Electrostatic interaction in the NH2-terminus accelerates inactivation of the Kv1.4 channel

Inactivation of potassium channels plays an important role in shaping the electrical signalling properties of nerve and muscle cells. While it has been assumed that the rapid inactivation of the Kv1.4 channel is controlled by a “ball and chain” inactivation mechanism, the chain structure of the channel has not been well defined. Here, by conducting electrophysiological studies on variants containing mutations of the positively charged and negatively charged segments of the NH₂-terminal of the channel protein, we show that neutralization or deletion of the positively charged segment (residues 83-98) significantly slowed the inactivation process. Replacement of this positively charged segment with the negatively charged segment (residues 123-137), and vice versa, so that both segments were simultaneously positively or negatively charged, also slowed the inactivation process. Furthermore, the inactivation process was not changed when the positively charged and the negatively charged segments were interchanged. In contrast, the voltage dependence of activation and inactivation of the channels was not significantly altered by these mutants. These results indicate that the electrostatic interaction between the positively and negatively charged segments plays a critical role in the inactivation process of the Kv1.4 channel. Taken together, we propose that the electrostatic interaction accelerates the inactivation of the Kv1.4 channel by making it easier for the inactivation ball to access its binding site.     

Interactions of a Reversible Ryanoid (21-Amino-9α-Hydroxy-Ryanodine) with Single Sheep Cardiac Ryanodine Receptor Channels

The binding of ryanodine to a high affinity site on the sarcoplasmic reticulum Ca²⁺-release channel results in a dramatic alteration in both gating and ion handling; the channel enters a high open probability, reduced-conductance state. Once bound, ryanodine does not dissociate from its site within the time frame of a single channel experiment. In this report, we describe the interactions of a synthetic ryanoid, 21-amino-9α-hydroxy-ryanodine, with the high affinity ryanodine binding site on the sheep cardiac sarcoplasmic reticulum Ca²⁺-release channel. The interaction of 21-amino-9α-hydroxy-ryanodine with the channel induces the occurrence of a characteristic high open probability, reduced-conductance state; however, in contrast to ryanodine, the interaction of this ryanoid with the channel is reversible under steady state conditions, with dwell times in the modified state lasting seconds. By monitoring the reversible interaction of this ryanoid with single channels under voltage clamp conditions, we have established a number of novel features of the ryanoid binding reaction. (a) Modification of channel function occurs when a single molecule of ryanoid binds to the channel protein. (b) The ryanoid has access to its binding site only from the cytosolic side of the channel and the site is available only when the channel is open. (c) The interaction of 21-amino-9α-hydroxy-ryanodine with its binding site is influenced strongly by transmembrane voltage. We suggest that this voltage dependence is derived from a voltage-driven conformational alteration of the channel protein that changes the affinity of the binding site, rather than the translocation of the ryanoid into the voltage drop across the channel.     

On the possible methods for the mathematical description of the ball and chain model of ion channel inactivation

Ion channels are large transmembrane proteins that are able to conduct small inorganic ions. They are characterized by high selectivity and the ability to gate, i.e. to modify their conductance in response to different stimuli. One of the types of gating follows the ball and chain model, according to which a part of the channel’s protein forms a ball connected with the intracellular side of the channel by a polypeptide chain. The ball is able to modify the conductance of the channel by properly binding to and plugging the channel pore. In this study, the polypeptide ball is treated as a Brownian particle, the movements of which are limited by the length of the chain. The probability density of the ball’s position is resolved by different diffusional operators — parabolic (including the case with drift), hyperbolic, and fractional. We show how those different approaches shed light on different aspects of the movement. We also comment on some features of the survival probabilities (which are ready to be compared with electrophysiological measurements) for issues based on the above operators. Paper authored by participants of the international conference: International Workshop on Ionic Channels, Szczyrk, Poland, May 27 – June 01, 2007. Publication cost was covered by the organisers of this meeting.     

Quantitative characterization of the thrombin-heparin interaction. Discrimination between specific and nonspecific binding models

Equilibrium binding of human alpha-thrombin to heparin was investigated at pH 7.4 as a function of thrombin and heparin concentrations, NaCl concentration, temperature, and heparin chain length with the extrinsic fluorescence probe, p-aminobenzamidine, or by quantitative affinity chromatography, in order to distinguish between sequence-specific and nonspecific electrostatic modes of binding. Analysis of binding data by a nonspecific binding model developed for protein-nucleic acid interactions, or by the discrete binding site model previously used to analyze the thrombin-heparin interaction, indicated that both models described the binding interaction equally well over the range of thrombin binding densities accessible to measurement. However, the strong dependence of the thrombin-heparin binding interaction on NaCl concentration, its minimal dependence on temperature, and the increase in apparent binding affinity with increasing heparin oligosaccharide chain length were best accounted for by a nonspecific electrostatic association of thrombin with 5 to 6 anionic residues contained in a 3-disaccharide binding site of heparin. This interaction was characterized by an intrinsic dissociation constant (KD,obs) of 6-10 microM at physiological ionic strength. Although the nonspecific binding model satisfactorily described the binding of thrombin to heparin chains ranging in size from 3 to approximately 13 disaccharides in terms of a single intrinsic KD,obs, deviations from this model were apparent with longer heparin chains (approximately 22 to approximately 35 disaccharides) from a progressive decrease in the intrinsic KD,obs of up to 4-fold. Sedimentation equilibrium analyses of thrombin-heparin complexes suggested a second weaker binding site on thrombin for heparin, which accounted for these deviations as well as the observed insolubility of thrombin-heparin complexes at high thrombin binding densities.     

Time-correlation analysis of simulated water motion in flexible and rigid gramicidin channels.

Molecular dynamics simulations have been done on a system consisting of the polypeptide membrane channel former gramicidin, plus water molecules in the channel and caps of waters at the two ends of the channel. In the absence of explicit simulation of the surrounding membrane, the helical form of the channel was maintained by artificial restraints on the peptide motion. The characteristic time constant of the artificial restraint was varied to assess the effect of the restraints on the channel structure and water motions. Time-correlation analysis was done on the motions of individual channel waters and on the motions of the center of mass of the channel waters. It is found that individual water molecules confined in the channel execute higher frequency motions than bulk water, for all degrees of channel peptide restraint. The center-of-mass motion of the chain of channel waters (which is the motion that is critical for transmembrane transport, due to the mandatory single filing of water in the channel) does not exhibit these higher frequency motions. The mobility of the water chain is dramatically reduced by holding the channel rigid. Thus permeation through the channel is not like flow through a rigid pipe; rather permeation is facilitated by peptide motion. For the looser restraints we used, the mobility of the water chain was not very much affected by the degree of restraint. Depending on which set of experiments is considered, the computed mobility of our water chain in the flexible channel is four to twenty times too high to account for the experimentally measured resistance of the gramicidin channel to water flow. From this result it appears likely that the peptide motions of an actual gramicidin channel embedded in a lipid membrane may be more restrained than in our flexible channel model, and that these restraints may be a significant modulator of channel permeability. For the completely rigid channel model the "trapping" of the water molecules in preferred positions throughout the molecular dynamics run precludes a reasonable assessment of mobility, but it seems to be quite low.     

Fluid Mechanics of DNA Double-Strand Filter Elution

Measurement of infrequent DNA double-strand breaks (DSB) in mammalian cells is essential for the understanding of cell damage by ionizing radiation and many DNA-reactive drugs. One of the most important assays for measuring DSB in cellular DNA is filter elution. This study is an attempt to determine whether standard concepts of fluid mechanics can yield a self-consistent model of this process. Major assumptions of the analysis are reptation through a channel formed by surrounding strands, with only strand ends captured by filter pores. Both viscosity and entanglement with surrounding strands are considered to determine the resistance to this motion. One important result is that the average elution time of a strand depends not only on its length, but also on the size distribution of the surrounding strands. This model is consistent with experimental observations, such as the dependence of elution kinetics upon radiation dose, but independence from the size of the DNA sample up to a critical filter loading, and possible overlap of elution times for strands of different length. It indicates how the dependence of elution time on the flow rate could reveal the relative importance of viscous and entanglement resistance, and also predicts the consequences of using different filters.     

NMR dynamics-derived insights into the binding properties of a peptide interacting with an SH2 domain

The signal transduction protein phospholipase C-gamma1 (PLC-gamma1) is activated when its C-terminal SH2 domain (PLCC) binds the phosphorylated Tyr-1021 site (pTyr-1021) in the beta-platelet-derived growth factor receptor (PDGFR). To better understand the contributions that dynamics make to binding, we have used NMR relaxation experiments to investigate the motional properties of backbone amide and side chain methyl groups in a peptide derived from the pTyr-1021 site of PDGFR, both free and in complex with the PLCC SH2 domain. The free peptide has relaxation properties that are typical for a small, unstructured polymer, while the backbone of the bound peptide is least flexible for residues in the central portion of the binding site with the amplitude of pico- to nanosecond time scale motions increasing toward the C-terminus of the peptide. The increase in large amplitude motion toward the end of the pY1021 peptide is consistent with the bound peptide existing as an ensemble of states with C-terminal residues having the broadest distribution of backbone conformations, while residues in the central binding site are the most restricted. Deuterium spin relaxation experiments establish that the protein-peptide interface is highly dynamic, and this mobility may play an important role in modulating the affinity of the interaction.     

On the role of ball and chain interactions in recovery from the inactivation of the shaker potassium channel

We describe a new factor in the recovery from inactivation in the ball and chain model. We propose a model in which the tension from the chain may help pull the ball away from its binding site, reducing the duration of the inactivation period. A corresponding model was built and analysed. Paper authored by participants of the international conference: International Workshop on Ionic Channels, Szczyrk, Poland, May 27 – June 01, 2007. Publication cost was covered by the organisers of this meeting.     

Molecular dynamics study of ion transport in transmembrane protein channels

Ion transport through biological membranes often takes place via pore-like protein channels. The elementary process of this transport can be described as a motion of the ion in a quasi-periodic multi-well potential. In this study molecular dynamics simulations of ion transport in a model channel were performed in order to test the validity of reaction-rate theory for this process. The channel is modelled as a hexagonal helix of infinite length, and the ligand groups interacting with the ion are represented by dipoles lining the central hole of the channel. The dipoles interact electrostatically with each other and are allowed to oscillate around an equilibrium orientation. The coupled equations of motion for the ion and the dipoles were solved simultaneously with the aid of a numerical integration procedure. From the calculated ion trajectories it is seen that, particularly at low temperatures, the ion oscillates back and forth in the trapping site many times before it leaves the site and jumps over the barrier. The observed oscillation frequency was found to be virtually temperature-independent (nu 0 approximately equal to 2 X 10(12) s-1) so that the strong increase of transport rate with temperature results almost exclusively from the Arrhenius-type exponential dependence of jump probability w on 1/T. At higher temperatures simultaneous jumps over several barriers occasionally occur. Although the exponential form of w(T) was in agreement with the predictions of rate theory, the activation energy Ea as determined from w(T) was different from the barrier height which was calculated from the static potential of the ion in the channel; the actual transport rate was 1 X 10(3) times higher than the rate predicted from the calculated barrier height. This observation was interpreted by the notion that ion transport in the channel is strongly influenced by thermal fluctuations in the conformation of the ligand system which in turn give rise to fluctuations of barrier height.     

Ionic channels with conformational substates.

Recent studies of protein dynamics suggest that ionic channels can assume many conformational substates. Long-lived substates have been directly observed in single-channel current records. In many cases, however, the lifetimes of conformational states will be far below the theoretical limit of time resolution of single-channel experiments. The existence of such hidden substates may strongly influence the observable (time-averaged) properties of a channel, such as the concentration dependence of conductance. A channel exhibiting fast, voltage-dependent transitions between different conductance states may behave as an intrinsic rectifier. In the presence of more than one permeable ion species, coupling between ionic fluxes may occur, even when the channel has only a single ion-binding site. In special situations the rate of ion translocation becomes limited by the rate of conformational transitions, meaning that the channel approaches the kinetic behavior of a carrier. As a result of the strong coulombic interaction between an ion in a binding site and polar groups of the protein, rate constants of conformational transitions may depend on the occupancy of the binding site. Under this condition a nonequilibrium distribution of conformational states is created when ions are driven through the channel by an external force. This may lead to an apparent violation of microscopic reversibility, i.e., to a situation in which the frequency of transitions from state A to state B is no longer equal to the transition frequency from state B to state A.     

Tyrosine phosphorylation of the inactivating peptide of the shaker B potassium channel: a structural-functional correlate

A synthetic peptide patterned after the sequence of the inactivating "ball" domain of the Shaker B K(+) channel restores fast (N-type) inactivation in mutant deletion channels lacking their constitutive ball domains, as well as in K(+) channels that do not normally inactivate. We now report on the effect of phosphorylation at a single tyrosine in position 8 of the inactivating peptide both on its ability to restore fast channel inactivation in deletion mutant channels and on the conformation adopted by the phosphorylated peptide when challenged by anionic lipid vesicles, a model target mimicking features of the inactivation site in the channel protein. We find that the inactivating peptide phosphorylated at Y8 behaves functionally as well as structurally as the noninactivating mutant carrying the mutation L7E. Moreover, it is observed that the inactivating peptide can be phosphorylated by the Src tyrosine kinase either as a free peptide in solution or when forming part of the membrane-bound protein channel as the constitutive inactivating domain. These findings suggest that tyrosine phosphorylation-dephosphorylation of this inactivating ball domain could be of physiological relevance to rapidly interconvert fast-inactivating channels into delayed rectifiers and vice versa.     

Length of the antibody heavy chain complementarity determining region 3 as a specificity-determining factor

The antigen binding site of an antibody is made up of residues residing in six hypervariable loops of the heavy and light chains. In most cases several or all of these loops are required for the establishment of the antigen-binding surface. Five of these loops display a limited diversity in length and sequence while the third complementarity determining region (CDR) of the heavy chain is highly different between antibodies not only with respect to sequence but also with respect to length. Its extensive diversity is a key component in the establishment of binding sites allowing for the recognition of essentially any antigen by humoral immunity. The relative importance of its sequence vs its length diversity in this context is however, not very well established. To investigate this matter further we have used an approach employing combinatorial antibody libraries and antigen-specific selection in the search for CDRH3 length and sequence diversity compatible with a given antigen specificity, the major antigenic determinant on the tumour-associated antigen mucin-1. In this way we have now defined heavy chain CDR3 length as a critical parameter in the creation of an antigen-specific binding site. We also propose that this may reflect a dependence of a particular structure of this hypervariable loop, the major carrier of diversity in the binding site, for establishment of a given specificity.     

Blocking mechanisms of batrachotoxin-activated Na channels in artificial bilayers

The effects of various pharmacological agents that block single batrachotoxin-activated Na channels from rat muscle can be described in terms of three modes of action that correspond to at least three different binding sites. Guanidinium toxins such as tetrodotoxin, saxitoxin, and a novel polypeptide, mu-conotoxin GIIIA, act only from the extra-cellular side and induce discrete blocked states that correspond to residence times of individual toxin molecules. Such toxins apparently do not deeply penetrate the channel pore since the voltage dependence of block is insensitive to toxin charge and block is not relieved by internal Na+. Many nonspecific organic cations, including charged anesthetics, exhibit a voltage-dependent block that is enhanced by depolarization when present on the inside of the channel. This site is probably within the pore, but binding to this site is weak, as indicated by fast blockade that often appears as lowered channel conductance. A separate class of neutral and tertiary amine anesthetics such as benzocaine and procaine induce discrete closed states when added to either side of the membrane. This blocking effect can be explained by preferential binding to closed states of the channel and appears to be due to a modulation of channel gating.     

Inhibition of nicotinic acetylcholine receptor by philanthotoxin-343: kinetic investigations in the microsecond time region using a laser-pulse photolysis technique.

The mechanism of inhibition of a nicotinic acetylcholine receptor (nAChR) in BC(3)H1 muscle cells by philanthotoxin-343 (PhTX-343), a synthetic analogue of philanthotoxin-433, a wasp toxin, was investigated using a laser-pulse photolysis technique with microsecond time resolution and in a carbamoylcholine concentration range of 20-100 microM and PhTX-343 concentration range of 0-200 microM. The rate constant for nAChR channel opening determined by the chemical kinetic techniques decreased with increasing PhTX-343 concentration, whereas there was no significant effect on the rate constant for channel closing. The resulting decrease in the channel-opening equilibrium constant accounted quantitatively for the inhibition of the receptor by the toxin. Single-channel current measurements suggest an additional step in which the open channel:inhibitor complex isomerizes to a nonconducting receptor form. Cell-flow experiments with a time resolution of 10 ms indicate that this isomerization step is only important at very high inhibitor concentrations. The inhibitor binds to the open-channel receptor form, with an affinity that is at least 5 times smaller than that for the closed-channel form. This indicates that receptor inhibition mainly involves the binding of PhTX-343 to the closed-channel form of the receptor. PhTX-343, and an analogue of this polyamine, had no effect when applied to the inside of the cell membrane. However, there was significant inhibition of the nAChR when these compounds were applied to the outside of the cell membrane, indicating an extracellular site for inhibition. Furthermore, increasing the transmembrane potential results in a decrease in the ability of PhTX-343 to inhibit the receptor. This observation is related to the voltage dependence of the effect of PhTX-343 on the rate constant for nAChR channel opening with increasing transmembrane voltage (-60 to 50 mV). This suggests that the voltage dependence of inhibition mainly reflects the effect of transmembrane voltage on the rate constant of channel opening and, therefore, the channel-opening equilibrium constant. PhTX-343 competes with cocaine and procaine for its binding site. The finding that this toxin, which binds to a common inhibitory site with compounds such as cocaine, exerts its effect by decreasing the channel-opening equilibrium constant suggests an approach for the development of therapeutic agents. A compound that binds to this regulatory site but does not affect the channel-opening equilibrium constant may be developed. Such a compound can displace an abused drug such as cocaine and thereby alleviate the toxic effect of this compound on the organism.     

Probing an open CFTR pore with organic anion blockers

The cystic fibrosis transmembrane conductance regulator (CFTR) is an ion channel that conducts Cl- current. We explored the CFTR pore by studying voltage-dependent blockade of the channel by two organic anions: glibenclamide and isethionate. To simplify the kinetic analysis, a CFTR mutant, K1250A-CFTR, was used because this mutant channel, once opened, can remain open for minutes. Dose-response relationships of both blockers follow a simple Michaelis-Menten function with K(d) values that differ by three orders of magnitude. Glibenclamide blocks CFTR from the intracellular side of the membrane with slow kinetics. Both the on and off rates of glibenclamide block are voltage dependent. Removing external Cl- increases affinity of glibenclamide due to a decrease of the off rate and an increase of the on rate, suggesting the presence of a Cl- binding site external to the glibenclamide binding site. Isethionate blocks the channel from the cytoplasmic side with fast kinetics, but has no measurable effect when applied extracellularly. Increasing the internal Cl- concentration reduces isethionate block without affecting its voltage dependence, suggesting that Cl- and isethionate compete for a binding site in the pore. The voltage dependence and external Cl- concentration dependence of isethionate block are nearly identical to those of glibenclamide block, suggesting that these two blockers may bind to a common binding site, an idea further supported by kinetic studies of blocking with glibenclamide/isethionate mixtures. By comparing the physical and chemical natures of these two blockers, we propose that CFTR channel has an asymmetric pore with a wide internal entrance and a deeply embedded blocker binding site where local charges as well as hydrophobic components determine the affinity of the blockers.     

A kinetic analysis of protein transport through the anthrax toxin channel

Anthrax toxin is composed of three proteins: a translocase heptameric channel, (PA(63))(7), formed from protective antigen (PA), which allows the other two proteins, lethal factor (LF) and edema factor (EF), to translocate across a host cell's endosomal membrane, disrupting cellular homeostasis. (PA(63))(7) incorporated into planar phospholipid bilayer membranes forms a channel capable of transporting LF and EF. Protein translocation through the channel can be driven by voltage on a timescale of seconds. A characteristic of the translocation of LF(N), the N-terminal 263 residues of LF, is its S-shaped kinetics. Because all of the translocation experiments reported in the literature have been performed with more than one LF(N) molecule bound to most of the channels, it is not clear whether the S-shaped kinetics are an intrinsic characteristic of translocation kinetics or are merely a consequence of the translocation in tandem of two or three LF(N)s. In this paper, we show both in macroscopic and single-channel experiments that even with only one LF(N) bound to the channel, the translocation kinetics are S shaped. As expected, the translocation rate is slower with more than one LF(N) bound. We also present a simple electrodiffusion model of translocation in which LF(N) is represented as a charged rod that moves subject to both Brownian motion and an applied electric field. The cumulative distribution of first-passage times of the rod past the end of the channel displays S-shaped kinetics with a voltage dependence in agreement with experimental data.     

A model for the mediation of processivity of DNA-targeting proteins by nonspecific binding: dependence on DNA length and presence of obstacles

A physical and mathematical model is presented to explain processivity of proteins on DNA. In this model, a DNA-targeting protein such as a restriction enzyme can diffuse to the DNA surface and nonspecifically bind to it. Once on the DNA surface it will either move along the DNA or equilibrate with the surrounding region. Owing to the nonspecific binding, the search for a specific site on the DNA occurs in a reduced dimensionality, and the protein appears processive when moving from one specific site to another. The simplest version of this nonspecific-binding-facilitated diffusion model is solved and the results quantitatively explain experimentally observed dependence of the processivity ratio on the intervening DNA length between two specific sites.     

Purification and properties of theDrosophila zen protein

Summary The zen protein is encoded by the zerknullt gene required for normal early development inDrosophila. Like many regulatory proteins of this type, zen contains a 60 amino acid homeobox sequence. We have purified the zen protein and studied its solution behavior and its interaction with DNA. The zen protein exists as a monomer in solution with a molecular weight of about 40000. It binds specifically to a site about 900 bases upstream from thezen gene. Within this binding site DNase protection experiments indicate that binding is confined to two regions approximately 11 and 14 bases in length that are separated by about 30 base pairs. The protein concentration dependence of the binding curve suggests that protein binding is non cooperative.     

Characterization of the nucleoside-binding site inside the Tsx channel of Escherichia coli outer membrane. Reconstitution experiments with lipid bilayer membranes

Reconstitution of purified Tsx protein from Escherichia coli into lipid bilayer membranes showed that Tsx formed small ion-permeable channels with a single-channel conductance of 10 pS in 1 M KCl. The dependence of conductance versus salt concentration was linear, suggesting that Tsx has no binding site for ions. Conductance was inhibited by the addition of 20 mM adenosine. Titration of the Tsx-mediated membrane conductance with different solutes including free bases, nucleosides, and deoxynucleosides suggested that the channel contains a binding site for nucleosides but not for sugars or amino acids, and binding increased in the following order: free base, nucleoside, and deoxynucleoside. Among the five nucleosides the stability constant for the binding increased in the order of cytidine, guanosine, uridine, adenosine, and thymidine. Control experiments revealed that the binding of the nucleosides is independent of ion concentration in the aqueous phase, i.e. there was no competition between nucleosides and ions for the binding site inside the channel. The binding of the solutes to the channel interior can be explained by a one-site two-barrier model for the Tsx channel. The advantage of a binding site inside a specific porin for the permeation of solutes is discussed with respect to the properties of a general diffusion pore.