The nicotinic acetylcholine receptor is the prototype ligand-gated ion channel. A number of aromatic amino acids have been identified as contributing to the agonist binding site, suggesting that cation-interactions may be involved in binding the quaternary ammonium group of the agonist, acetylcholine. Here we show a compelling correlation between: (i) ab initio quantum mechanical predictions of cation-binding abilities and (ii) EC 50 values for acetylcholine at the receptor for a series of tryptophan derivatives that were incorporated into the receptor by using the in vivo nonsense-suppression method for unnatural amino acid incorporation. Such a correlation is seen at one, and only one, of the aromatic residues-tryptophan-149 of the ␣ subunit. This finding indicates that, on binding, the cationic, quaternary ammonium group of acetylcholine makes van der Waals contact with the indole side chain of ␣ tryptophan-149, providing the most precise structural information to date on this receptor. Consistent with this model, a tethered quaternary ammonium group emanating from position ␣149 produces a constitutively active receptor.
Recent studies show that heterochromatin‐associated protein‐1 (HP1) recognizes a ‘histone code’ involving methylated Lys9 (methyl‐K9) in histone H3. Using in situ immunofluorescence, we demonstrate that methyl‐K9 H3 and HP1 co‐localize to the heterochromatic regions of Drosophila polytene chromosomes. NMR spectra show that methyl‐K9 binding of HP1 occurs via its chromo (chromosome organization modifier) domain. This interaction requires methyl‐K9 to reside within the proper context of H3 sequence. NMR studies indicate that the methylated H3 tail binds in a groove of HP1 consisting of conserved residues. Using fluorescence anisotropy and isothermal titration calorimetry, we determined that this interaction occurs with a KD of ∼100 μM, with the binding enthalpically driven. A V26M mutation in HP1, which disrupts its gene silencing function, severely destabilizes the H3‐binding interface, and abolishes methyl‐K9 H3 tail binding. Finally, we note that sequence diversity in chromo domains may lead to diverse functions in eukaryotic gene regulation. For example, the chromo domain of the yeast histone acetyltransferase Esa1 does not interact with methyl‐ K9 H3, but instead shows preference for unmodified H3 tail.
Brain-derived neurotrophic factor (BDNF), like other neurotrophins, has long-term effects on neuronal survival and differentiation; furthermore, recent work has shown that BDNF also can induce rapid changes in synaptic efficacy. We have investigated the mechanism(s) of these synaptic effects on cultured embryonic hippocampal neurons. In the presence of the GABAA receptor antagonist, picrotoxin, the application of BDNF (100 ng/ml) for 1-5 min increased the amplitude of evoked synaptic currents by 48 +/- 9% in 10 of 15 pairs of neurons and increased the frequency of EPSC bursts to 205 +/- 20% of the control levels. There was no detectable effect of BDNF on various measures of electrical excitability, including the resting membrane potential, input resistance, action potential threshold, and action potential amplitude. In addition, BDNF did not change the postsynaptic currents induced by the exogenous application of glutamate. BDNF did increase the frequency of miniature EPSCs (mEPSCs) (268.0 +/- 46.8% of control frequency), however, without affecting the mEPSC amplitude. The effect of BDNF on mEPSC frequency was blocked by the tyrosine kinase inhibitor K252a and also by the removal of extracellular calcium ([Ca2+]o). Fura-2 recordings showed that BDNF elicited an increase in intracellular calcium concentration ([Ca2+]c). This effect was dependent on [Ca2+]o; it was blocked by K252a and by thapsigargin, but not by caffeine. The results demonstrate that BDNF enhances glutamatergic synaptic transmission at a presynaptic locus and that this effect is accompanied by a rise in [Ca2+]c that requires the release of Ca2+ from IP3-gated stores.
Prolongation of relaxation is a hallmark of diabetic cardiomyopathy. Most studies attribute this defect to decreases in sarco(endo)plasmic reticulum Ca 2؉ -ATPase (SERCA2a) expression and SERCA2a-to-phospholamban (PLB) ratio. Since its turnover rate is slow, SERCA2a is susceptible to posttranslational modifications during diabetes. These modifications could in turn compromise conformational rearrangements needed to translocate calcium ions, also leading to a decrease in SERCA2a activity. In the present study one such modification was investigated, namely advanced glycation end products (AGEs). Hearts from 8-week streptozotocin-induced diabetic (8D) rats showed typical slowing in relaxation, confirming cardiomyopathy. Hearts from 8D animals also expressed lower levels of SERCA2a protein and higher levels of PLB. Analysis of matrix-assisted laser desorption/ionization time-of-flight mass data files from trypsin-digested SERCA2a revealed several cytosolic SERCA2a peptides from 8D modified by single noncrosslinking AGEs. Crosslinked AGEs were also found. Lysine residues within actuator and phosphorylation domains were cross-linked to arginine residues within the nucleotide binding domain via pentosidine AGEs. Two weeks of insulin-treatment initiated after 6 weeks of diabetes attenuated these changes. These data demonstrate for the first time that AGEs are formed on SERCA2a during diabetes, suggesting a novel mechanism by which cardiac relaxation can be slowed during diabetes. Diabetes 53: [463][464][465][466][467][468][469][470][471][472][473] 2004 R eductions in rate and force of cardiac contractions are root causes for the increased incidence of morbidity and mortality among diabetic patients (1-3). Studies show that this "diabetic cardiomyopathy" is independent of coronary vascular diseases and is brought about by shifts in metabolism, cellular biochemistry, and structure (4 -8). At the molecular level, decreases in chronotropy and inotropy result from alterations in expression and/or function of several sarcolemmal membrane receptors and associated signal transduction proteins as well as other key proteins involved in regulating/maintaining intracellular ionic homeostasis (9 -11). Of particular interest is a transport protein on the sarcoplasmic reticular membrane that plays an integral role in cardiac relaxation. This protein, referred to as sarco(endo)plasmic reticulum Ca 2ϩ -ATPase (SERCA2a), is responsible for replenishing intracellular calcium stores following release and in so doing terminate contraction.SERCA2a is a member of a large family of P-type ATPase enzymes that utilizes the energy generated from hydrolysis of terminal phosphate bond of ATP to pump calcium against its electrochemical gradient (12,13). SERCA1a is the best studied of these single polypeptides. It consists of 10 transmembrane helixes (M1 through M10) and three cytoplasmic domains, referred to as A (actuator), N (nucleotide binding) and P (phosphorylation) domains (14). Translocation of calcium ions from the cytosol to the lumen of...
An approach to identify backbone conformational changes underlying nicotinic acetylcholine receptor (nAChR) gating was developed. Specific backbone peptide bonds were replaced with an ester, which disrupts backbone hydrogen bonds at the site of mutation. At a conserved proline residue (alphaPro221) in the first transmembrane (M1) domain, the amide-to-ester mutation provides receptors with near-normal sensitivity, although the natural amino acids tested other than Pro produce receptors that gate with a much larger EC50 than normal. Therefore, a backbone hydrogen bond at this site may interfere with normal gating. In the alphaM2 domain, the amide-to-ester mutation yielded functional receptors at 15 positions, 3 of which provided receptors with >10-fold lower EC50 than wild type. These results support a model for gating that includes significant changes of backbone conformation within the M2 domain.
Cyclic nucleotide-gated (cng) channels are important components of signaling systems mediating sensory transduction. In vertebrate photoreceptors, light activates a signaling cascade that causes a decrease in intracellular cGMP concentrations, closing retinal cng channels. Signal transduction in olfactory receptor neurons is believed to proceed via G-proteinmediated elevation of intracellular cAMP in response to odorant binding by 7-helix receptors. cAMP opens the olfactory cng channel, which is highly permeable to Ca 2ϩ . Here we demonstrate by in situ hybridization and immunohistochemistry with subunit-specific antibodies that both subunits of the heteromeric rat olfactory cng channel are also widely expressed in the brain. Expression of the retinal rod cng channel, however, can be detected only in the eye. In the adult hippocampus, the olfactory cng channel is expressed on cell bodies and processes of CA1 and CA3 neurons. In cultured embryonic hippocampal neurons, the channel is localized to a subset of growth cones and processes. We recorded conductances with the electrophysiological characteristics of the heteromeric olfactory cng channel in excised inside-out patches from these cultured neurons. We also show that Ca 2ϩ influx into hippocampal neurons in response to cyclic nucleotide elevation can be detected using fura-2 imaging. Cyclic nucleotide elevation has been implicated in several mechanisms of synaptic plasticity in the hippocampus, and these mechanisms also require elevation of intracellular Ca 2ϩ . Our results suggest that the "olfactory" cng channel could regulate synaptic efficacy in brain neurons by modulating Ca 2ϩ levels in response to changes in cyclic nucleotide concentrations. Key words: cyclic nucleotide-gated channels; cAMP; cGMP; olfaction; sensory transduction; hippocampus; synaptic plasticityCyclic nucleotide-gated (cng) channels open in response to binding of the intracellular cyclic nucleotides cGMP and cAMP. They resemble voltage-gated channels, having six transmembrane domains and a pore region, but they also contain a cytoplasmic region with homology to the cyclic nucleotide binding domains of cGMP-and cAMP-dependent protein kinases (PKG and PKA). The native retinal rod and olfactory cng channels are believed to be heterotetramers composed of two different subunits. The ␣ subunits [also known in the rat as rRCNC1 (retinal) and rOCNC1 (olfactory)] can form homomeric channels (for review, see Zagotta and Siegelbaum, 1996). The  subunits (rRCNC2 and rOCNC2) cannot form functional channels on their own, but they oligomerize with the ␣ subunits to create channels with modified properties (Chen et al., 1993;Bradley et al., 1994;Liman and Buck, 1994;Korschen et al., 1995). Unlike most voltage-gated and ligand-gated channels, cng channels do not exhibit desensitization but remain open continuously in the presence of cyclic nucleotide.The incorporation of the rOCNC2 subunit into the olfactory cng channel dramatically affects cyclic nucleotide sensitivity. The heteromeric rOCNC1/rOCNC2 c...
Decrease in cardiac contractility is a hallmark of chronic diabetes. Previously we showed that this defect results, at least in part, from a dysfunction of the type 2 ryanodine receptor calcium-release channel (RyR2). The mechanism(s) underlying RyR2 dysfunction is not fully understood. The present study was designed to determine whether non-cross-linking advanced glycation end products (AGEs) on RyR2 increase with chronic diabetes and if formation of these post-translational complexes could be attenuated with insulin treatment. Overnight digestion of RyR2 from 8-week control animals (8C) with trypsin afforded 298 peptides with monoisotopic mass (M؉H ؉ ) >500. Digestion of RyR2 from 8-week streptozotocin-induced diabetic animals (8D) afforded 21% fewer peptides, whereas RyR2 from 6-week diabetic/2-week insulin-treated animals generated 304 peptides. Using an in-house PERLscript algorithm, search of matrix-assisted laser desorption ionization-time of flight mass data files identified several M؉H ؉ peaks corresponding to theoretical RyR2 peptides with single N ⑀ -(carboxymethyl)-lysine, imidazolone A, imidazone B, pyrraline, or 1-alkyl-2-formyl-3,4-glycosyl pyrrole modification that were present in 8D but not 8C. Insulin treatment minimized production of some of these nonenzymatic glycation products. These data show for the first time that AGEs are formed on intracellular RyR2 during diabetes. Because AGE complexes are known to compromise protein activity, these data suggest a potential mechanism for diabetesinduced RyR2 dysfunction. Diabetes 52:1825-1836, 2003 A significant percentage of patients with diabetes (both type 1 and type 2) develop a unique cardiomyopathy that is independent of coronary atherosclerosis (1-3). This "diabetic cardiomyopathy" as it is termed starts off with asymptomatic left ventricular diastolic dysfunction (slowing of relaxation kinetics). As the disease progresses, systolic function becomes compromised, leading to an increase in incidence of morbidity and mortality (4 -6).The release of calcium ions from internal sarcoplasmic reticulum via the type 2 ryanodine receptor calciumrelease channel (RyR2) is an integral step in the cascade of events leading to cardiac muscle contraction (7). We and others have shown that expression of this protein decreases in hearts of chronic diabetic patients (8,9) as well as in the streptozotocin (STZ)-induced diabetic rats (10 -13). Using the latter model, we found that in addition to a decrease in expression of RyR2, its functional integrity is also compromised in diabetes (14,15). This dysfunction is manifested as a decrease in RyR2 ability to bind the specific ligand [ 3 H]ryanodine and a slowing in its electrophoretic mobility using denaturing SDS-PAGE.Two distinct and separate types of post-translational modifications are likely to be induced by diabetes. First, it is well known that metabolic changes brought about by diabetes increase production of reactive oxygen species (e.g., -18]). These free radical and nonradical species react with several a...
1. The kinetic properties of cloned mouse embryonic nicotinic acetylcholine receptors (AChRs) expressed in HEK 293 cells or Xenopus oocytes were examined using high concentrations of acetylcholine (ACh), carbamylcholine (CCh), or tetramethylammonium (TMA). The rate constants of agonist binding and channel gating were estimated by fitting kinetic models to idealized open and closed intervals over a range of agonist concentrations. 2. Once doubly liganded, TMA‐activated receptors open at approximately 3000 s‐1. The equilibrium binding constants for TMA are 525 and 12,800 microM. Doubly liganded CCh‐activated receptors open at approximately 11,500 s‐1; the equilibrium binding constants for this agonist are 14 and 570 microM. If we assume that doubly liganded, ACh‐activated receptors open at 60,000 s‐1, then the equilibrium binding constants for ACh are 20 and > 650 microM, similar to those for CCh. For all three agonists the higher affinity site both binds and releases agonists more slowly than does the lower affinity site. 3. ACh and CCh bind to the two sites equally rapidly, at approximately 2 x 10(7) and 4 x 10(7) M‐1 s‐1 at the first and second binding sites, respectively. Compared with ACh, the TMA association rate is approximately 100 times slower at the first binding site, and approximately 30 times slower at the second binding site. These results indicate that at both binding sites the association rate of TMA is not limited by diffusional or steric factors. 4. All three agonists dissociate from the receptor binding sites at similar rates. The dissociation rate for all agonists was approximately 40 times slower at the first binding site than at the second. These results suggest that the interaction of the quarternary amine moiety with the receptor determines the rate of release of the agonist, and that the nature of this interaction is quite different at the two binding sites. 5. Although the channel opening rates for the three agonists varied approximately 20‐fold, the channel closing rates were not strongly agonist dependent, and varied less than 3‐fold. We speculate that the ester moiety of the agonist promotes both rapid binding and fast opening of the ligand receptors, and that interactions of the quarternary amine moiety of the agonist with the receptor determine the channel closing rate constant.
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