Biomedical subjects

Fan Gao

Publications and source records attributed to Fan Gao.

At least 19 recordsLinked to original sources

PubMed2006

Cross-talk between ON and OFF channels in the salamander retina: indirect bipolar cell inputs to ON-OFF ganglion cells.

It has been widely accepted that ON and OFF channels in the visual system are segregated with little cross-communication, except for the mammalian rod bipolar cell-AII amacrine cell-ganglion cell pathway. Here, we show that in the tiger salamander retina the light responses of a subpopulation of ON-OFF ganglion cells are mediated by crossing the ON and OFF bipolar cell pathways. Although the majority of ON-OFF ganglion cells (type I cells) receive direct excitatory inputs from depolarizing and hyperpolarizing bipolar cells (DBCs and HBCs), about 5% (type II cells) receive indirect excitatory inputs from DBCs and 20% (type III cells) receive indirect excitatory inputs from HBCs. These indirect bipolar cell inputs are likely to be mediated by a subpopulation of amacrine cells that exhibit transient hyperpolarizing light responses (AC(H)s) and make GABAergic/glycinergic synapses on DBC or HBC axon terminals. GABA and glycine receptor antagonists enhanced the ON and OFF excitatory cation current (DeltaI(C)) in type I ganglion cells, but completely suppressed the ON DeltaI(C) mediated by DBCs in type II cells and the OFF DeltaI(C) mediated by HBCs in types III cells. Dendrites of type I cells ramify in both sublamina A and B, type II cells exclusively in sublamina A, and type III cells exclusively in sublamina B of the inner plexiform layer. These results demonstrate that indirect, amacrine cell-mediated bipolar cell-ganglion cell synaptic pathways exist in a non-mammalian retina, and that bidirectional cross-talk between ON and OFF channels is present in the vertebrate retina.

Amacrine Cells↗

PubMed2006

Solution NMR of acetylcholine binding protein reveals agonist-mediated conformational change of the C-loop.

Previous X-ray crystallography, molecular dynamics simulation, fluorescence spectroscopy, and deuterium-hydrogen exchange of acetylcholine binding protein (AChBP) suggest that after binding of the agonist, the C-loop at the periphery of the binding site draws inward to cap the site and envelop the agonist. In this study, we use high-resolution solution NMR to monitor changes in the chemical environment of the C-loop without and with acetylcholine (ACh) bound. Substitution of [15N]cysteine for the native cysteines 123, 136, 187, and 188 provided intrinsic monitors of the chemical environments of the Cys- and C-loops, respectively. Two-dimensional transverse relaxation-optimized spectroscopy 15N-1H HSQC spectroscopy of apo-AChBP revealed seven well resolved cross-peaks for the group of cysteines. The spectrum of AChBP with Ser substituted for Cys 187 and 188 shows only two main cross-peaks, corresponding to Cys 123 and 136 from the Cys-loop, enabling resonance assignments. After binding of ACh, the five cross-peaks associated with cysteines from the C-loop condense into two predominant cross-peaks not observed in the spectrum from the apo protein, indicating a restricted range of conformations and change in chemical environment of the C-loop. The results show that isotopic cysteine can be incorporated into specified positions of AChBP expressed from a eukaryotic source, that the C-loop assumes multiple conformations without ACh, but that its conformation becomes restricted with ACh bound. The collective findings suggest a structural mechanism for agonist recognition in AChBP and related Cys-loop receptors.

Acetylcholine↗

PubMed2005

Prehension stability: experiments with expanding and contracting handle.

We studied adjustments in digit forces and moments during holding a vertically oriented handle under slow, externally imposed changes in the width of the grasp. Subjects (n = 8) grasped a customized motorized handle with five digits and held it statically in the air. The handle width either increased (expanded) or decreased (contracted) at a rate of 1.0, 1.5, or 2.0 mm/s, while the subjects were asked to ignore the handle width changes, and their attention was distracted. External torques of 0.0, 0.25, and 0.5 Nm were applied to the handle in two directions. Forces and moments at the digit tips were measured with six-component sensors. The analysis was performed at the virtual finger (VF) and individual finger (IF) levels (VF is an imagined finger that produces the same wrench, i.e., the force and moment, as several fingers combined). In all the tasks, the normal VF and thumb forces increased with the handle expansion and decreased with the handle contraction. Similar behavior was seen for the thumb tangential force. In contrast, the VF tangential force decreased with the handle expansion and increased with the handle contraction. The changes in the tangential forces assisted the perturbations in the tasks requiring exertion of the supination moments and annulled the perturbation in the pronation effort tasks. In the former tasks, the equilibrium was maintained by the changes of the moments of normal forces, whereas in the latter tasks, the equilibrium was maintained by the changes of the moments of the tangential forces. Analysis at the IF level has shown that the resultant force and moment exerted on the object could arise from dissimilar adjustments of individual fingers to the same handle width change. The complex adjustments of digit forces to handle width change may be viewed as coming from two sources. First, there are local spring-like adjustments of individual digit forces and moments caused by both mechanical properties of the digits and the action of spinal reflexes. These stiffness-like reactions mainly assist in perturbing the rotational equilibrium of the object rather than in maintaining it. Second, there are tilt-preventing adjustments defined by the common task constraints that unite the digits into a task-specific synergy. The "virtual springs theory" developed in robotics literature is insufficient for describing the phenomena observed in human grasping.

Adaptation, Physiological↗

PubMed2005

Maintaining rotational equilibrium during object manipulation: linear behavior of a highly non-linear system.

We address issues of simultaneous control of the grasping force and the total moment of forces applied to a handheld object during its manipulation. Six young healthy male subjects grasped an instrumented handle and performed its cyclic motion in the vertical direction. The handle allowed for setting different clockwise (negative) or counterclockwise torques. Three movement frequencies: 1, 1.5 and 2 Hz, and five different torques: -1/3, -1/6, 0, 1/6 and 1/3 Nm, were used. The rotational equilibrium was maintained by two means: (1) Concerted changes of the moments produced by the normal and tangential forces, specifically anti-phase changes of the moments during the tasks with zero external torque and in-phase changes during the non-zero-torque tasks, and (2) Redistribution of the normal forces among individual fingers such that the agonist fingers--the fingers that resist external torque--increased the force in phase with the acceleration, while the forces of the antagonist fingers--those that assist the external torque--especially, the fingers with the large moment arms, the index and little fingers, stayed unchanged. The observed effects agree with the principle of superposition--according to which some complex actions, for example, prehension, can be decomposed into elemental actions controlled independently--and the mechanical advantage hypothesis according to which in moment production the fingers are activated in proportion to their moment arms with respect to the axis of rotation. We would like to emphasize the linearity of the observed relations, which was not prescribed by the task mechanics and seems to be produced by specific neural control mechanisms.

Adult↗

PubMed2005

Internal forces during object manipulation.

Internal force is a set of contact forces that does not disturb object equilibrium. The elements of the internal force vector cancel each other and, hence, do not contribute to the resultant (manipulation) force acting on the object. The mathematical independence of the internal and manipulation forces allows for their independent (decoupled) control realized in robotic manipulators. To examine whether in humans internal force is coupled with the manipulation force and what grasping strategy the performers utilize, the subjects (n=6) were instructed to make cyclic arm movements with a customized handle. Six combinations of handle orientation and movement direction were tested. These involved: parallel manipulations (1) VV task (vertical orientation and vertical movement) and (2) HH task (horizontal orientation and horizontal movement); orthogonal manipulations (3) VH task (vertical orientation and horizontal movement) and (4) HV task (horizontal orientation and vertical movement); and diagonal manipulations (5) DV task (diagonal orientation and vertical movement) and (6) DH task (diagonal orientation and horizontal movement). Handle weight (from 3.8 to 13.8 N), and movement frequency (from 1 to 3 Hz) were systematically changed. The analysis was performed at the thumb-virtual finger level (VF, an imaginary finger that produces a wrench equal to the sum of wrenches produced by all the fingers). At this level, the forces of interest could be reduced to the internal force and internal moment. During the parallel manipulations, the internal (grip) force was coupled with the manipulation force (producing object acceleration) and the thumb-VF forces increased or decreased in phase: the thumb and VF worked in synchrony to grasp the object more strongly or more weakly. During the orthogonal manipulations, the thumb-VF forces changed out of phase: the plots of the internal force vs. object acceleration resembled an inverted letter V. The HV task was the only task where the relative phase (coupling) between the normal forces of the thumb and VF depended on oscillation frequency. During the diagonal manipulations, the coupling was different in the DV and DH tasks. A novel observation of substantial internal moments is described: the moments produced by the normal finger forces were counterbalanced by the moments produced by the tangential forces such that the resultant moments were close to zero. Implications of the findings for the notion of grasping synergies are discussed.

Adult↗

PubMed2005

Characterization of displaced bipolar cells in the tiger salamander retina.

In immunocytochemical studies of the tiger salamander retina, 17% of neurons in the outer nuclear layer did not label for recoverin, a photoreceptor marker. Lucifer yellow injection showed a population of cells in the ONL to be displaced bipolar cells, with axon terminals that stratified exclusively in the OFF sublamina of the inner plexiform layer (IPL), and predominately within the cone-dominated region of the OFF sublamina. Glutamate generated a dendritic cationic conductance increase in all displaced bipolar cells tested, and typical cone-dominated bipolar cell light responses were observed among displaced cells that stratified in the central IPL. We conclude that displaced bipolar cells in the tiger salamander retina are entirely OFF-center cells, and predominately cone-dominated cells.

Animals↗

PubMed2004

Alpha-conotoxins ImI and ImII target distinct regions of the human alpha7 nicotinic acetylcholine receptor and distinguish human nicotinic receptor subtypes.

The Conus peptides alpha-conotoxin ImI (alpha-ImI) and ImII (alpha-ImII) differ by only three of 11 residues in their primary sequences and yet are shown to inhibit the human alpha7 nicotinic acetylcholine receptor (nAChR) by targeting different sites. Mutations at both faces of the classical ligand binding site of the alpha7 nAChR strongly affect antagonism by alpha-ImI but not alpha-ImII. The effects of the mutations on alpha-ImI binding and functional antagonism are explained by computational docking of the NMR structure of alpha-ImI to a homology model of the ligand binding domain of the alpha7 nAChR. A distinct binding site for alpha-ImII is further demonstrated by its weakened antagonism for a chimeric receptor in which the membrane-spanning domains and intervening linkers of the alpha7 nAChR are replaced with the corresponding sequence from the serotonin type-3 receptor (5HT(3)). The two toxins also discriminate between different subtypes of human nicotinic receptors; alpha-ImII most strongly blocks the human alpha7 and alpha1beta1deltaepsilon receptor subtypes, while alpha-ImI most potently blocks the human alpha3beta2 subtype. Collectively, the data show that while alpha-ImI targets the classical competitive ligand binding site in a subtype selective manner, alpha-ImII is a probe of a novel inhibitory site in homomeric alpha7 nAChRs.

Animals↗

PubMed2004

Agonist-mediated conformational changes in acetylcholine-binding protein revealed by simulation and intrinsic tryptophan fluorescence.

We delineated acetylcholine (ACh)-dependent conformational changes in a prototype of the nicotinic receptor ligand binding domain by molecular dynamics simulation and changes in intrinsic tryptophan (Trp) fluorescence. Prolonged molecular dynamics simulation of ACh-binding protein showed that binding of ACh establishes close register of Trps from adjacent subunits, Trp(143) and Trp(53), and draws the peripheral C-loop inward to occlude the entrance to the binding cavity. Close register of Trp(143) and Trp(53) was demonstrated by ACh-mediated quenching of intrinsic Trp fluorescence, elimination of quenching by mutation of one or both Trps to Phe, and decreased lifetime of Trp fluorescence by bound ACh. Occlusion of the binding cavity by the C-loop was demonstrated by restricted access of an extrinsic quencher of binding site Trp fluorescence by ACh. The collective findings showed that ACh initially establishes close register of conserved Trps from adjacent subunits and then draws the C-loop inward to occlude the entrance to the binding cavity.

Acetylcholine↗

PubMed2004

Motor control goes beyond physics: differential effects of gravity and inertia on finger forces during manipulation of hand-held objects.

According to basic physics, the local effects induced by gravity and acceleration are identical and cannot be separated by any physical experiment. In contrast-as this study shows-people adjust the grip forces associated with gravitational and inertial forces differently. In the experiment, subjects oscillated a vertically-oriented handle loaded with five different weights (from 3.8 N to 13.8 N) at three different frequencies in the vertical plane: 1 Hz, 1.5 Hz and 2.0 Hz. Three contributions to the grip force-static, dynamic, and stato-dynamic fractions-were quantified. The static fraction reflects grip force related to holding a load statically. The stato-dynamic fraction reflects a steady change in the grip force when the same load is moved cyclically. The dynamic fraction is due to acceleration-related adjustments of the grip force during oscillation cycles. The slope of the relation between the grip force and the load force was steeper for the static fraction than for the dynamic fraction. The stato-dynamic fraction increased with the frequency and load. The slope of the dynamic grip force-load force relation decreased with frequency, and as a rule, increased with the load. Hence, when adjusting grip force to task requirements, the central controller takes into account not only the expected magnitude of the load force but also such factors as whether the force is gravitational or inertial and the contributions of the object mass and acceleration to the inertial force. As an auxiliary finding, a complex finger coordination pattern aimed at preserving the rotational equilibrium of the object during shaking movements was reported.

Acceleration↗

PubMed2004

Control of finger force direction in the flexion-extension plane.

We have examined the interaction among individual finger forces in tasks that required the production of the total force by a subset of fingers in a particular direction in the flexion-extension plane. Nine subjects produced fingertip forces in a prescribed direction with a maximum voluntary contraction (MVC) effort and held the peak force for two seconds. Six finger combinations were tested, four single-finger tasks--Index (I), Middle (M), Ring (R) and Little (L)--one two-digit task (IM), and one four-digit task (IMRL). The subjects were asked to generate the finger forces in two directions, 0 degrees (perpendicular to the surface of the transducer) and 15 degrees toward the palm. In all task conditions, there were two experimental sessions, with and without visual feedback on the task force vector. The main findings were: 1. The target direction significantly affected the constant error (CE) but not the variable error (VE) while removal of the feedback resulted in an increase in VE. 2. The direction of the forces produced by fingers that were not explicitly required to produce force (enslaved fingers) depended on the target direction. 3. In multi-finger tasks, the individual fingers produced force in directions that could differ significantly from the target direction, while the resultant force pointed in the target direction. There was a negative co-variation among the deviations of the directions of the individual finger forces from the target direction. If a finger force vector deviated from the target, another finger force vector was likely to deviate in the opposite direction. We conclude that a multi-finger synergy is involved in the control of the finger force direction.

Adult↗

PubMed2004

Structural basis for epibatidine selectivity at desensitized nicotinic receptors.

The agonist binding sites of the fetal muscle nicotinic acetylcholine receptor are formed at the interfaces of alpha-subunits and neighboring gamma- and delta-subunits. When the receptor is in the nonconducting desensitized state, the alpha-gamma site binds the agonist epibatidine 200-fold more tightly than does the alpha-delta site. To determine the structural basis for this selectivity, we constructed gamma/delta-subunit chimeras, coexpressed them with complementary wild-type subunits in HEK 293 cells, and determined epibatidine affinity of the resulting complexes. The results reveal three determinants of epibatidine selectivity: gamma104-117/delta106-delta119, gamma164-171/delta166-177, and gammaPro190/deltaAla196. Point mutations reveal that three sequence differences within the gamma104-117/delta106-delta119 region are determinants of epibatidine selectivity: gammaLys104/deltaTyr106, gammaSer111/deltaTyr113, and gammaTyr117/deltaTyr119. In the delta-subunit, simultaneous mutation of these residues to their gamma equivalent produces high affinity, gamma-like epibatidine binding. However, converting gamma to delta affinity requires replacement of the gamma104-117 segment with delta sequence, suggesting interplay of residues in this region. The structural basis for epibatidine selectivity is explained by computational docking of epibatidine to a homology model of the alpha-gamma binding site.

Amino Acid Sequence↗

PubMed2004

Proteomics shows Hsp70 does not bind peptide sequences indiscriminately in vivo.

Heat shock protein 70 (Hsp70) binds peptide and has several functions that include protein folding, protein trafficking, and involvement with immune function. However, endogenous Hsp70-binding peptides had not previously been identified. Therefore, we eluted and identified several hundred endogenously bound peptides from Hsp70 using liquid chromatography ion trap mass spectrophotometry (LC-ITMS). Our work shows that the peptides are capable of binding Hsp70 as previously described. They are generally 8-26 amino acids in length and correspond to specific regions of many proteins. Through computationally assisted analysis of peptides eluted from Hsp70 we determined variable amino acid sequences, including a 5 amino acid core sequence that Hsp70 favorably binds. We also developed a computer algorithm that predicts Hsp70 binding within proteins. This work helps to define what peptides are bound by Hsp70 in vivo and suggests that Hsp70 facilitates peptide selection by aiding a funneling mechanism that is flexible but allows only a limited number of peptides to be processed.

Algorithms↗

PubMed2004

Light-evoked current responses in rod bipolar cells, cone depolarizing bipolar cells and AII amacrine cells in dark-adapted mouse retina.

Light-evoked excitatory cation current (DeltaI(C)) and inhibitory chloride current (DeltaI(Cl)) of rod and cone depolarizing bipolar cells (DBC(R)s and DBC(C)s) and AII amacrine cells (AIIACs) in dark-adapted mouse retinal slices were studied by whole-cell voltage-clamp recording techniques, and the cell morphology was revealed by Lucifer yellow fluorescence with a confocal microscope. DeltaI(C) of all DBC(R)s exhibited similar high sensitivity to 500 nm light, but two patterns of DeltaI(Cl) were observed in DBC(R)s with slightly different axon morphology. At least two types of DBC(C)s were identified: one with axon terminals ramified in 70-85% of the depth of the inner plexiform layer (IPL) and DBC(R)-like DeltaI(C) sensitivity, whereas the other with axon terminals ramified in 55-75% of IPL depth and much lower DeltaI(C) sensitivity. The relative rod/cone inputs to DBCs and AIIACs were analysed by comparing the DeltaI(C) and DeltaI(Cl) thresholds and dynamic ranges with the corresponding values of rods and cones. On average, the sensitivity of a DBC(R) to the 500 nm light is about 20 times higher than that of a rod. The sensitivity of an AIIAC is more than 1000 times higher than that of a rod, suggesting that AIIAC responses are pooled through a coupled network of about 40 AIIACs. Interactions of rod and cone signals in dark-adapted mouse retina appear asymmetrical: rod signals spread into the cone system more efficiently than cone signals into the rod system. The mouse synaptic circuitry allows small rod signals to be highly amplified, and effectively transmitted to the cone system via rod-cone and AIIAC-DBC(C) coupling.

Amacrine Cells↗

PubMed2004

Stratum-by-stratum projection of light response attributes by retinal bipolar cells of Ambystoma.

The visual system processes light images by projecting various representations of the visual world to segregated regions in the brain through parallel channels. Retinal bipolar cells constitute the first parallel channels that carry different light response attributes to different parts of the inner plexiform layer (IPL). Here we present a systematic study on detailed axonal morphology and light response characteristics of over 200 bipolar cells in dark-adapted salamander retinal slices by the whole-cell voltage clamp and Lucifer yellow fluorescence (with a confocal microscope) techniques. Four major groups of bipolar cells were identified according to the patterns of axon terminal ramification in the IPL: 36% were narrowly monostratified (whose axon terminals ramified in one of the 10 strata of the IPL), 27% were broadly monostratified, 19% were multistratified, and 18% bore pyramidally branching axons. By analysing the bipolar cells with narrowly monostratified axon terminals in each of the 10 strata of the IPL, we found that several key light response attributes are highly correlated with the strata in which the cells' axon terminals ramify. The 10 IPL strata appear to be the basic building blocks for attributes of light-evoked signal outputs in all bipolar cells, and several general stratum-by-stratum rules were identified by analysing the broadly monostratified, multistratified and pyramidally branching cells. These rules not only uncover mechanisms by which third-order retinal cells integrate and compute bipolar cell signals, but also shed considerable light on how bipolar cells in other vertebrates process visual information and how physiological signals may shape the morphology and projection of output synapses of visual neurones during development.

Adaptation, Ocular↗

PubMed2004

ABCC9 mutations identified in human dilated cardiomyopathy disrupt catalytic KATP channel gating.

Stress tolerance of the heart requires high-fidelity metabolic sensing by ATP-sensitive potassium (K(ATP)) channels that adjust membrane potential-dependent functions to match cellular energetic demand. Scanning of genomic DNA from individuals with heart failure and rhythm disturbances due to idiopathic dilated cardiomyopathy identified two mutations in ABCC9, which encodes the regulatory SUR2A subunit of the cardiac K(ATP) channel. These missense and frameshift mutations mapped to evolutionarily conserved domains adjacent to the catalytic ATPase pocket within SUR2A. Mutant SUR2A proteins showed aberrant redistribution of conformations in the intrinsic ATP hydrolytic cycle, translating into abnormal K(ATP) channel phenotypes with compromised metabolic signal decoding. Defective catalysis-mediated pore regulation is thus a mechanism for channel dysfunction and susceptibility to dilated cardiomyopathy.

ATP-Binding Cassette Transporters↗

PubMed2004

Synaptic circuitry mediating light-evoked signals in dark-adapted mouse retina.

Light-evoked excitatory cation current (DeltaIC) and inhibitory chloride current (DeltaICl) of rod and cone bipolar cells and AII amacrine cells (AIIACs) were recorded from slices of dark-adapted mouse retinas, and alpha ganglion cells were recorded from flatmounts of dark-adapted mouse retinas. The cell morphology was revealed by Lucifer yellow fluorescence with a confocal microscope. DeltaIC of all rod depolarizing bipolar cells (DBCRs) exhibited similar high sensitivity to 500 nm light, but two patterns of DeltaICl were observed with slightly different axon morphologies. At least two types of cone depolarizing bipolar cells (DBCCs) were identified: one with axon terminals ramified in 70-85% of IPL depth and DBCR-like DeltaIC sensitivity, and the other with axon terminals ramified in 55-75% of IPL depth and much lower DeltaIC sensitivity. The relative rod/cone inputs to DBCs and AIIACs were analyzed by comparing the DeltaIC and DeltaICl thresholds and dynamic ranges with the corresponding values of rods and cones. On average, the sensitivity of a DBCR to the 500 nm light is about 20 times higher than that of a rod. The sensitivity of an AIIAC is more than 1000 times higher than that of a rod, suggesting that AIIAC responses are pooled through a coupled network of about 40 AIIACs. Interactions of rod and cone signals in dark-adapted mouse retinas appear asymmetrical: rod signals spread into the cone system more efficiently than cone signals into the rod system. The mouse synaptic circuitry allows small rod signals to be highly amplified and effectively transmitted to the cone system via rod/cone and AIIAC/DBCC coupling. Three types of alpha ganglion cells (alphaGCs) were identified. (1) ONGCs exhibits no spike activity in darkness, increased spikes in light, sustained inward DeltaIC, sustained outward DeltaICl of varying amplitude, and large soma (20-25 microm in diameter) with an alpha-cell-like dendritic field about 180-350 microm stratifying near 70% of the IPL depth. (2) Transient OFFalphaGCs (tOFFalphaGCs) exhibit no spike activity in darkness, transient increased spikes at light offset, small sustained outward DeltaIC in light, a large transient inward DeltaIC at light offset, a sustained outward DeltaICl, and a morphology similar to the ONalphaGCs except for that their dendrites stratified near 30% of the IPL depth. (3) Sustained OFFalpha GCs (sOFFalphaGCs) exhibit maintained spike activity of 5-10 Hz in darkness, sustained decrease of spikes in light, sustained outward DeltaIC, sustained outward DeltaICl, and a morphology similar to the tOFFalphaGCs. By comparing the response thresholds and dynamic ranges of alphaGCs with those of the pre-ganglion cells, our data suggest that the light responses of each type of alphaGCs are mediated by different sets of bipolar cells and amacrine cells.

Amacrine Cells↗

PubMed2004

Rotational equilibrium during multi-digit pressing and prehension.

We review a series of studies that show stabilization of the moment of a couple produced by a set of digits in many maximal and submaximal accurate force production tasks that have no requirements for the moment. In particular, an unusual and novel multi-digit force production task shows stabilization of the total moment while the total force requires extensive practice to be stabilized. Similar results were obtained in persons with Down syndrome during easier tasks. During prehension, changes in digit forces and coordinates of their points of application suggest the presence of two multi-digit synergies whose purpose is to assure a certain grip force and a certain total moment, respectively. Elderly persons show impaired production of both maximal and submaximal moments that goes beyond their documented loss of muscle force. We conclude that moment production (keeping rotational equilibrium) is a central constraint in a variety of multi-digit tasks that has received little attention. Analysis of digit interaction for moment production during handwriting could signify a major step towards understanding the control of this action.

Fingers↗

PubMed2004

Toward atomic-scale understanding of ligand recognition in the muscle nicotinic receptor.

The nicotinic receptor at the motor endplate has served as a prototype for understanding structure, function and ligand recognition in the superfamily of pentameric ligand-gated ion channels. Yet despite this advanced state of knowledge, atomic-scale understanding of such elementary processes as ligand recognition has remained elusive owing to the lack of a high-resolution x-ray structure. However, the field has recently entered a state of rapid advancement following the discovery and atomic structural determination of the water-soluble acetylcholine binding protein (AChBP), a homolog of the receptor ligand binding domain. The AChBP structure provides the theoretical foundation for generating homology models of the corresponding receptor ligand binding domains within this structural family of receptors. Experimental assignment of residue equivalence between AChBP and receptor subunits subsequently yielded homology models ready for experimental testing. One such test is computational determination of ligand docking orientation in conjunction with mutagenesis of predicted contact residues and measurements of ligand binding affinity. Applied to different analogs of the competitive antagonist curare, docking computations that incorporate intrinsic protein flexibility reveal fundamentally distinct orientations of each analog bound to AChBP. The different contact residues predicted for each analog were tested and confirmed by mutagenesis of AChBP followed by measurements of ligand binding. By applying the same computational and experimental approaches to the adult human muscle AChR, we find that the two curare analogs also dock in distinctly different orientations. Thus subtle structural changes in the ligand, and by extension, structural differences in non-conserved residues among receptor subtypes and species, can dramatically alter the orientation of the bound ligand. The results have important implications for design of drugs targeting nicotinic receptors and members of the superfamily of pentameric ligand-gated ion channels.

Amino Acid Sequence↗