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A Auerbach

Publications and source records attributed to A Auerbach.

At least 37 records · Page 2Linked to original sources
PubMed1999

Length-dependent gametic CAG repeat instability in the Huntington's disease knock-in mouse.

The CAG repeats in the human Huntington's disease (HD) gene exhibit striking length-dependent intergenerational instability, typically small size increases or decreases of one to a few CAGs, but little variation in somatic tissues. In a subset of male transmissions, larger size increases occur to produce extreme HD alleles that display somatic instability and cause juvenile onset of the disorder. Initial efforts to reproduce these features in a mouse model transgenic for HD exon 1 with 48 CAG repeats revealed only mild intergenerational instability ( approximately 2% of meioses). A similar pattern was obtained when this repeat was inserted into exon 1 of the mouse Hdh gene. However, lengthening the repeats in Hdh to 90 and 109 units produced a graded increase in the mutation frequency to >70%, with instability being more evident in female transmissions. No large jumps in CAG length were detected in either male or female transmissions. Instead, size changes were modest increases and decreases, with expansions typically emanating from males and contractions from females. Limited CAG variation in the somatic tissues gave way to marked mosaicism in liver and striatum for the longest repeats in older mice. These results indicate that gametogenesis is the primary source of inherited instability in the Hdh knock-in mouse, as it is in man, but that the underlying repeat length-dependent mechanism, which may or may not be related in the two species, operates at higher CAG numbers. Moreover, the large CAG repeat increases seen in a subset of male HD transmissions are not reproduced in the mouse, suggesting that these arise by a different fundamental mechanism than the small size fluctuations that are frequent during gametogenesis in both species.

Age Factors↗
PubMed1998

A distinct contribution of the delta subunit to acetylcholine receptor channel activation revealed by mutations of the M2 segment.

Acetylcholine receptor (AChR) channels with proline (P) mutations in the putative pore-forming domain (at the 12' position of the M2 segment) were examined at the single-channel level. For all subunits (alpha, beta, epsilon, and delta), a 12'P mutation increased the open channel lifetime >5-fold. To facilitate the estimation of binding and gating rate constants, subunits with 12'P mutations were co-expressed with alpha subunits having a binding site mutation that slows channel opening (alphaD200N). In these AChRs, a 12'P mutation in epsilon or beta slowed the closing rate constant approximately 6-fold but had no effect on either the channel opening rate constant or the equilibrium dissociation constant for ACh (Kd). In contrast, a 12'P mutation in delta slowed the channel closing rate constant only approximately 2-fold and significantly increased both the channel opening rate constant and the Kd. Pairwise expression of 12'P subunits indicates that mutations in epsilon and beta act nearly independently, but one in delta reduces the effect of a homologous mutation in epsilon or beta. The results suggest that a 12'P mutation in epsilon and beta has mainly local effects, whereas one in delta has both local and distributed effects that influence both agonist binding and channel gating.

Amino Acid Sequence↗
PubMed1998

Desensitization of mouse nicotinic acetylcholine receptor channels. A two-gate mechanism.

The rate constants of acetylcholine receptor channels (AChR) desensitization and recovery were estimated from the durations and frequencies of clusters of single-channel currents. Diliganded-open AChR desensitize much faster than either unliganded- or diliganded-closed AChR, which indicates that the desensitization rate constant depends on the status of the activation gate rather than the occupancy of the transmitter binding sites. The desensitization rate constant does not change with the nature of the agonist, the membrane potential, the species of permeant cation, channel block by ACh, the subunit composition (epsilon or gamma), or several mutations that are near the transmitter binding sites. The results are discussed in terms of cyclic models of AChR activation, desensitization, and recovery. In particular, a mechanism by which activation and desensitization are mediated by two distinct, but interrelated, gates in the ion permeation pathway is proposed.

Acetylcholine↗
PubMed1998

Drosophila engrailed can substitute for mouse Engrailed1 function in mid-hindbrain, but not limb development.

The Engrailed-1 gene, En1, a murine homologue of the Drosophila homeobox gene engrailed (en), is required for midbrain and cerebellum development and dorsal/ventral patterning of the limbs. In Drosophila, en is involved in regulating a number of key patterning processes including segmentation of the epidermis. An important question is whether, during evolution, the biochemical properties of En proteins have been conserved, revealing a common underlying molecular mechanism to their diverse developmental activities. To address this question, we have replaced the coding sequences of En1 with Drosophila en. Mice expressing Drosophila en in place of En1 have a near complete rescue of the lethal En1 mutant brain defect and most skeletal abnormalities. In contrast, expression of Drosophila en in the embryonic limbs of En1 mutants does not lead to repression of Wnt7a in the embryonic ventral ectoderm or full rescue of the embryonic dorsal/ventral patterning defects. Furthermore, neither En2 nor en rescue the postnatal limb abnormalities that develop in rare En1 null mutants that survive. These studies demonstrate that the biochemical activity utilized in mouse to mediate brain development has been retained by Engrailed proteins across the phyla, and indicate that during evolution vertebrate En proteins have acquired two unique functions during embryonic and postnatal limb development and that only En1 can function postnatally.

Animals↗
PubMed1997

Maximum likelihood estimation of aggregated Markov processes.

We present a maximum likelihood method for the modelling of aggregated Markov processes. The method utilizes the joint probability density of the observed dwell time sequence as likelihood. A forward-backward recursive procedure is developed for efficient computation of the likelihood function and its derivatives with respect to the model parameters. Based on the calculated forward and backward vectors, analytical formulae for the derivatives of the likelihood function are derived. The method exploits the variable metric optimizer for search of the likelihood space. It converges rapidly and is numerically stable. Numerical examples are given to show the effectiveness of the method.

Markov Chains↗
PubMed1997

Subconductance states of a mutant NMDA receptor channel kinetics, calcium, and voltage dependence.

The kinetic properties of main and subconductance states of a mutant mouse N-methyl-D-aspartate (NMDA) receptor channel were examined. Recombinant receptors made of zeta-epsilon 2 (NR1-NR2B) subunits having asparagine-to-glutamine mutations in the M2 segment (zeta N598Q/epsilon 2N589Q) were expressed in Xenopus oocytes. Single channel currents recorded from outside-out patches were analyzed using hidden Markov model techniques. In Ca(2+)-free solutions, an open receptor channel occupies a main conductance (93 pS) and a subconductance (62 pS) with about equal probability. There are both brief and long-lived subconductance states, but only a single main level state. At -80 mV, the lifetime of the main and the longer-lived sub level are both approximately 3.3 ms. The gating of the pore and the transition between conductance levels are essentially independent processes. Surprisingly, hyperpolarization speeds both the sub-to-main and main-to-sub transition rate constants (approximately 120 mV/e-fold change), but does not alter the equilibrium occupancies. Extracellular Ca2+ does not influence the transition rate constants. We conclude that the subconductance levels arise from fluctuations in the energetics of ion permeation through a single pore, and that the voltage dependence of these fluctuations reflects the modulation by the membrane potential of the barrier between the main and subconductance conformations of the pore.

Animals↗
PubMed1997

Mutation in the M1 domain of the acetylcholine receptor alpha 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) alpha subunit that causes a slow channel congenital myasthenic syndrome (SCCMS). We previously showed that receptors containing alpha N217K 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 alpha N217K. The apparent affinity of ACh binding, measured by competition against the rate of 125I-alpha-bungarotoxin binding, is also enhanced 20-fold by alpha N217K. 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 beta, epsilon, or delta 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.

Acetylcholine↗
PubMed1997

Stoichiometry of recombinant N-methyl-D-aspartate receptor channels inferred from single-channel current patterns.

Single-channel currents were recorded from mouse NR1-NR2B (zeta-epsilon2) receptors containing mixtures of wild-type and mutant subunits expressed in Xenopus oocytes. Mutant subunits had an asparagine-to-glutamine (N-to-Q) mutation at the N0 site of the M2 segment (NR1:598, NR2B:589). Receptors with pure N or Q NR1 and NR2 subunits generated single-channel currents with distinctive current patterns. Based on main and sublevel amplitudes, occupancy probabilities, and lifetimes, four patterns of current were identified, corresponding to receptors with the following subunit compositions (NR1/NR2): N/N, N/Q, Q/N, and Q/Q. Only one current pattern was apparent for each composition. When a mixture of N and Q NR2 subunits was coexpressed with pure mutant NR1 subunits, three single-channel current patterns were apparent. One pattern was the same as Q/Q receptors and another was the same as Q/N receptors. The third, novel pattern presumably arose from hybrid receptors having both N and Q NR2 subunits. When a mixture of N and Q NR1 subunits was coexpressed with pure mutant NR2 subunits, six single-channel current patterns were apparent. One pattern was the same as Q/Q receptors and another was the same as N/Q receptors. The four novel patterns presumably arose from hybrid receptors having both N and Q NR1 subunits. The relative frequency of NR1 hybrid receptor current patterns depended on the relative amounts of Q and N subunits that were injected into the oocytes. The number of hybrid receptor patterns suggests that there are two NR2 subunits per receptor and is consistent with either three or five NR1 subunits per receptor, depending on whether or not the order of mutant and wild-type subunits influences the current pattern. When considered in relation to other studies, the most straightforward interpretation of the results is that N-methyl-D-aspartate receptors are pentamers composed of three NR1 and two NR2 subunits.

Amino Acid Sequence↗
PubMed1996

Binding sites contribute unequally to the gating of mouse nicotinic alpha D200N acetylcholine receptors.

1. Single channel currents were recorded from HEK 293 cells expressing recombinant mouse adult (alpha 2 beta delta gamma) acetylcholine receptors (AChRs) containing a mutation at residue D200 of the alpha-subunit. Rate and equilibrium constants for AChR activation were estimated from open and closed time obtained over a range of ACh concentrations. 2. Mutation of alpha D200 to asparagine (alpha D200N) dramatically slows the rate constant of channel opening, with adult AChRs slowing 100-fold and embryonic AChRs slowing 400-fold. the rate constant of channel closing increased 3-fold, resulting in a decrease of the gating equilibrium constant of up to 1200-fold. In contrast to channel gating steps, ACh-binding steps are only modestly effected by alpha D200N. 3. Introduction of a potential glycosylation site in alpha D200N cannot account for the effect on channel gating because eliminating the consensus for glycosylation with the mutation alpha D200N + T202V fails to restore efficient gating. Gating is similarly impaired with the substitutions of E, K and Q at position alpha 200. 4. the agonists carbamylcholine and tetramethylammonium also activate the alpha D200N AChR, but with channel opening rates even slower than with ACh. The agonist dependence of the opening rate constant is similar in alpha D200N and wild type AChRs. 5. AChRs containing D200N at just one of the two alpha-subunits show either small or large changes in the gating equilibrium constant, presumably due to the presence of the mutation at either the alpha delta or alpha epsilon/alpha gamma sites. The changes in free energy of channel gating show that the contribution of each binding site is nearly independent. However, the sites do not contribute equally to gating, as an alpha D200N mutation at the alpha epsilon or alpha gamma binding site slows channel opening relatively more than at the alpha delta site.

Amino Acid Sequence↗
PubMed1996

Voltage dependence of mouse acetylcholine receptor gating: different charge movements in di-, mono- and unliganded receptors.

1. The voltage dependence of binding and gating in wild-type and mutant recombinant mouse nicotinic acetylcholine receptors (AChRs) was examined at the single-channel level. 2. The closing rate constant of diliganded receptors decreased e-fold with approximately 66 mV hyperpolarization in both wild-type (adult and embryonic) and mutant receptors. The opening rate constant of a mutant receptor (alpha Y93F) was not voltage dependent. 3. The voltage dependence of closing in monoliganded receptors was examined in several receptors having a mutation in the binding site (alpha G153S) or pore region (alpha L251C and epsilon T264P). The closing rate constant of these monoliganded receptors decreased e-fold with approximately 124 mV hyperpolarization. 4. The voltage dependence of closing and opening in unliganded receptors was examined in two receptors having a mutation in the pore region (alpha L251C and epsilon T264P). Neither the closing nor the opening rate constants of unliganded receptors were voltage dependent. 5. If z if the amount of charge that moves during channel closure and delta is the distance (as a fraction of the electric field) that the charge moves, we conclude that z delta = 0.4 in diliganded receptors, 0.2 in monoliganded receptors, and 0.0 in unliganded receptors. It is likely that charges on the protein, rather than the agonist molecule, move z delta = 0.2 after each ACh molecule has bound. 6. The results suggest that unliganded openings arise from a local, concerted change in the structure of the pore (channel opening) that does not involve the net movement of charged residues. We speculate that as a consequence of agonist binding, charged moieties in the protein change their disposition so that they move with respect to the electric field when the channel gates. The results are consistent with the idea that there is semi-independent movement of distinct domains during AChR gating.

Acetylcholine↗
PubMed1996

Estimating single-channel kinetic parameters from idealized patch-clamp data containing missed events.

We present here a maximal likelihood algorithm for estimating single-channel kinetic parameters from idealized patch-clamp data. The algorithm takes into account missed events caused by limited time resolution of the recording system. Assuming a fixed dead time, we derive an explicit expression for the corrected transition rate matrix by generalizing the theory of Roux and Sauve (1985, Biophys. J. 48:149-158) to the case of multiple conductance levels. We use a variable metric optimizer with analytical derivatives for rapidly maximizing the likelihood. The algorithm is applicable to data containing substates and multiple identical or nonidentical channels. It allows multiple data sets obtained under different experimental conditions, e.g., concentration, voltage, and force, to be fit simultaneously. It also permits a variety of constraints on rate constants and provides standard errors for all estimates of model parameters. The algorithm has been tested extensively on a variety of kinetic models with both simulated and experimental data. It is very efficient and robust; rate constants for a multistate model can often be extracted in a processing time of approximately 1 min, largely independent of the starting values.

Algorithms↗
PubMed1996

Inorganic, monovalent cations compete with agonists for the transmitter binding site of nicotinic acetylcholine receptors.

The properties of adult mouse recombinant nicotinic acetylcholine receptors activated by acetylcholine (ACh+) or tetramethylammonium (TMA+) were examined at the single-channel level. The midpoint of the dose-response curve depended on the type of monovalent cation present in the extracellular solution. The shifts in the midpoint were apparent with both inward and outward currents, suggesting that the salient interaction is with the extracellular domain of the receptor. Kinetic modeling was used to estimate the rate constants for agonist binding and channel gating in both wild-type and mutant receptors exposed to Na+, K+, or Cs+. The results indicate that in adult receptors, the two binding sites have the same equilibrium dissociation constant for agonists. The agonist association rate constant was influenced by the ionic composition of the extracellular solution whereas the rate constants for agonist dissociation, channel opening, and channel closing were not. In low-ionic-strength solutions the apparent association rate constant increased in a manner that suggests that inorganic cations are competitive inhibitors of ACh+ binding. There was no evidence of an electrostatic potential at the transmitter binding site. The equilibrium dissociation constants for inorganic ions (Na+, 151 mM; K+, 92 mM; Cs+, 38 mM) and agonists (TMA+, 0.5 mM) indicate that the transmitter binding site is hydrophobic. Under physiological conditions, about half of the binding sites in resting receptors are occupied by Na+.

Acetylcholine↗
PubMed1996

Identification of a high affinity divalent cation binding site near the entrance of the NMDA receptor channel.

Single channel currents from recombinant N-methyl-D-aspartate (NMDA) receptors having an N-to-Q mutation in M2 reveal a divalent cation binding site that is near the entrance of the pore (approximately 0.2 through the electric field). Ca2+ rapidly binds to this site and readily permeates the channel, while Mg2+ binds more slowly and does not permeate as readily. In wild-type receptors, Mg2+ also blocks the current by occupying a site that is approximately 0.6 through the field. When the more external site is occupied by Ca2+, the conductance of the pore to NA+ is reduced but not abolished, perhaps by an electrostatic blocking mechanism. The site serves to enrich the fraction of NMDA receptor current carried by CA2+.

Animals↗
PubMed1996

Inactivation of Fac in mice produces inducible chromosomal instability and reduced fertility reminiscent of Fanconi anaemia.

Fanconi anaemia (FA) is an autosomal recessive disease characterized by bone marrow failure, variable congenital malformations and predisposition to malignancies. Cells derived from FA patients show elevated levels of chromosomal breakage and an increased sensitivity to bifunctional alkylating agents such as mitomycin C (MMC) and diepoxybutane (DEB). Five complementation groups have been identified by somatic cell methods, and we have cloned the gene defective in group C (FAC)(7). To understand the in vivo role of this gene, we have disrupted murine Fac and generated mice homozygous for the targeted allele. The -/- mice did not exhibit developmental abnormalities nor haematologic defects up to 9 months of age. However, their spleen cells had dramatically increased numbers of chromosomal aberrations in response to MMC and DEB. Homozygous male and female mice also had compromised gametogenesis, leading to markedly impaired fertility, a characteristic of FA patients. Thus, inactivation of Fac replicates some of the features of the human disease.

Animals↗