Location Of Ion Binding Sites Research Articles

Block of Nachbac by Cadmium and Lanthanum Ions

The prokaryotic, voltage-gated sodium channel, NaChBac, has features common to both eukaryotic Nav and Cav channels, but also differs in important details. NaChBac assembles as a homo-tetramer, and it achieves Na+-selective conduction with a pseudo-symmetric selectivity filter possessing a 4-glutamate motif (EEEE), which is common also to eukaryotic Cav channels, but is distinctly different from the asymmetric selectivity filter (DEKA) of eukaryotic Nav channels. Block by impermeant, or weakly permeant, ions offers functional clues as to the nature and location of ion-binding sites within the channel's conducting pathway. Here, we examine block by a transition metal (Cd2+), or a lanthanide (La3+), in the extracellular solution. Whole-cell patch-clamp experiments were performed using tsA201 cells transiently expressing NaChBac. Onset of block and washout were observed using repeated depolarizations to −10 mV, from a holding potential of −120mV (IC50s (mM, mean±SEM): Cd, 2.0±0.06; La, 0.81±0.03. Two analyses examined voltage dependence of block and provided estimates of the “apparent electrical distance”, δ, from external solution to the blocking site based on the analysis of Woodhull (J. Gen. Physiol., 1973). First, peak current-voltage relations were determined in the presence and absence of the blocker. Second, instantaneous I-V relations allowed calculation of fractional block of the conductance activated by a fixed, activating prepulse. In each analysis, a fit of fractional block, fb, vs V yielded an estimate of δ, and the IC50(V=0). IC50 values were similar to those obtained directly from dose-response data (see above). Values for δ appeared to vary slightly with the method, but indicated only weak V dependence of block: Cd, 0.02 - 0.08; La, 0.02 - 0.13. These data are consistent with a superficial binding site, and only weak coupling of block to movements of permeant ions within the channel. Supported by: MOP-10053 (CIHR).

Structural and functional insights into alphavirus polyprotein processing and pathogenesis

Alphaviruses, a group of positive-sense RNA viruses, are globally distributed arboviruses capable of causing rash, arthritis, encephalitis, and death in humans. The viral replication machinery consists of four nonstructural proteins (nsP1-4) produced as a single polyprotein. Processing of the polyprotein occurs in a highly regulated manner, with cleavage at the P2/3 junction influencing RNA template use during genome replication. Here, we report the structure of P23 in a precleavage form. The proteins form an extensive interface and nsP3 creates a ring structure that encircles nsP2. The P2/3 cleavage site is located at the base of a narrow cleft and is not readily accessible, suggesting a highly regulated cleavage. The nsP2 protease active site is over 40 Å away from the P2/3 cleavage site, supporting a trans cleavage mechanism. nsP3 contains a previously uncharacterized protein fold with a zinc-coordination site. Known mutations in nsP2 that result in formation of noncytopathic viruses or a temperature sensitive phenotype cluster at the nsP2/nsP3 interface. Structure-based mutations in nsP3 opposite the location of the nsP2 noncytopathic mutations prevent efficient cleavage of P23, affect RNA infectivity, and alter viral RNA production levels, highlighting the importance of the nsP2/nsP3 interaction in pathogenesis. A potential RNA-binding surface, spanning both nsP2 and nsP3, is proposed based on the location of ion-binding sites and adaptive mutations. These results offer unexpected insights into viral protein processing and pathogenesis that may be applicable to other polyprotein-encoding viruses such as HIV, hepatitis C virus (HCV), and Dengue virus.

Study of conductance with pericardium: effect of binding site asymmetry on conductance of ions

The observed values of specific conductance of different uni-univalent and uni-bivalent electrolytes were measured with the uptake side, discharge side and across the pericardial membrane in an experimental setup developed in our laboratory. The values of specific conductance (K) were found to be increased with the increase in concentration of the different electrolyte and attain maximum limiting value. The sequence of observed specific conductance for the alkali chlorides and sulphate under the same condition was found to be Na2SO4 > KCl > NaCl > LiCl which is in accordance with their ionic concentration and ionic radii order. The values of specific conductance of all the electrolytes were found different for the uptake side (Kup), discharge side (Kdis) and across the membrane (Kac). The order of three kinds under identical condition being: Kup > Kdis > Kac. This sequence is in good accordance with the higher ion binding affinity of the uptake side than discharge side of the membrane and the asymmetric location of ion binding site with respect to the two sides of pericardium. This asymmetricity of binding site is most likely to cause the variation in the values of Kup and Kdis. The corresponding values of Kac is the lowest which seems to be due to the lower value of dielectric constant of the channel medium of the protein membrane interior than the aqueous phase on both sides of the membrane. In this study the outer layer of pericardium behaved as an uptake side and inner layer as a discharge side.

Theoretical and computational models of biological ion channels

The goal of this review is to establish a broad and rigorous theoretical framework to describe ion permeation through biological channels. This framework is developed in the context of atomic models on the basis of the statistical mechanical projection-operator formalism of Mori and Zwanzig. The review is divided into two main parts. The first part introduces the fundamental concepts needed to construct a hierarchy of dynamical models at different level of approximation. In particular, the potential of mean force (PMF) as a configuration-dependent free energy is introduced, and its significance concerning equilibrium and non-equilibrium phenomena is discussed. In addition, fundamental aspects of membrane electrostatics, with a particular emphasis on the influence of the transmembrane potential, as well as important computational techniques for extracting essential information from all-atom molecular dynamics (MD) simulations are described and discussed. The first part of the review provides a theoretical formalism to 'translate' the information from the atomic structure into the familiar language of phenomenological models of ion permeation. The second part is aimed at reviewing and contrasting results obtained in recent computational studies of three very different channels: the gramicidin A (gA) channel, which is a narrow one-ion pore (at moderate concentration), the KcsA channel from Streptomyces lividans, which is a narrow multi-ion pore, and the outer membrane matrix porin F (OmpF) from Escherichia coli, which is a trimer of three beta-barrel subunits each forming wide aqueous multi-ion pores. Comparison with experiments demonstrates that current computational models are approaching semi-quantitative accuracy and are able to provide significant insight into the microscopic mechanisms of ion conduction and selectivity. We conclude that all-atom MD with explicit water molecules can represent important structural features of complex biological channels accurately, including such features as the location of ion-binding sites along the permeation pathway. We finally discuss the broader issue of the validity of ion permeation models and an outlook to the future.

Affinity and Location of an Internal K+ Ion Binding Site in Shaker K Channels

We have examined the interaction between TEA and K+ ions in the pore of Shaker potassium channels. We found that the ability of external TEA to antagonize block of Shaker channels by internal TEA depended on internal K+ ions. In contrast, this antagonism was independent of external K+ concentrations between 0.2 and 40 mM. The external TEA antagonism of internal TEA block increased linearly with the concentration of internal K+ ions. In addition, block by external TEA was significantly enhanced by increases in the internal K+ concentration. These results suggested that external TEA ions do not directly antagonize internal TEA, but rather promote increased occupancy of an internal K+ site by inhibiting the emptying of that site to the external side of the pore. We found this mechanism to be quantitatively consistent with the results and revealed an intrinsic affinity of the site for K+ ions near 65 mM located approximately 7% into the membrane electric field from the internal end of the pore. We also found that the voltage dependence of block by internal TEA was influenced by internal K+ ions. The TEA site (at 0 internal K+) appeared to sense approximately 5% of the field from the internal end of the pore (essentially colocalized with the internal K+ site). These results lead to a refined picture of the number and location of ion binding sites at the inner end of the pore in Shaker K channels.

Recent Advances in the High Resolution Structures of Bacterial Channels: Gramicidin A

Gramicidin is a polypeptide antibiotic which forms dimeric channels specific for the transport of monovalent cations across membranes. It adopts several different conformations, most notably double helical (pore) and helical dimer (channels) forms, which have very different structural and functional characteristics. This review focuses on recent high resolution structure determinations of both the pore and channel forms of the molecule by X-ray crystallographic and/or NMR spectroscopic techniques. It discusses the structural consequences of binding ions and the location of ion binding sites and how the structures are related to the conductance properties of the molecule. This relatively simple molecule is probably the best characterized ion channel (both structurally and functionally) and has, to date, been the principal proving-ground for many of our ideas about the molecular nature of ion conduction in membranes.

28] Estimating binding constants for site-specific interactions between monovalent ions and proteins

This chapter focuses on estimating binding constants for site-specific interactions among monovalent ions and proteins. The observed effects of salts on the thermodynamic and kinetic properties of proteins can be classified as either specific or aspecific according to their dependence or independence, respectively, on the nature of the salts studied. The widespread availability of computational methods for quantitation of aspecific salt effects from crystallographic structures of macromolecules contrasts with the paucity of approaches for quantitating specific effects. Despite the weak nature of site-specific interactions among monovalent ions and proteins, they play important regulatory roles in many proteins by modulating protein structure and functional energetics. The approach described in this chapter invokes the concept of site-specific ion binding to quantitate specific salt effects in proteins. Owing to the lack of detailed structural information describing the location of ion binding sites on the surfaces of proteins, it is necessary to locate putative binding sites before quantitating the energetics of binding. Identification is based on the assumption that the energy of interaction between monovalent ions and proteins is determined primarily by the electrostatic potential on the surface of the protein.

Location of ion-binding sites in the gramicidin channel by X-ray diffraction

We report the first X-ray diffraction on gramicidin in its membrane-active form by using uniformly aligned multilayer samples of membranes containing gramicidin and ions (Tl +, K +, Ba 2+, Mg 2+ or without ions). Prom the difference electron density profiles, we found a pair of symmetrically located ion-binding sites for Tl + at 9.6(±0.3) Å and for Ba 2+ at 13.0(±0.2) Å from the midpoint of the gramicidin channel. The location of Ba 2+-binding sites is near the ends of the channel, consistent with the experimental observation that divalent cations do not permeate but block the channel. The location of Tl +-binding sites is somewhat of a surprise. It was generally thought that monovalent cations bind to the first turn of the helix from the mouth of the channel. (It is now generally accepted that the gramicidin channel is a cylindrical pore formed by two monomers, each a single-stranded β 6.3 helix and hydrogen-bonded head-to-head at their N termini.) But our experiment shows that the Tl +-binding site is either near the bottom of or below the first helix turn.

Ion interactions in (1-13C)D-Val8 and D-Leu14 analogs of gramicidin A, the helix sense of the channel and location of ion binding sites.

Ion-induced chemical shifts in the carbonyl carbon resonances of synthesized ad verified (1-13C)D-Val8 gramicidin A and (1-13C)D-Leu14 gramicidin A are utilized in combination with the previously determined location of the ion binding sites of the gramicidin A channel (using the carbonyls of L-residues) to determine that the helix sense of the gramicidin A channel) is left-handed. Having resolved the handedness issue, the location of the ion binding sites (which are fundamental to understanding the mechanism of ion transport) are further delineated with the results indicating two sites separated by just over 20 A. Furthermore, the demonstration that the divalent barium ion interacts at the binding site while not being transported through the channel is used to argue that the mechanism of monovalent vs. divalent cation selectivity is due to the positive image force contribution to the central barrier.