in excitation-contraction coupling.     

Acetylcholine Receptor Gating is Influenced by the Polarity of Amino Acids at Position 9′ in the M2 Domain

Ligand-gated ion channels contain a conserved leucine at position 9′ (L9′) in the M2 transmembrane domain. We used multiple substitutions at this position in the γ subunit of the mouse acetylcholine receptor (AChR) (γL9′) to examine the role of residue polarity at this position in the gating process at both the macroscopic and single-channel levels. The midpoint of the macroscopic dose-response relationship (EC₅₀) and the channel closing rate constant, α, decreased as the polarity of the residue at that position increased, suggesting a stabilization of the open state of the channel. Both parameters showed similar dependencies on the polarity of the substituted residue. These data support the notion that during AChR gating, the amino acid at the 9′ position moves into a polar environment, and that interactions between this residue and the polar environment determine the stability of the open state. Since this residue is conserved in all other members of the ligand-gated ion channel family, we suggest that a similar mechanism applies to the other members of the family. Received: 17 September 1999/Revised: 15 December 1999     

Antimutator Mutations in the α Subunit of Escherichia Coli DNA Polymerase III: Identification of the Responsible Mutations and Alignment with Other DNA Polymerases

The dnaE gene of Escherichia coli encodes the DNA polymerase (α subunit) of the main replicative enzyme, DNA polymerase III holoenzyme. We have previously identified this gene as the site of a series of seven antimutator mutations that specifically decrease the level of DNA replication errors. Here we report the nucleotide sequence changes in each of the different antimutator dnaE alleles. For each a single, but different, amino acid substitution was found among the 1,160 amino acids of the protein. The observed substitutions are generally nonconservative. All affected residues are located in the central one-third of the protein. Some insight into the function of the regions of polymerase III containing the affected residues was obtained by amino acid alignment with other DNA polymerases. We followed the principles developed in 1990 by M. Delarue et al. who have identified in DNA polymerases from a large number of prokaryotic and eukaryotic sources three highly conserved sequence motifs, which are suggested to contain components of the polymerase active site. We succeeded in finding these three conserved motifs in polymerase III as well. However, none of the amino acid substitutions responsible for the antimutator phenotype occurred at these sites. This and other observations suggest that the effect of these mutations may be exerted indirectly through effects on polymerase conformation and/or DNA/polymerase interactions.     

The β1-Subunit of the MaxiK Channel Associates with the Thromboxane A2 Receptor and Reduces Thromboxane A2 Functional Effects

The large conductance voltage- and Ca²⁺-activated K⁺ channel (MaxiK, BK_Ca, BK) is composed of four pore-forming α-subunits and can be associated with regulatory β-subunits. One of the functional roles of MaxiK is to regulate vascular tone. We recently found that the MaxiK channel from coronary smooth muscle is trans-inhibited by activation of the vasoconstricting thromboxane A₂ prostanoid receptor (TP), a mechanism supported by MaxiK α-subunit (MaxiKα)-TP physical interaction. Here, we examined the role of the MaxiK β1-subunit in TP-MaxiK association. We found that the β1-subunit can by itself interact with TP and that this association can occur independently of MaxiKα. Subcellular localization analysis revealed that β1 and TP are closely associated at the cell periphery. The molecular mechanism of β1-TP interaction involves predominantly the β1 extracellular loop. As reported previously, TP activation by the thromboxane A₂ analog U46619 caused inhibition of MaxiKα macroscopic conductance or fractional open probability (FP_o) as a function of voltage. However, the positive shift of the FP_o versus voltage curve by U46619 relative to the control was less prominent when β1 was coexpressed with TP and MaxiKα proteins (20 ± 6 mV, n = 7) than in cells expressing TP and MaxiKα alone (51 ± 7 mV, n = 7). Finally, β1 gene ablation reduced the EC₅₀ of the U46619 agonist in mediating aortic contraction from 18 ± 1 nm (n = 12) to 9 ± 1 nm (n = 12). The results indicate that the β1-subunit can form a tripartite complex with TP and MaxiKα, has the ability to associate with each protein independently, and diminishes U46619-induced MaxiK channel trans-inhibition as well as vasoconstriction.     

Alternative-Splicing in the Exon-10 Region of GABAA Receptor β2 Subunit Gene: Relationships between Novel Isoforms and Psychotic Disorders

Background

Non-coding single nucleotide polymorphisms (SNPs) in GABRB2, the gene for β₂-subunit of gamma-aminobutyric acid type A (GABA_A) receptor, have been associated with schizophrenia (SCZ) and quantitatively correlated to mRNA expression and alternative splicing.

Methods and Findings

Expression of the Exon 10 region of GABRB2 from minigene constructs revealed this region to be an “alternative splicing hotspot” that readily gave rise to differently spliced isoforms depending on intron sequences. This led to a search in human brain cDNA libraries, and the discovery of two novel isoforms, β_2S1 and β_2S2, bearing variations in the neighborhood of Exon-10. Quantitative real-time PCR analysis of postmortem brain samples showed increased β_2S1 expression and decreased β_2S2 expression in both SCZ and bipolar disorder (BPD) compared to controls. Disease-control differences were significantly correlated with SNP rs187269 in BPD males for both β_2S1 and β_2S2 expressions, and significantly correlated with SNPs rs2546620 and rs187269 in SCZ males for β_2S2 expression. Moreover, site-directed mutagenesis indicated that Thr³⁶⁵, a potential phosphorylation site in Exon-10, played a key role in determining the time profile of the ATP-dependent electrophysiological current run-down.

Conclusion

This study therefore provided experimental evidence for the importance of non-coding sequences in the Exon-10 region in GABRB2 with respect to β₂-subunit splicing diversity and the etiologies of SCZ and BPD.     

Down Regulated Expression of the β1 Subunit of the Big-conductance Ca^2＋ Sensitive K＋ Channel in Sphincter of Oddi Cells from Rabbits Fed with a High Cholesterol Diet

Hypercholesterolemia,which is closely related to gallbladder bile stasis,can cause sphincter ofOddi dysfunction (SOD) by increasing the tension of sphincter of Oddi (SO).Intracellular calcium ionconcentration ([Ca~(2 )]_i) could influence the tension of SO.The β1 subunit of the big-conductance Ca~(2 )sensitive K~ channel (BK_(Ca) can enhance the sensitivity of the BK_(Ca) channel to [Ca~(2 )]_i.Absence and decline ofthe BK_(Ca) channel subunit β1 could lead to many diseases.However,the relationship betweenhypercholesterolemia and the expression of β1 subunit is not well understood.In this study,we successfullyexpressed and purified the rabbit BK_(Ca) β1 subunit protein and prepared its polyclonal antibody.The specificityof the prepared antibody was determined by western blotting.A SOD rabbit model induced by a high cholesteroldiet was established and the expression of the β1 subunit of SO was determined by immunohistochemicalstaining and western blotting.Compared with the controls,our results demonstrated that hypercholesterolemiacould decrease the expression of the β1 subunit in the SO cells from rabbits.This indicates that lowerexpression of BK_(Ca) channel β1 subunit might induce SOD.     

Conformational Studies of the 313-320 and 313-332 Peptide Fragments Derived from the αIIb Subunit of Integrin Receptor with Molecular Dynamics Simulations

The peptide sequence YMESRADRKLAEVGRVYLFL, derived from 313-332 region of the α_IIb, has been identified as a potent inhibitor of platelet aggregation and fibrinogen binding to α_IIbβ₃. More detailed studies have revealed that the Y₃₁₃MESRADR₃₂₀ sequence is the shortest octapeptide with strong inhibitory activity. This work provides insight of the solution conformation of these peptides, by performing extensive molecular dynamics simulations of 100 ns. The 8mer peptide has no stable conformation in water while the 20mer peptide retains a relative conformational stability. Analysis of side chain orientation of the RAD fragment revealed the synplanar arrangement of guanidinium and β-carboxylic groups providing a framework for explaining the similar biological activity of the two peptides, despite their differences in sequence and conformation.     

Serine 216 Phosphorylation of?Estrogen Receptor α in Neutrophils: Migration and Infiltration into the Mouse Uterus

Background

Whereas estrogen receptors are present in immune cells, it is not known if they are phosphorylated to regulate immune cell functions. Here we determined the phosphorylation status of estrogen receptor α (ERα) at residue serine 216 in mouse neutrophils and examined its　role in migration and infiltration. Serine 216 is the conserved phosphorylation site within the DNA binding domains found in the majority of nuclear receptors.

Methodology/Principal Findings

A phospho-peptide antibody specific to phosphorylated serine 216 and ERα KO mice were utilized in immunohistochemistry, double immuno-staining or Western blot to detect phosphorylation of ERα in peripheral blood as well as infiltrating neutrophils in the mouse uterus. Transwell assays were performed to examine migration of neutrophils. An anti-Ly6G antibody identified neutrophils. About 20% of neutrophils expressed phosphorylated ERα at serine 216 in peripheral white blood cells (WBC) from C3H/HeNCrIBR females. Phosphorylation was additively segregated between C3H/HeNCrIBR and C57BL/6 females. Only neutrophils that expressed phosphorylated ERα migrated in Transwell assays as well as infiltrated the mouse uterus during normal estrous cycles.

Conclusions/Significance

ERα was phosphorylated at serine 216 in about 20% of mouse peripheral blood neutrophils. Only those that express phosphorylated ERα migrate and infiltrate the mouse uterus. This phosphorylation was the first to be characterized in endogenous ERα found in normal tissues and cells. Phosphorylated ERα may have opened a novel research direction for biological roles of phosphorylation in ERα actions and can be developed as a drug target for treatment of immune-related diseases.     

5-Fluorotryptophan-Containing N-Terminal Domain of the α-Subunit of the Torpedo californica Acetylcholine Receptor: Preparation in Escherichia coli and 19F NMR Study

A protein corresponding to the extracellular 1–209 domain of the -subunit of the nicotine acetylcholine receptor from the electric organ of Torpedo californica was prepared using the corresponding cDNA domain by culturing Escherichia coli cells on a synthetic medium supplemented with 5-fluoro-L-tryptophan. The presence of a (His)₆ fragment preceding the 1–209 sequence allowed purification of the protein isolated from inclusion bodies by affinity chromatography on Ni-NTA Agarose. The incorporation of 5-fluorotryptophan residues was found by ¹⁹F NMR to be 50%. The spectrum of the protein reduced in the denaturing conditions and subsequently reoxidized in a dilute solution under denaturing conditions in the presence of 0.05% SDS was sufficiently resolved, which allowed partial assignment of ¹⁹F resonances using the Trp60Phe mutant protein. The ability of the prepared domains to specifically bind snake -neurotoxins was demonstrated with the use of radioiodinated -bungarotoxin and trifluoroacetylated -cobratoxin.