Potassium and sodium binding to the outer mouth of the K+ channel.

Molecular dynamics simulations of the K+ channel from Streptomyces lividans (KcsA channel) were performed in a membrane-mimetic environment with Na+ and K+ in different initial locations. The structure of the channel remained stable and well preserved for simulations lasting up to 1.5 ns. Salt bridges between Asp80 and Arg89 of neighboring subunits, not detected in the X-ray structure, enhanced the stability of the tetrameric structure. Na+ or K+ ions located in the channel vestibule lost part of their hydration shell and diffused into the channel inner pore in less than a few hundred picoseconds. This powerful catalytic action was caused by strong electrostatic interactions with Asp80 and Glu71. The hydration state of the metal ions turned out to depend significantly on the conformational flexibility of the channel. Furthermore, Na+ entered the channel inner pore bound to more water molecules than K+. The different hydration state of the two ions may be a determinant factor in the ion selectivity of the channel.     

An inactivation stabilizer of the Na+ channel acts as an opportunistic pore blocker modulated by external Na+

The Na+ channel is the primary target of anticonvulsants carbamazepine, phenytoin, and lamotrigine. These drugs modify Na+ channel gating as they have much higher binding affinity to the inactivated state than to the resting state of the channel. It has been proposed that these drugs bind to the Na+ channel pore with a common diphenyl structural motif. Diclofenac is a widely prescribed anti-inflammatory agent that has a similar diphenyl motif in its structure. In this study, we found that diclofenac modifies Na+ channel gating in a way similar to the foregoing anticonvulsants. The dissociation constants of diclofenac binding to the resting, activated, and inactivated Na+ channels are approximately 880 microM, approximately 88 microM, and approximately 7 microM, respectively. The changing affinity well depicts the gradual shaping of a use-dependent receptor along the gating process. Most interestingly, diclofenac does not show the pore-blocking effect of carbamazepine on the Na+ channel when the external solution contains 150 mM Na+, but is turned into an effective Na+ channel pore blocker if the extracellular solution contains no Na+. In contrast, internal Na+ has only negligible effect on the functional consequences of diclofenac binding. Diclofenac thus acts as an "opportunistic" pore blocker modulated by external but not internal Na+, indicating that the diclofenac binding site is located at the junction of a widened part and an acutely narrowed part of the ion conduction pathway, and faces the extracellular rather than the intracellular solution. The diclofenac binding site thus is most likely located at the external pore mouth, and undergoes delicate conformational changes modulated by external Na+ along the gating process of the Na+ channel.     

A structural model of the tetrodotoxin and saxitoxin binding site of the Na+ channel.

Biophysical evidence has placed the binding site for the naturally occurring marine toxins tetrodotoxin (TTX) and saxitoxin (STX) in the external mouth of the Na+ channel ion permeation pathway. We developed a molecular model of the binding pocket for TTX and STX, composed of antiparallel beta-hairpins formed from peptide segments of the four S5-S6 loops of the voltage-gated Na+ channel. For TTX the guanidinium moiety formed salt bridges with three carboxyls, while two toxin hydroxyls (C9-OH and C10-OH) interacted with a fourth carboxyl on repeats I and II. This alignment also resulted in a hydrophobic interaction with an aromatic ring of phenylalanine or tyrosine residues for the brainII and skeletal Na+ channel isoforms, but not with the cysteine found in the cardiac isoform. In comparison to TTX, there was an additional interaction site for STX through its second guanidinium group with a carboxyl on repeat IV. This model satisfactorily reproduced the effects of mutations in the S5-S6 regions and the differences in affinity by various toxin analogs. However, this model differed in important ways from previously published models for the outer vestibule and the selectivity region of the Na+ channel pore. Removal of the toxins from the pocket formed by the four beta-hairpins revealed a structure resembling a funnel that terminated in a narrowed region suitable as a candidate for the selectivity filter of the channel. This region contained two carboxyls (Asp384 and Glu942) that substituted for molecules of water from the hydrated Na+ ion. Simulation of mutations in this region that have produced Ca2+ permeation of the Na+ channel created a site with three carboxyls (Asp384, Glu942, and Glu1714) in proximity.     

Locations of local anesthetic dibucaine in model membranes and the interaction between dibucaine and a Na+ channel inactivation gate peptide as studied by 2H- and 1H-NMR spectroscopies.

To study the molecular mechanisms of local anesthesia, locations of local anesthetic dibucaine in model membranes and the interactions of dibucaine with a Na+ channel inactivation gate peptide have been studied by 2H- and 1H-NMR spectroscopies. The 2H-NMR spectra of dibucaine-d9 and dibucaine-d1, which are deuterated at the butoxy group and at the 3 position in its quinoline ring, respectively, have been observed in multilamellar dispersions of the lipid mixture composed of phosphatidylcholine, phosphatidylserine, and phosphatidylethanolamine. 2H-NMR spectra of deuterated palmitic acids incorporated, as a probe, into the lipid mixture containing cholesterol have also been observed. An order parameter, SCD, for each carbon segment was calculated from the observed quadrupole splittings. Combining these results, we concluded that first, the butoxy group of dibucaine is penetrating between the acyl chains of lipids in the model membranes, and second, the quinoline ring of dibucaine is located at the polar region of lipids but not at the hydrophobic acyl chain moiety. These results mean that dibucaine is situated in a favorable position that permits it to interact with a cluster of hydrophobic amino acids (Ile-Phe-Met) within the intracellular linker between domains III and IV of Na+ channel protein, which functions as an inactivation gate. To confirm whether the dibucaine molecule at the surface region of lipids can really interact with the hydrophobic amino acids, we synthesized a model peptide that includes the hydrophobic amino acids (Ac-GGQDIFMTEEQK-OH, MP-1), the amino acid sequence of which corresponds to the linker part of rat brain type IIA Na+ channel, and the one in which Phe has been substituted by Gln (MP-2), and measured 1H-NMR spectra in both phosphate buffer and phosphatidylserine liposomes. It was found that the quinoline ring of dibucaine can interact with the aromatic ring of Phe by stacking of the rings; moreover, the interaction can be reinforced by the presence of lipids. In conclusion, we wish to propose that local anesthesia originates from the pi-stacking interaction between aromatic rings of an anesthetic molecule located at the polar headgroup region of the so-called boundary lipids and of the Phe in the intracellular linker between domains III and IV of the Na+ channel protein, prolonging the inactivated state and consequently making it impossible to proceed to the resting state.     

Structural study of the sodium channel inactivation gate peptide including an isoleucine-phenylalanine-methionine motif and its analogous peptide (phenylalanine/glutamine) in trifluoroethanol solutions and SDS micelles.

In order to gain insight into the gating mechanisms of Na+ channels, in particular their inactivation mechanisms, we studied the structures of the Na+ channel inactivation gate related peptide which includes the IFM (Ile-Phe-Met) motif (Ac-KKKFGGQDIFMTEEQKK-NH2; K1480-K1496 in rat brain type-IIA Na+ channels, MP-3A) and its F/Q(Gln) substituted one (MP-4A) in trifluoroethanol (TFE) solutions and sodium dodecyl sulfate (SDS) micelles using circular dichroism (CD) and 1H-NMR spectroscopies. Based on observed nuclear Overhauser effect constraints, three-dimensional structures of MP-3A and MP-4A were determined using simulated annealing molecular dynamics/ energy minimization calculations. In TFE solutions, no appreciable differences in the structure were observed using either CD or NMR spectra. In SDS micelles, however, the two peptides exhibited definitely different structures from each other. It was found that in MP-3A, residues 11488 and T1491 were spatially proximate with each other owing to hydrogen bonding between the amide proton of 11488 and the hydroxyl oxygen atom of T1491, whereas in MP-4A, F/Q substitution separated them owing to conformational changes. The solvent-accessible surfaces calculated for the structures of MP-3A and MP-4A showed that the former has a smoother interaction surface to the hydrophobic docking site than the latter. In conclusion, the conformational changes, as well as decreased hydrophobicity around the IFM motif owing to the F/Q mutation, may be one reason why F1489Q mutated channels cannot inactivate almost completely.     

Structural differences of bacterial and mammalian K+ channels

Using a peptide toxin, kaliotoxin (KTX), we gained new insight into the topology of the pore region of a voltage-gated potassium channel, mKv1.1. In order to find new interactions between mKv1.1 and KTX, we investigated the pH dependence of KTX block which was stronger at pH(o) 6.2 compared with pH(o) 7.4. Using site-directed mutagenesis on the channel and the toxin, we found that protonation of His(34) in KTX caused the pH(o) dependence of KTX block. Glu(350) and Glu(353) in mKv1.1, which interact with His(34) in KTX, were calculated to be 4 and 7 A away from His(34)/KTX, respectively. Docking of KTX into a homology model of mKv1.1 based on the KcsA crystal structure using this and other known interactions as constraints showed structural differences between mKv1.1 and KcsA within the turret (amino acids 348-357). To satisfy our data, we would have to modify the KcsA crystal structure for the mKv1.1 channel orienting Glu(350) 7 A and Glu(353) 4 A more toward the center of the pore compared with KcsA. This would place Glu(350) 15 A and Glu(353) 11 A away from the center of the pore instead of the distances for the equivalent KcsA residues with 22 A for Gly(53) and 15 A for Gly(56), respectively. Bacterial and mammalian potassium channels may have structural differences regarding the turret of the outer pore vestibule. This topological difference between both channel types may have substantial influence on structure-guided development of new drugs for mammalian potassium channels by rational drug design.     

Voltage dependence of partial reactions of the Na+/K+ pump: predictions from microscopic models

A theoretical treatment of the voltage dependence of electroneutral Na+-Na+ and K+-K+ exchange mediated by the Na+/K+ pump is given. The analysis is based on the Post-Albers reaction scheme in which the overall transport process is described as a sequence of conformational transitions and ion-binding and ion-release steps. The voltage dependence of the exchange rate is determined by a set of 'dielectric coefficients' reflecting the magnitude of charge translocations associated with individual reaction steps. Charge movement may result from conformational changes of the transport protein and/or from migration of ions in an access channel connecting the binding sites with the aqueous medium. It is shown that valuable mechanistic information may be obtained by studying the voltage dependence of transport rates at different (saturating and nonsaturating) ion concentrations.     

The molecular basis of the temperature- and pH-induced conformational transitions in elastin-based peptides

Elastin undergoes an inverse temperature transition and collapses at high temperatures in both simulation and experiment. We investigated a pH-dependent modification of this transition by simulating a glutamic acid (Glu)-substituted elastin at varying pHs and temperatures. The Glu-substituted peptide collapsed at higher temperature than the unsubstituted elastin when Glu was charged. The charge effects could be reversed by neutralization of the Glu carboxyl groups at low pH, and in that case the peptide collapsed at a lower temperature. The collapse was accompanied by the formation of beta-turns and short distorted beta-sheets. Formation of contacts between hydrophobic side chains drives the collapse at high temperature, but interactions between water and polar groups (Glu and main chain) can attenuate this effect at high pH. The overall competition and balance of the polar and nonpolar groups determined the conformational states of the peptide. Water hydration contributed to the conformational transition, and the peptide and its hydration shell must be considered. Structurally, waters near polar residues mainly formed hydrogen bonds with the protein atoms, while waters around the hydrophobic side chains tended to be parallel to the peptide groups to maximize water-water interactions.     

Interactions of monovalent cations with sodium channels in squid axon. II. Modification of pharmacological inactivation gating

The time-, frequency-, and voltage-dependent blocking actions of several cationic drug molecules on open Na channels were investigated in voltage-clamped, internally perfused squid giant axons. The relative potencies and time courses of block by the agents (pancuronium [PC], octylguanidinium [C8G], QX-314, and 9-aminoacridine [9-AA]) were compared in different intracellular ionic solutions; specifically, the influences of internal Cs, tetramethylammonium (TMA), and Na ions on block were examined. TMA+ was found to inhibit the steady state block of open Na channels by all of the compounds. The time-dependent, inactivation-like decay of Na currents in pronase-treated axons perfused with either PC, 9-AA, or C8G was retarded by internal TMA+. The apparent dissociation constants (at zero voltage) for interaction between PC and 9-AA with their binding sites were increased when TMA+ was substituted for Cs+ in the internal solution. The steepness of the voltage dependence of 9-AA or PC block found with internal Cs+ solutions was greatly reduced by TMA+, resulting in estimates for the fractional electrical distance of the 9-AA binding site of 0.56 and 0.22 in Cs+ and TMA+, respectively. This change may reflect a shift from predominantly 9-AA block in the presence of Cs+ to predominantly TMA+ block. The depth, but not the rate, of frequency-dependent block by QX-314 and 9-AA is reduced by internal TMA+. In addition, recovery from frequency-dependent block is not altered. Elevation of internal Na produces effects on 9-AA block qualitatively similar to those seen with TMA+. The results are consistent with a scheme in which the open channel blocking drugs, TMA (and Na) ions, and the inactivation gate all compete for a site or for access to a site in the channel from the intracellular surface. In addition, TMA ions decrease the apparent blocking rates of other drugs in a manner analogous to their inhibition of the inactivation process. Multiple occupancy of Na channels and mutual exclusion of drug molecules may play a role in the complex gating behaviors seen under these conditions.     

Single and combined effects of carbamazepine and vinpocetine on depolarization-induced changes in Na+, Ca2+ and glutamate release in hippocampal isolated nerve endings

The single and combined effects of carbamazepine and vinpocetine on the release of the excitatory amino acid neurotransmitter glutamate, on the rise in internal Na+ (Na(i), as determined with SBFI), and on the rise in internal Ca2+ (Ca(i), as determined with fura-2) induced by an increased permeability of presynaptic Na+ channels, with veratridine, or by an increased permeability of presynaptic Ca2+ channels with high K+, were investigated in isolated hippocampal nerve endings. The present study shows that carbamazepine and vinpocetine, both inhibit dose dependently the release of preloaded [3H]Glu induced by veratridine. However, carbamazepine is two orders of magnitude less potent than vinpocetine. The calculated IC(50)'s for carbamazepine and vinpocetine to inhibit veratridine-induced [3H]Glu release are 200 and 2 microM, respectively. Consistently 150 microM carbamazepine and 1.5 microM vinpocetine reduce the veratridine-induced rise in Na(i) in a similar extent. The single effects of carbamazepine and of vinpocetine on the presynaptic Na+ channel mediated responses, namely the rise in Na(i) and the release of Glu induced by veratridine, are additive. Responses that depend on the entrance of external Ca2+ via presynaptic Ca2+ channels, such as the release of [3H]Glu and the rise in Ca(i) induced by high K+, are insensitive to 300 microM carbamazepine and slightly reduced by 5 microM vinpocetine. It is concluded that the additive effects of carbamazepine, which is one of the most common antiepileptic drugs, and vinpocetine that besides its known neuroprotective action and antiepileptic potential is a memory enhancer, may perhaps be advantageous in the treatment of epileptic patients.     

A delta-conotoxin from Conus ermineus venom inhibits inactivation in vertebrate neuronal Na+ channels but not in skeletal and cardiac muscles

We have isolated delta-conotoxin EVIA (delta-EVIA), a conopeptide in Conus ermineus venom that contains 32 amino acid residues and a six-cysteine/four-loop framework similar to that of previously described omega-, delta-, microO-, and kappa-conotoxins. However, it displays low sequence homology with the latter conotoxins. delta-EVIA inhibits Na+ channel inactivation with unique tissue specificity upon binding to receptor site 6 of neuronal Na+ channels. Using amphibian myelinated axons and spinal neurons, we showed that delta-EVIA increases the duration of action potentials by inhibiting Na+ channel inactivation. delta-EVIA considerably enhanced nerve terminal excitability and synaptic efficacy at the frog neuromuscular junction but did not affect directly elicited muscle action potentials. The neuronally selective property of delta-EVIA was confirmed by showing that a fluorescent derivative of delta-EVIA labeled motor nerve endings but not skeletal muscle fibers. In a heterologous expression system, delta-EVIA inhibited inactivation of rat neuronal Na+ channel subtypes (rNaV1.2a, rNaV1.3, and rNaV1.6) but did not affect rat skeletal (rNaV1.4) and human cardiac muscle (hNaV1.5) Na+ channel subtypes. delta-EVIA, in the range of concentrations used, is the first conotoxin found to affect neuronal Na+ channels without acting on Na+ channels of skeletal and cardiac muscle. Therefore, it is a unique tool for discriminating voltage-sensitive Na+ channel subtypes and for studying the distribution and modulation mechanisms of neuronal Na+ channels, and it may serve as a lead to design new drugs adapted to treat diseases characterized by defective nerve conduction.     

External pore collapse as an inactivation mechanism for Kv4.3 K+ channels

Kv4 channels are thought to lack a C-type inactivation mechanism (collapse of the external pore) and to inactivate as a result of a concerted action of cytoplasmic regions of the channel. To investigate whether Kv4 channels have outer pore conformational changes during the inactivation process, the inactivation properties of Kv4.3 were characterized in 0 mM and in 2 mM external K+ in whole-cell voltage-clamp experiments. Removal of external K+ increased the inactivation rates and favored cumulative inactivation by repetitive stimulation. The reduction in current amplitude during repetitive stimulation and the faster inactivation rates in 0 mM external K+ were not due to changes in the voltage dependence of channel opening or to internal K+ depletion. The extent of the collapse of the K+ conductance upon removal of external K+ was more pronounced in NMG+-than in Na+-containing solutions. The reduction in the current amplitude during cumulative inactivation by repetitive stimulation is not associated with kinetic changes, suggesting that it is due to a diminished number of functional channels with unchanged gating properties. These observations meet the criteria for a typical C-type inactivation, as removal of external K+ destabilizes the conducting state, leading to the collapse of the pore. A tentative model is presented, in which K+ bound to high-affinity K+-binding sites in the selectivity filter destabilizes an outer neighboring K+ modulatory site that is saturated at approximately 2 mM external K+. We conclude that Kv4 channels have a C-type inactivation mechanism and that previously reported alterations in the inactivation rates after N- and C- termini mutagenesis may arise from secondary changes in the electrostatic interactions between K+-binding sites in the selectivity filter and the neighboring K+-modulatory site, that would result in changes in its K+ occupancy.     

Sodium flux ratio through the amiloride-sensitive entry pathway in frog skin

The sodium flux ratio of the amiloride-sensitive Na+ channel in the apical membrane of in vitro Rana catesbeiana skin has been evaluated at different sodium concentrations and membrane potentials in sulfate Ringer solution. Amiloride-sensitive unidirectional influxes and effluxes were determined as the difference between bidirectional 22Na and 24Na fluxes simultaneously measured in the absence and presence of 10(-4) M amiloride in the external bathing solution. Amiloride- sensitive Na+ effluxes were induced by incorporation of cation- selective ionophores (amphotericin B or nystatin) into the normally Na+- impermeable basolateral membrane. Apical membrane potentials (Va) were measured with intracellular microelectrodes. We conclude that since the flux ratio exponent, n', is very close to 1, sodium movement through this channel can be explained by a free-diffusion model in which ions move independently. This result, however, does not necessarily preclude the possibility that this transport channel may contain one or more ion binding sites.     

Batrachotoxin-modified sodium channels in planar lipid bilayers. Characterization of saxitoxin- and tetrodotoxin-induced channel closures

The guanidinium toxin-induced inhibition of the current through voltage-dependent sodium channels was examined for batrachotoxin-modified channels incorporated into planar lipid bilayers that carry no net charge. To ascertain whether a net negative charge exists in the vicinity of the toxin-binding site, we studied the channel closures induced by tetrodotoxin (TTX) and saxitoxin (STX) over a wide range of [Na+]. These toxins carry charges of +1 and +2, respectively. The frequency and duration of the toxin-induced closures are voltage dependent. The voltage dependence was similar for STX and TTX, independent of [Na+], which indicates that the binding site is located superficially at the extracellular surface of the sodium channel. The toxin dissociation constant, KD, and the rate constant for the toxin-induced closures, kc, varied as a function of [Na+]. The Na+ dependence was larger for STX than for TTX. Similarly, the addition of tetraethylammonium (TEA+) or Zn++ increased KD and decreased kc more for STX than for TTX. These differential effects are interpreted to arise from changes in the electrostatic potential near the toxin-binding site. The charges giving rise to this potential must reside on the channel since the bilayers had no net charge. The Na+ dependence of the ratios KDSTX/KDTTX and kcSTX/kcTTX was used to estimate an apparent charge density near the toxin-binding site of about -0.33 e X nm-2. Zn++ causes a voltage-dependent block of the single-channel current, as if Zn++ bound at a site within the permeation path, thereby blocking Na+ movement. There was no measurable interaction between Zn++ at its blocking site and STX or TTX at their binding site, which suggests that the toxin-binding site is separate from the channel entrance. The separation between the toxin-binding site and the Zn++ blocking site was estimated to be at least 1.5 nm. A model for toxin-induced channel closures is proposed, based on conformational changes in the channel subsequent to toxin binding.     

Kinetically distinguishable AMPA receptors in rat hippocampus are associated with the loss of glutamate-sensitive conformational transitions

We describe a stopped-flow method to study alpha-amino-7-hydroxy-5-methyl-4-isoxazole propionate (AMPA)-kainate receptor-mediated Na+ ion flux through native membranes. Resealed plasmalemma vesicles and nerve endings from the rat hippocampus were mixed rapidly with a membrane impermeant form of the fluorescence indicator, sodium binding benzofurane oxazole and the changes in fluorescence intensity in response to various [Glu] on the time scale of 0.04 ms-10 s were monitored at a sampling rate of 6.55 kHz. Inhibitors like ouabain (1 mM) and 5-methyl-10,11-dihydro-5H-dibenzo[a,d]cyclohepten-5,10-imine maleate (dizocilpine, 50 microM) enhanced Na+ ion translocation under low-[Na+] and physiological conditions, respectively. Dependence of AMPA-kainate receptor kinetics on [Glu] was described in a model of channel activation by faster and slower desensitizing receptors. The model accounted for almost all of the Na+ ion flux activity in the 30 microM-10 mM range of [Glu]. We found that the values of the initial rate constant for Na+ ion influx, JA, and rate constant for desensitization, alpha, for the faster desensitizing receptor were dependent on data sampling rate, whereas the initial rate constant for Na+ ion flux through the slower desensitizing receptor, JB, varied much less with the sampling rate. These phenomena can be described by (1) a fractal model of short-lived AMPA-kainate receptor channel with many closely spaced states (fractal dimension approximately 1.8) and (2) a model of long-lived AMPA-kainate receptor channel with two discrete states.     

N-type inactivation and the S4-S5 region of the Shaker K+ channel

The intracellular segment of the Shaker K+ channel between transmembrane domains S4 and S5 has been proposed to form at least part of the receptor for the tethered N-type inactivation "ball." We used the approach of cysteine substitution mutagenesis and chemical modification to test the importance of this region in N-type inactivation. We studied N-type inactivation or the block by a soluble inactivation peptide ("ball peptide") before and after chemical modification by methanethiosulfonate reagents. Particularly at position 391, chemical modification altered specifically the kinetics of ball peptide binding without altering other biophysical properties of the channel. Results with reagents that attach different charged groups at 391 C suggested that there are both electrostatic and steric interactions between this site and the ball peptide. These findings identify this site to be in or near the receptor site for the inactivation ball. At many of the other positions studied, modification noticeably inhibited channel current. The accessible cysteines varied in the state-dependence of their modification, with five- to tenfold changes in reactions rate depending on the gating state of the channel.     

Redox properties of local anesthetics: A structural determinant of closed channel blockers in BTX-modified Na+ channels

Single channel analyses and macroscopic current measurements have shown that benzocaine is a predominantly closed channel blocker in BTX-modified Na+ channels; cocaine is an open channel blocker; and tetracaine, a dual channel blocker (Wang & Wang, 1994; Wang et al., 1994). The reason for such a selective state-dependent block by local anesthetics in BTX-modified Na+ channels is not clear. We assessed the redox properties of tetracaine, benzocaine, cocaine, and various derivatives by their ability to donate electrons to radical intermediates of eosin dye excited by visible light. Electron-donor properties of the drugs were previously proposed to be involved in Na+ channel blockade (Marinov, 1991). Our results provide evidence that redox properties of tetracaine, benzocaine, and their homologs correlate with their ability to enhance Na+ channel inactivation in BTX-modified Na+ channels. This correlation may be explained in terms of the previously proposed redox model of ion channels.     

Molecular Dynamics Simulation and Molecular Docking Studies of Angiotensin Converting Enzyme with Inhibitor Lisinopril and Amyloid Beta Peptide

Angiotensin converting enzyme (ACE) cleaves amyloid beta peptide. So far this cleavage mechanism has not been studied in detail at atomic level. Keeping this view in mind, we performed molecular dynamics simulation of crystal structure complex of testis truncated version of ACE (tACE) and its inhibitor lisinopril along with Zn²⁺ to understand the dynamic behavior of active site residues of tACE. Root mean square deviation results revealed the stability of tACE throughout simulation. The residues Ala 354, Glu 376, Asp 377, Glu 384, His 513, Tyr 520 and Tyr 523 of tACE stabilized lisinopril by hydrogen bonding interactions. Using this information in subsequent part of study, molecular docking of tACE crystal structure with Aβ-peptide has been made to investigate the interactions of Aβ-peptide with enzyme tACE. The residues Asp 7 and Ser 8 of Aβ-peptide were found in close contact with Glu 384 of tACE along with Zn²⁺. This study has demonstrated that the residue Glu 384 of tACE might play key role in the degradation of Aβ-peptide by cleaving peptide bond between Asp 7 and Ser 8 residues. Molecular basis generated by this attempt could provide valuable information towards designing of new therapies to control Aβ concentration in Alzheimer’s patient.     

Differences in saxitoxin and tetrodotoxin binding revealed by mutagenesis of the Na+ channel outer vestibule.

The marine guanidinium toxins, saxitoxin (STX) and tetrodotoxin (TTX), have played crucial roles in the study of voltage-gated Na+ channels. Because they have similar actions, sizes, and functional groups, they have been thought to associate with the channel in the same manner, and early mutational studies supported this idea. Recent experiments by. Biophys. J. 67:2305-2315) have suggested that the toxins bind differently to the isoform-specific domain I Phe/Tyr/Cys location. In the adult skeletal muscle Na+ channel isoform (microliter), we compared the effects on both TTX and STX affinities of mutations in eight positions known to influence toxin binding. The results permitted the assignment of energies contributed by each amino acid to the binding reaction. For neutralizing mutations of Asp400, Glu755, and Lys1237, all thought to be part of the selectivity filter of the channel, the loss of binding energy was identical for the two toxins. However, the loss of binding energy was quite different for vestibule residues considered to be more superficial. Specifically, STX affinity was reduced much more by neutralizations of Glu758 and Asp1532. On the other hand, mutation of Tyr401 to Cys reduced TTX binding energy twice as much as it reduced STX binding energy. Kinetic analysis suggested that all outer vestibule residues tested interacted with both toxins early in the binding reaction (consistent with larger changes in the binding than unbinding rates) before the transition state and formation of the final bound complex. We propose a revised model of TTX and STX binding in the Na+ channel outer vestibule in which the toxins have similar interactions at the selectivity filter, TTX has a stronger interaction with Tyr401, and STX interacts more strongly with the more extracellular residues.     

Functional role of the conserved active site proline of triosephosphate isomerase

The importance of the fully conserved active site proline, Pro168, for the reaction mechanism of triosephosphate isomerase (TIM) has been investigated by studying the enzymatic and crystallographic properties of the P168A variant of trypanosomal TIM. In TIM, Pro168 follows the key catalytic residue Glu167, situated at the beginning of the flexible active site loop (loop 6). Turnover numbers of the P168A variant for its substrates are reduced approximately 50-fold, whereas the Km values are approximately 2 times lower. The affinity of the P168A variant for the transition state analogue 2-phosphoglycolate (2PG) is reduced 5-fold. The crystal structures of unliganded and liganded (2PG) P168A show that the phosphate moiety of 2PG is bound similarly as in wild-type TIM, whereas the interactions of the carboxylic acid moiety with the side chain of the catalytic Glu167 differ. The unique properties of the proline side chain at position 168 are required to transmit ligand binding to the conformational change of Glu167: the side chain of Glu167 flips from the inactive swung-out to the active swung-in conformation on ligand binding in wild-type TIM, whereas in the mutant this conformational change does not occur. Further structural comparisons show that in the wild-type enzyme the concerted movement of loop 6 and loop 7 from unliganded-open to liganded-closed appears to be facilitated by the interactions of the phosphate moiety with loop 7. Apparently, the rotation of 90 degrees of the Gly211-Gly212 peptide plane of loop 7 plays a key role in this concerted movement.