The electric potential at the entrance of the amphotericin channel

The dependence of the blockage of amphotericin B channel conductance on the concentration of inorganic anions is shown. A model of the interaction of amphotericin B channel with blocker ion is proposed. According to the model the blocker ion is bound to the charged groups of the channel entrance. The inorganic anions also interact with these groups and affect the blocker concentration near the channel entrance.     

Voltage-controlled gating at the intracellular entrance to a hyperpolarization-activated cation channel.

Hyperpolarization-activated cation (HCN) channels regulate pacemaking activity in cardiac cells and neurons. Our previous work using the specific HCN channel blocker ZD7288 provided evidence for an intracellular activation gate for these channels because it appears that ZD7288, applied from the intracellular side, can enter and leave HCN channels only at voltages where the activation gate is opened (Shin, K.S., B.S. Rothberg, and G. Yellen. 2001. J. Gen. Physiol. 117:91-101). However, the ZD7288 molecule is larger than the Na(+) or K(+) ions that flow through the open channel. In the present study, we sought to resolve whether the voltage gate at the intracellular entrance to the pore for ZD7288 also can be a gate for permeant ions in HCN channels. Single residues in the putative pore-lining S6 region of an HCN channel (cloned from sea urchin; spHCN) were substituted with cysteines, and the mutants were probed with Cd(2+) applied to the intracellular side of the channel. One mutant, T464C, displayed rapid irreversible block when Cd(2+) was applied to opened channels, with an apparent blocking rate of approximately 3 x 10(5) M(-1)s(-1). The blocking rate was decreased for channels held at more depolarized voltages that close the channels, which is consistent with the Cd(2+) access to this residue being gated from the intracellular side of the channel. 464C channels could be recovered from Cd(2+) inhibition in the presence of a dithiol applied to the intracellular side. The rate of this recovery also was reduced when channels were held at depolarized voltages. Finally, Cd(2+) could be trapped inside channels that were composed of WT/464C tandem-linked subunits, which could otherwise recover spontaneously from Cd(2+) inhibition. Thus, Cd(2+) escape is also gated at the intracellular side of the channel. Together, these results are consistent with a voltage-controlled structure at the intracellular side of the spHCN channel that can gate the flow of cations through the pore.     

Ion coordination in the amphotericin B channel.

The antifungal polyene antibiotic amphotericin B forms channels in lipid membranes that are permeable to ions, water, and nonelectrolytes. Anion, cation, and ion pair coordination in the water-filled pore of the "barrel" unit of the channels was studied by molecular dynamics simulations. Unlike the case of the gramicidin A channel, the water molecules do not create a single-file configuration in the pore, and some cross sections of the channel contain three or four water molecules. Both the anion and cation are strongly bound to ligand groups and water molecules located in the channel. The coordination number of the ions is about six. The chloride has two binding sites in the pore. The binding with water is dominant; more than four water molecules are localized in the anion coordination sphere. Three motifs of the ion coordination were monitored. The dominant motif occurs when the anion is bound to one ligand group. The ion is bound to two or three ligand groups in the less favorable configurations. The strong affinity of cations to the channel is determined by the negatively charged ligand oxygens, whose electrostatic field dominates over the field of the hydrogens. The ligand contribution to the coordination number of the sodium ion is noticeably higher than in the case of the anion. As in the case of the anion, there are three motifs of the cation coordination. The favorable one occurs when the cation is bound to two ligand oxygens. In the less favorable cases, the cation is bound to three or four oxygens. In the contact ion pair, the cation and anion are bound to two ligand oxygens and one ligand hydrogen, respectively. There exist intermediate solvent-shared states of the ion pair. The average distances between ions in these states are twice as large as that of the contact ion pair. The stability of the solvent-shared state is defined by the water molecule oriented along the electrostatic field of both ions.     

Modulation of MthK potassium channel activity at the intracellular entrance to the pore

We used a bacterial complementation screen with the LB2003 K(+) uptake-deficient strain of Escherichia coli to analyze residues that are critical to Methanobacterium thermoautotrophicum potassium channel (MthK) function. Channel expression and relative structural integrity of mutants were analyzed by SDS-PAGE and Western blot, and mechanisms underlying altered mutant channel function were analyzed using single-channel recording. We observed that wild-type MthK expression complements K(+) uptake deficiency. Although MthK function was previously thought to require Ca(2+) in the millimolar range, we demonstrate that at elevated temperatures the requirement for Ca(2+) becomes much lower. Mutations at the cytoplasmic mouth of the MthK pore can blunt complementation, indicating that those mutant channels cannot support K(+) uptake. In contrast, substitutions at the Ca(2+)-binding site in the MthK RCK domain did not decrease complementation compared with wild-type MthK. We focused on mutations to residues Glu-92 and Glu-96, which may form the narrowest part of the pore in the channel's closed state. Mutations at these residues can yield slight changes in single-channel conductance that do not necessarily correlate with effects on bacterial complementation. However, mutations at Glu-92 could also change channel open probability, and these changes correlated with complementation effects. The most striking of these mutations was E92A, which nearly eliminated bacterial complementation by decreasing the open probability of MthK. Our results suggest that the small, hydrophobic alanine side chain at the K(+) channel bundle crossing may generate an intrinsically stable structure, which in turn shifts the closed-to-open-state equilibrium toward the closed state.     

Antibody interaction with the amphotericin channel

The monoclonal antibodies against asymmetric channel formed in the lipid bilayer of polyene antibiotic amphotericin B and cholesterol after addition of the antibiotic to the compartment from the cis side of the membrane were obtained. The effect of the antibodies on ion conductance of the channel depends on the distribution of cholesterol in the membrane. When cholesterol was present on both sides of the lipid bilayer, three antibody molecules bound to the channel from the trans side of the membrane, thus markedly increasing the lifetime of the open state of the channel. When cholesterol was present in the cis monolayer only, the antibodies, added to the trans compartment of the cell, reduced the membrane conduction.     

A microscopic electrostatic model for the amphotericin B channel.

A microscopic model of an amphotericin B channel is proposed. The structure of the pores is generated using the atomic coordinates of the molecule in the structure determined experimentally by X-ray diffraction. The net charges of the atoms are determined by Mulliken analysis. With these charges the electrostatic energy profiles are calculated for a monovalent ion passing through the channels formed by different number of antibiotic molecules having different radii. The water inside the channel was considered through a continuum medium using the dielectric constant of the bulk, and the membrane contribution was included using the virtual images of the pore in a dielectric slab of epsilon = 3. The model satisfactorily explains the permeability and selectivity characteristics as well as other observations yet unexplained. The electrostatic profiles obtained reinforce the hypothesis of the existence of channels formed by a variable number of units.     

Computer analysis of the spatial structure of the amphotericin channel

Energy of Amphotericin B cholesterol complex in a membrane was calculated by the method of atom--atomic potentials. The complex is shown to have two stable states. One of them is stabilized by electrostatic interactions between charged groups of neighbouring antibiotic molecules due to a decline of the molecules to the pore radius. Another state with radial orientation of antibiotic molecules and smaller pore diameter is stabilized mainly by van-der-Waals forces. A conclusion is made that transitions between open and closed states may result from small shifts and turn of all the antibiotic molecules in the complex.     

Ion passage pathways and thermodynamics of the amphotericin B membrane channel

Amphotericin B is a polyene macrolide antibiotic used to treat systemic fungal infections. Amphotericin B's chemotherapeutic action requires the formation of transmembrane channels, which are known to transmit monovalent ions. We have investigated the ion passage pathways through the pore of a realistic model structure of the channel and computed the associated thermodynamic properties. Our calculations combined the free energy computations using the Poisson equation with a continuum solvent model and the molecular simulations in which solvent molecules were present explicitly. It was found that there are no substantial structural barriers to a single sodium or chloride ion passage. Thermodynamic free energy calculations showed that the path along which the ions prefer to move is off center from the channel's central axis. In accordance with experiments, Monte Carlo molecular simulations established that sodium ions can pass through the pore. When it encounters a chloride anion in the channel, the sodium cation prefers to form a solvent-bridged pair configuration with the anion.     

HIV-1 frameshift efficiency is primarily determined by the stability of base pairs positioned at the mRNA entrance channel of the ribosome

The human immunodeficiency virus (HIV) requires a programmed −1 ribosomal frameshift for Pol gene expression. The HIV frameshift site consists of a heptanucleotide slippery sequence (UUUUUUA) followed by a spacer region and a downstream RNA stem–loop structure. Here we investigate the role of the RNA structure in promoting the −1 frameshift. The stem–loop was systematically altered to decouple the contributions of local and overall thermodynamic stability towards frameshift efficiency. No correlation between overall stability and frameshift efficiency is observed. In contrast, there is a strong correlation between frameshift efficiency and the local thermodynamic stability of the first 3–4 bp in the stem–loop, which are predicted to reside at the opening of the mRNA entrance channel when the ribosome is paused at the slippery site. Insertion or deletions in the spacer region appear to correspondingly change the identity of the base pairs encountered 8 nt downstream of the slippery site. Finally, the role of the surrounding genomic secondary structure was investigated and found to have a modest impact on frameshift efficiency, consistent with the hypothesis that the genomic secondary structure attenuates frameshifting by affecting the overall rate of translation.     

Far-field analysis of coupled bulk and boundary layer diffusion toward an ion channel entrance.

We present a far-field analysis of ion diffusion toward a channel embedded in a membrane with a fixed charge density. The Smoluchowski equation, which represents the 3D problem, is approximated by a system of coupled three- and two-dimensional diffusions. The 2D diffusion models the quasi-two-dimensional diffusion of ions in a boundary layer in which the electrical potential interaction with the membrane surface charge is important. The 3D diffusion models ion transport in the bulk region outside the boundary layer. Analytical expressions for concentration and flux are developed that are accurate far from the channel entrance. These provide boundary conditions for a numerical solution of the problem. Our results are used to calculate far-field ion flows corresponding to experiments of Bell and Miller (Biophys. J. 45:279, 1984).     

Ionic specificity of the gramicidin channel and the thallous ion

The thallous ion was found to interact very specifically with gramicidin channels in black lipid membranes. Although the ion itself carries current through the channel better than Na⁺ or K⁺, it blocks Na⁺ currents at concentrations which are two orders of magnitude lower than the Na⁺ concentrations.     

Metal ion specificity at the catalytic site of yeast enolase.

A new, more gentle enzyme purification for yeast enolase was developed. A series of kinetic experiments was performed with yeast enolase where the concentration of Mg(II) is kept constant and at the Km' level; the addition of Mn(II), Zn(II), or Cu(II) gives a hyperbolic decrease in the enzyme activity. The final velocity of these mixed-metal systems is the same as the velocity obtained only with Mn(II), Zn(II), or Cu(II), respectively. The concentration of the second metal that gives half-maximal effect in the presence of Mg(II) is approximately the same as the apparent Km (Km') value measured for that cation alone. Direct binding of Mn(II) to apoenolase in the absence and presence of Mg(II) shows that Mn(II) and Mg(II) compete for the same metal site on enolase. In the presence of D-2-phosphoglycerate (PGA) and Mg(II), only a single cation site per monomer is occupied by Mn(II). Water proton relaxation rate (PRR) studies of enzyme-ligand complexes containing Mn(II) and Mn(II) in the presence of Mg(II) are consistent with Mn(II) binding at site I under both conditions. PRR titrations of ligands such as the substrate PGA or the inhibitors orthophosphate or fluoride to the enolase-Mn(II)-Mg(II) complex are similar to those obtained for the enolase-Mn(II) complex, also indicating that Mn(II) is at site I in the presence of Mg(II). High-resolution 1H and 31P NMR was used to determine the paramagnetic effect of enolase-bound Mn(II) on the relaxation rates of the nuclei of the competitive inhibitor phosphoglycolate. The distances between the bound Mn(II) and the nuclei were calculated.(ABSTRACT TRUNCATED AT 250 WORDS)     

Organization of polyene antibiotic amphotericin B at the argon-water interface

Amphotericin B (AmB) is a life-saving antibiotic, used to treat deep-seated mycotic infections. Both the therapeutic and toxic side effects of AmB are directly dependent on its molecular organization. Organization of AmB was studied in monocomponent monomolecular layers formed at the argon-water interface, by means of polarized and non-polarized electronic absorption spectroscopy and analyzed in terms of the exciton splitting theory. The results provide direct indication that AmB forms spontaneously dimers that can be assembled into molecular structures characterized by homogeneous orientational distribution in the monolayer, interpreted as cylindrical pores. The structures are not stable at surface pressures higher than 20 mN/m and therefore dimers are concluded as abundant molecular organization forms of AmB in biomembranes. Possibility of stabilization of the cylindrical structures, at higher surface pressures, by other molecules, e.g. sterols, is also discussed.     

Measurement of calcium channel inactivation is dependent upon the test pulse potential.

We have developed two methods to measure Ca2+ channel inactivation in Lymnaea neurons-one method, based upon the conventional double-pulse protocol, uses currents during a moderately large depolarizing pulse, and the other uses tail currents after a very strong activating pulse. Both methods avoid contamination by proton currents and are unaffected by rundown of Ca2+ current. The magnitude of inactivation measured differs for the two methods; this difference arises because the measurement of inactivation is inherently dependent upon the test pulse voltage used to monitor the Ca2+ channel conductance. We discuss two models that can generate such test pulse dependence of inactivation measurements-a two-channel model and a two-open-state model. The first model accounts for this by assuming the existence of two types of Ca2+ channels, different proportions of which are activated by the different test pulses. The second model assumes only one Ca2+ channel type, with two closed and open states; in this model, the test pulse dependence is due to the differential activation of channels in the two closed states by the test pulses. Test pulse dependence of inactivation measurements of Ca2+ channels may be a general phenomenon that has been overlooked in previous studies.     

TRPC channel lipid specificity and mechanisms of lipid regulation

TRPC channels are a subset of the transient receptor potential (TRP) proteins widely expressed in mammalian cells. They are thought to be primarily involved in determining calcium or sodium entry and have broad-ranging functions that include regulation of cell proliferation, motility and contraction. The channels do not respond to a single stimulator but rather are activated or modulated by a multiplicity of factors, potentially existing as integrators at the plasma membrane. This review considers the sensitivity of TRPCs to lipid factors, with focus on sensitivities to diacylglycerols, lysophospholipids, arachidonic acid and its metabolites, sphingosine-1-phosphate (S1P), cholesterol and derivatives, and other lipid factors such as gangliosides. Promiscuous and selective lipid-sensing are apparent. In many cases the lipids stimulate channel function or increase insertion of channels in the membrane. Both direct and indirect (receptor-dependent) lipid effects are evident. Although information is limited, the lipid profiles are consistent with TRPCs having close working relationships with phospholipase C and A2 enzymes. We need much more information about lipid-sensing by TRPCs if we are to fully appreciate its significance, but the available data suggest that lipid-sensing is a key, but not exclusive, aspect of TRPC biology.     

Dissecting independent channel and scaffolding roles of the Drosophila transient receptor potential channel

Drosophila transient receptor potential (TRP) serves dual roles as a cation channel and as a molecular anchor for the PDZ protein, INAD (inactivation no afterpotential D). Null mutations in trp cause impairment of visual transduction, mislocalization of INAD, and retinal degeneration. However, the impact of specifically altering TRP channel function is not known because existing loss-of-function alleles greatly reduce protein expression. In the current study we describe the isolation of a set of new trp alleles, including trp(14) with an amino acid substitution juxtaposed to the TRP domain. The trp(14) flies stably express TRP and display normal molecular anchoring, but defective channel function. Elimination of the anchoring function alone in trp(Delta)(1272), had minor effects on retinal morphology whereas disruption of channel function caused profound light-induced cell death. This retinal degeneration was greatly suppressed by elimination of the Na(+)/Ca(2+) exchanger, CalX, indicating that the cell death was due primarily to deficient Ca(2+) entry rather than disruption of the TRP-anchoring function.