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W Nonner

Publications and source records attributed to W Nonner.

At least 19 recordsLinked to original sources
PubMed2000

Binding and selectivity in L-type calcium channels: a mean spherical approximation.

L-type calcium channels are Ca(2+) binding proteins of great biological importance. They generate an essential intracellular signal of living cells by allowing Ca(2+) ions to move across the lipid membrane into the cell, thereby selecting an ion that is in low extracellular abundance. Their mechanism of selection involves four carboxylate groups, containing eight oxygen ions, that belong to the side chains of the "EEEE" locus of the channel protein, a setting similar to that found in many Ca(2+)-chelating molecules. This study examines the hypothesis that selectivity in this locus is determined by mutual electrostatic screening and volume exclusion between ions and carboxylate oxygens of finite diameters. In this model, the eight half-charged oxygens of the tethered carboxylate groups of the protein are confined to a subvolume of the pore (the "filter"), but interact spontaneously with their mobile counterions as ions interact in concentrated bulk solutions. The mean spherical approximation (MSA) is used to predict ion-specific excess chemical potentials in the filter and baths. The theory is calibrated using a single experimental observation, concerning the apparent dissociation constant of Ca(2+) in the presence of a physiological concentration of NaCl. When ions are assigned their independently known crystal diameters and the carboxylate oxygens are constrained, e.g., to a volume of 0.375 nm(3) in an environment with an effective dielectric coefficient of 63.5, the hypothesized selectivity filter produces the shape of the calcium binding curves observed in experiment, and it predicts Ba(2+)/Ca(2+) and Na(+)/Li(+) competition, and Cl(-) exclusion as observed. The selectivities for Na(+), Ca(2+), Ba(2+), other alkali metal ions, and Cl(-) thus can be predicted by volume exclusion and electrostatic screening alone. Spontaneous coordination of ions and carboxylates can produce a wide range of Ca(2+) selectivities, depending on the volume density of carboxylate groups and the permittivity in the locus. A specific three-dimensional structure of atoms at the binding site is not needed to explain Ca(2+) selectivity.

Binding Sites↗
PubMed1998

Ion permeation and glutamate residues linked by Poisson-Nernst-Planck theory in L-type calcium channels.

L-type Ca channels contain a cluster of four charged glutamate residues (EEEE locus), which seem essential for high Ca specificity. To understand how this highly charged structure might produce the currents and selectivity observed in this channel, a theory is needed that relates charge to current. We use an extended Poisson-Nernst-Planck (PNP2) theory to compute (mean) Coulombic interactions and thus to examine the role of the mean field electrostatic interactions in producing current and selectivity. The pore was modeled as a central cylinder with tapered atria; the cylinder (i.e., "pore proper") contained a uniform volume density of fixed charge equivalent to that of one to four carboxyl groups. The pore proper was assigned ion-specific, but spatially uniform, diffusion coefficients and excess chemical potentials. Thus electrostatic selection by valency was computed self-consistently, and selection by other features was also allowed. The five external parameters needed for a system of four ionic species (Na, Ca, Cl, and H) were determined analytically from published measurements of thre limiting conductances and two critical ion concentrations, while treating the pore as a macroscopic ion-exchange system in equilibrium with a uniform bath solution. The extended PNP equations were solved with these parameters, and the predictions were compared to currents measured in a variety of solutions over a range of transmembrane voltages. The extended PNP theory accurately predicted current-voltage relations, anomalous mole fraction effects in the observed current, saturation effects of varied Ca and Na concentrations, and block by protons. Pore geometry, dielectric permittivity, and the number of carboxyl groups had only weak effects. The successful prediction of Ca fluxes in this paper demonstrates that ad hoc electrostatic parameters, multiple discrete binding sites, and logistic assumptions of single-file movement are all unnecessary for the prediction of permeation in Ca channels over a wide range of conditions. Further work is needed, however, to understand the atomic origin of the fixed charge, excess chemical potentials, and diffusion coefficients of the channel. The Appendix uses PNP2 theory to predict ionic currents for published "barrier-and-well" energy profiles of this channel.

Biophysical Phenomena↗
PubMed1998

Anomalous mole fraction effect, electrostatics, and binding in ionic channels.

Ionic channels bathed in mixed solutions of two permeant electrolytes often conduct less current than channels bathed in pure solutions of either. For many years, this anomalous mole fraction effect (AMFE) has been thought to occur only in single-file pores containing two or more ions at a time. Most thinking about channels incorporates this view. We show here that the AMFE arises naturally, as an electrostatic consequence of localized ion specific binding, if the average current through a channel is described by a theory (Poisson-Nernst-Planck, PNP) that computes the average electric field from the average concentration of charges in and near the channel. The theory contains only those ion-ion interactions mediated by the mean field, and it does not enforce single filing. The AMFE is predicted by PNP over a wide range of mean concentrations of ions in the channel; for example, it is predicted when (on the average) less, or much less, than one ion is found in the channel's pore. In this treatment, the AMFE arises, in large measure, from a depletion layer produced near a region of ion-specific binding. The small excess concentration of ions in the binding region repels all nearby ions of like charge, thereby creating a depletion layer. The overall conductance of the channel arises in effect from resistors in series, one from the binding region, one from the depletion zone, and one from the unbinding region. The highest value resistor (which occurs in the depletion zone) limits the overall series conductance. Here the AMFE is not the result of single filing or multiple occupancy, and so previous views of permeation need to be revised: the presence of an AMFE does not imply that ions permeate single file through a multiply occupied pore.

Binding Sites↗
PubMed1994

Attempts to define functional domains of gap junction proteins with synthetic peptides.

To map the binding sites involved in channel formation, synthetic peptides representing sequences of connexin 32 were tested for their ability to inhibit cell-cell channel formation. Both large peptides representing most of the two presumed extracellular loops of connexin32 and shorter peptides representing subsets of these larger peptides were found to inhibit cell-cell channel formation. The properties of the peptide inhibition suggested that the binding site is complex, involving several segments of both extracellular loops. One of the peptides (a 12-mer) did not inhibit but instead was found to form channels in membranes. Both in oocyte membranes and in bilayers, the channels formed by the peptide were asymmetrically voltage dependent. Their unit conductances ranged from 20 to 160 pS. These data are discussed in the form of a model in which the connexin sequence represented by the peptide is part of a beta structure providing the lining of the channel pore.

Amino Acid Sequence↗
PubMed1994

Anion-cation interactions in the pore of neuronal background chloride channels.

Background Cl channels in neurons and skeletal muscle are significantly permeable for alkali cations when tested with asymmetrical concentrations of the same salt. Both anion and cation permeation were proposed to require binding of an alkali cation with the pore (Franciolini, F., and W. Nonner. 1987. Journal of General Physiology. 90:453-478). We tested this hypothesis by bilaterally substituting large alkali cations for Na and found no significant changes of unitary conductance at 300 mM symmetrical concentrations. In addition, all organic cations examined were permeant in a salt gradient test (1,000 mM internal@300 mM external), including triethanolamine, benzyltrimethylamine, and bis-tris-propane (BTP, which is divalent at the tested pH of 6.2). Inward currents were detected following substitution of internal NaCl by the Na salts of the divalent anions of phosphoric, fumaric, and malic acid. Zero-current potentials in gradients of the Na and BTP salts of varied anions (propionate, F, Br, nitrate) that have different permeabilities under bi-ionic conditions, were approximately constant, as if the permeation of either cation were coupled to the permeation of the anion. These results rule out our earlier hypothesis of anion permeation dependent on a bound alkali cation, but they are consistent with the idea that the tested anions and cations form mixed complexes while traversing the Cl channel.

Animals↗
PubMed1994

A multi-ion permeation mechanism in neuronal background chloride channels.

Unitary current/voltage relationships of background Cl channels of rat hippocampal neurons were determined for varied gradients and absolute concentrations of NaCl. The channels revealed permeabilities for both Cl and Na ions. A hyperlinear increase of unitary conductance, observed for a symmetrical increase of salt concentration from 300 and 600 mM, indicated a multi-ion permeation mechanism. A variety of kinetic models of permeation were tested against the experimental current/voltage relationships. Models involving a pore occupied by mixed complexes of up to five ions were necessary to reproduce all measurements. A minimal model included four equilibrium states and four rate-limiting transitions, such that the empty pore accepts first an anion and then can acquire one or two cation/anion pairs. Three transport cycles are formed: a slow anion cycle (between the empty and single-anion states), a slow cation cycle (between the one- and three-ion states), and a fast anion cycle (between the three- and five-ion states). Thus, permeant anions are required for cation permeation, and several bound anions and cations promote a high rate of anion permeation. The optimized free-energy and electrical charge parameters yielded a self-consistent molecular interpretation, which can account for the particular order in which the pore accepts ions from the solutions. Although the model describes the mixed anion/cation permeability of the channel observed at elevated concentrations, it predicts a high selectivity for Cl anion at physiological ionic conditions.

Animals↗
PubMed1992

Transient K current in the somatic membrane of cultured central neurons of embryonic rat.

1. Somatic K currents of cultured hippocampal, striatal, and spinal cord neurons of embryonic rat were recorded under voltage clamp in membrane spheres ("blebs") excised by means of a tight-seal pipette. 2. The somatic K current in blebs was subject to rapid and near complete inactivation during 300-ms depolarizations, whereas whole-cell K currents included a substantial maintained component. Size and kinetic properties of bleb and whole-cell currents were stable throughout the recording period. 3. The steady-state inactivation of somatic A current was steeply voltage dependent and complete near voltage levels that activated current, whereas peak conductances did not saturate during depolarizations up to +90 mV. Activation started with a delay. Half-times of activation decreased with depolarization, but half-times of inactivation varied little with depolarization. Recovery from inactivation followed a sigmoidal time course with half-times of approximately 50 ms. 4. Half-times of activation and inactivation varied over more than an order of magnitude between individual neurons. Midpoint potentials of inactivation and peak conductance varied over approximately 40 mV. The parameter ranges of hippocampal, striatal, and spinal cord neurons overlapped. 5. Individual soma membranes revealed signs of K channel heterogeneity in their 4-aminopyridine block, current fluctuations, and current kinetics. On the other hand, currents elicited after conditioning pulses that established varied degrees of steady-state inactivation or of recovery from full inactivation had superimposable time courses. 6. The described characteristics of the somatic A channels are compared with those reported for the RCK4, Raw3, and mShal products expressed in Xenopus oocytes. Whereas the ranges of voltage dependencies and of most kinetic characteristics are compatible among native and cloned channels, these three cloned channels recover much more slowly from inactivation. In addition, inactivation in native channels, unlike that in RCK4 and Raw3 channels, was stable after excision in a subcellular fragment.

4-Aminopyridine↗
PubMed1987

Anion and cation permeability of a chloride channel in rat hippocampal neurons.

The ionic permeability of a voltage-dependent Cl channel of rat hippocampal neurons was studied with the patch-clamp method. The unitary conductance of this channel was approximately 30 pS in symmetrical 150 mM NaCl saline. Reversal potentials interpreted in terms of the Goldman-Hodgkin-Katz voltage equation indicate a Cl:Na permeability ratio of approximately 5:1 for conditions where there is a salt gradient. Many anions are permeant; permeability generally follows a lyotropic sequence. Permeant cations include Li, Na, K, and Cs. The unitary conductance does not saturate for NaCl concentrations up to 1 M. No Na current is observed when the anion Cl is replaced by the impermeant anion SO4. Unitary conductance depends on the cation species present. The channel is reversibly blocked by extracellular Zn or 9-anthracene carboxylic acid. Physiological concentrations of Ca or Mg do not affect the Na:Cl permeability ratio. The permeability properties of the channel are consistent with a permeation mechanism that involves an activated complex of an anionic site, an extrinsic cation, and an extrinsic anion.

Animals↗
PubMed1984

Non-stationary fluctuations of the potassium conductance at the node of ranvier of the frog.

Potassium currents were recorded from voltage-clamped nodes of isolated, myelinated axons of Rana pipiens. Nodes were maintained in a modified Ringer solution containing tetrodotoxin to block sodium current and 47.5 mM-potassium to minimize effects of extracellular potassium accumulation. Voltage protocols included depolarizing pulses lasting a few milliseconds to several seconds. Fluctuations about the ensemble average of the current were characterized in terms of non-stationary variance and autocovariance. The fluctuations had a Gaussian amplitude distribution and were virtually free of contaminations from systematic variations of the membrane current. Corrections for background noise were based on measurements done while potassium current was blocked with tetraethylammonium, and on simulations of extrinsic current fluctuations expected to arise from noise in the actual membrane voltage. The fluctuations were attributed to variations of nodal potassium conductance, since they were absent at the reversal potential of the potassium current and at membrane voltages that do not activate potassium current. Covariances indicated that voltage steps that reversed a macroscopic potassium current also reversed the sign of the fluctuation. Plots of the conductance variance versus the mean potassium conductance were generated from both the activation and deactivation (tail) phases of the potassium currents at various voltages between -80 and +70 mV. When the current was activated by a small depolarization (-50 mV) the trajectories from both phases were indistinguishable and were fitted by the parabola expected for a single population of channels with only one open-channel conductance. Apparent single-channel conductance from the early activation phase averaged 24 pS and was not significantly voltage dependent. In contrast, experiments with large depolarizations (+10 to +70 mV) gave significantly different variance--mean trajectories during activation and deactivation and these trajectories were poorly fitted by parabola. This result indicates that the fluctuations reflect several populations of channels and/or a population of channels that can have several levels of non-zero conductance. Projections of the fluctuation covariance showed long correlations, as well as the rapidly decaying component expected from the activation gating of channels. A slow fluctuation arose at a time slightly later than the rise of potassium current, spanned the entire length of brief depolarizations, and extended up to 880 ms during long depolarizations.(ABSTRACT TRUNCATED AT 400 WORDS)

Action Potentials↗
PubMed1981

Structure of the axolemma of frog myelinated nerve: relaxation experiments with a lipophilic probe ion.

Asymmetrical displacement currents are measured in the absence and in the presence of the lipophilic anion dipicrylamine (DPA) in the extracellular solution of nerve fibers of the frog Rana esculenta. DPA (30 nM--3 microM) enhances the current by a component that has the properties expected for a translocation current of DPA ion across the lipid membrane. Analysis in terms of a single-barrier model yields the translocation rate constant (k), the total surface density of DPA absorbed to the membrane (Nt), and the equidistribution voltage (psi). The value of kappa of about 10(4) s-1 is similar to that for a solvent-free artificial bilayer formed by the Montal-Mueller method. The surface density Nt varies with the DPA concentration as it does in the artificial bilayer, but is about tenfold smaller at all concentrations. The DPA ions sense an intrinsic electric field that is offset by a transmembrane voltage between 0 and 30 mV (inside positive). The part of the axolemma probed by the DPA ion appears as a thin ( less than 2.5 nm), fluid bilayer of lipids. DPA ions seem, however, to be excluded from the major part of the axolemma as if this area is occupied by integral proteins or negative charges.

Animals↗
PubMed1981

Block of endplate channels by permeant cations in frog skeletal muscle.

Motor endplates of frog semitendinosus muscles were studied under voltage clamp. Current fluctuations induced by iontophoretic application of acetylcholine were analyzed to give the elementary conductance, gamma , and mean open time, tau , of endplate channels. Total replacement of the external Na+ ion by several other metal ions and by many permeant organic cations changed both gamma and tau . Except with NH4+ ions, the gamma values with foreign test ions were all smaller than expected from the independence relation and their previously measured permeability ratios. The more hydrophobic ions gave the smallest gamma values. Foreign permeant cations also depress gamma when mixed with Na+ ions. These effects could be interpreted in terms of binding of ions to a saturable site within the endplate channel as they pass through. The site for organic ions would have a hydrophobic component. Similar evidence is given for a metal ion binding site on the cytoplasmic end of the channel accessible to internal ions. Most foreign cations also shortened tau when applied externally. The changes of gating did not seem to be correlated with changes in gamma . Thus there is no evidence for control of tau by ions bound within the pore.

Anesthetics, Local↗
PubMed1980

Low intracellular pH and chemical agents slow inactivation gating in sodium channels of muscle.

Excitation of nerve or muscle requires an orderly opening and closing of molecular pores, the ionic channels, in the plasma membrane. During the action potential, Na channels are opened (activated) by the advancing wave of depolarisation, contributing a pulse of inward sodium current, and then are closed again (inactivated) by the continued depolarisation. As one approach both to obtaining molecular information on the Na channel and towards further defining the recently discovered kinetic interactions of the inactivation and activation gating steps, we have surveyed here the effects of chemical agents reported to slow or prevent Na channel inactivation. We find that many of the agents studied by others on invertebrate giant axons or vertebrate nerve act on our frog skeletal muscle preparation. In addition, we have discovered that simply lowering the intracellular pH nearly eliminates inactivation. The activation mechanism seems to resist modification.

Animals↗
PubMed1980

Relations between the inactivation of sodium channels and the immobilization of gating charge in frog myelinated nerve.

1. Single, voltage-clamped nerve fibres of Rana esculenta were stimulated with ;P/2' pulse patterns for measuring Na and gating currents at 13 degrees C.2. Gating currents during test pulses to - 122 or + 10 mV were measured after 45 msec conditioning steps to voltages between - 122 and - 18 mV. As the conditioning voltage was made more positive than - 80 mV, the movable gating charge diminished along a sigmoid curve, approaching a value of nearly one third of the maximum charge. On the other hand, Na inactivation began at a more negative potential and proceeded to undetectable levels.3. After a depolarizing prepulse, both time constant and size of the charge movment depended less steeply on the test voltage than normally. The prepulse reduced gating currents associated with steps from - 122 to test voltages >/= - 40 mV, but enhanced gating currents obtained with test voltages < - 40 mV.4. Increasing the duration of a depolarizing pulse (- 54 to + 42 mV) reduced the fast ;off' gating current at the end of the pulse and enhanced a slow component. Their total charge corresponded approximately to that carried during the pulse. During depolarization, Na current inactivated in a fast and a slow phase. The fast phase was also reflected in the loss of fast charge movement (immobilization) as seen after the pulse was interrupted at various durations.5. The available Na current and the fast movement of gating charge diminished in parallel during prepulses more positive than - 54 mV, and recovered in parallel upon repolarization to levels between - 102 and - 46 mV. During prepulses between - 62 and - 78 mV, however, Na inactivation occurred up to 4 times faster than charge immobilization. Also, at - 78 mV, Na current was inactivated 3 times faster than it recovered.6. These findings indicate that Na inactivation and charge immobilization are linked, but proceed with high-order kinetics. The simplest scheme that accounts for their relation is [Formula: see text] Depending on voltage, either state h(2) (E > - 45 mV) or h(3) (E < - 45 mV) becomes kinetically undetectable.7. A model of the Na channel is developed in which inactivation gains most of its voltage dependence by a coupling to the fast charge movement (activation). The model is shown to be quantitatively consistent with the results. In particular, the change of kinetics observed near - 45 mV can be explained as an effect of the redistribution of charges on the inactivation process.

Animals↗
PubMed1980

Conductance fluctuations from the inactivation process of sodium channels in myelinated nerve fibres.

1. Na currents and fluctuations of Na currents were studied under voltage clamp in the same myelinated nerve fibres of Rana esculenta at 13 degrees C. The results were used to test several kinetic models for the gating process of Na channels.2. Long voltage pulses, depolarizing the membrane by 16-48 mV from a hyperpolarizing holding level of - 28 mV, were applied in 4 sec intervals. The d.c. and a.c. components of the membrane current were recorded during the last 328 msec of the 473 msec pulses. For each depolarization, ninety-six trials were made with the node in Ringer solution and, again, after adding 300 nm-tetrodotoxin (TTX) in that solution.3. The TTX-sensitive d.c. component declined during the 328 msec records by 14-51% of its time average. The a.c. component was corrected for this trend by subtracting the first from the second of each pair of subsequent records. The TTX-sensitive part of its variance declined, on the average, in parallel to the current, as if the open probability rather than the conductance of the individual Na channels was reduced by a slow process.4. Single-channel conductances, gamma, were calculated on the assumption that Na channels have only one non-zero conductance and were corrected for the limited band width (5 kHz) of the a.c. records. Values of gamma increased slightly (< 30% from 16 to 40 mV), and averaged 8.85 +/- 0.7 pS (s.e. of mean, seventeen measurements on ten fibres). This small degree of change in gamma suggests that deviations from the all-or-none gating are very small.5. Power spectral densities of the fluctuations between 3 Hz and 5 kHz were calculated from the trend-free a.c. records and corrected for the TTX-insensitive noise component. Control calculations showed that the only effect of the nonstationarity in the Na current was to enhance the low-frequency points of such spectra by less than 10%. The spectra revealed at least two Lorentzian components with cut-off frequencies in the range expected from the activation and inactivation kinetics. The low-frequency component became dominant as depolarization was increased.6. Na currents recorded during brief (< 40 msec) depolarizations were analysed in terms of various all-or-none gating models, in which inactivation either was independent of activation (Hodgkin-Huxley (HH) model) or could occur only from the partly or fully activated states (coupled models). The transient Na currents were reproduced by all models.7. With the parameters from such fits, the fluctuation spectra expected for each model were calculated. The predictions differed in the fraction, r(h), of the variance contributed by the slow (inactivation) fluctuations; r(h) was larger in the coupled models than in the HH model.8. The experimental spectra were divided into two spectral components to yield empirical values for r(h). We used as templates the spectral curves derived for the fast and for the slow fluctuations of the HH model. The empirical r(h) values were one (48 mV) to four (16 mV) times larger than those expected for the HH model. They were also larger than the theoretical r(h) of the coupled models at the small depolarizations, but became equal or smaller than those at the largest depolarization. Direct comparison of the measured and theoretical spectra revealed the same discrepancies.9. We conclude that all of the simple gating models considered in this paper are inconsistent with the fluctuation measurements, the coupled models giving slightly smaller deviations than the model with independent activation and inactivation.

Animals↗
PubMed1978

Asymmetrical displacement currents in the membrane of frog myelinated nerve: early time course and effects of membrane potential.

1. Asymmetrical displacement currents were studied in myelinated nerve fibres from Rana esculenta with a voltage clamp technique. 2. For brief pulses symmetrical with respect to a holding potential of--97mV, the asymmetry current flowing during pulses (on-response) exhibited a rising phase to a peak followed by an approximately exponential decline. After the pulses the rising phase in the off-response could not be resolved; the time constant varied about 2-fold with either size or duration of the pulse. 3. For longer pulses a second slower component could be detected both in on- and off-responses. 4. The rapidly declining on- and off-responses associated with brief pulses carried about the same charges Qon and Qoff. Increasing the duration of the pulse reduced Qoff. For all pulses tested Qoff approached about one fifth of Qmax. The reduction of Qoff was roughly characterised by time constants ranging between 1.5 and 0.5 ms for potentials between--25 and + 23 mV. Analysis of individual membrane currents confirmed that the capacity current after depolarizing pulses decreased with pulse length. 5. The effects of membrane potential on asymmetry current were studied by varying the level from which pulses were applied during 46.9ms prepulses in the range from--97 to--29mV. The fast and slow components of asymmetry current were affected differently. For potentials more positive than--90mV the fast on-response was reduced and reversed its sign at a potential 25mV more negative than the potential estimated from the steady-state charge distribution measured from--97mV.

Animals↗
PubMed1976

Block of gating currents by ultraviolet radiation in the membrane of myelinated nerve.

The effect of ultraviolet radiation on the asymmetrical displacement currents in the membrane of the node of Ranvier was measured and compared with the ultraviolet blocking of the sodium current. Ultraviolet radiation irreversibly reduced the peak sodium current and the charge displaced during a depolarizing test pulse, the relative reduction being independent of potential. The ratio of the ultraviolet sensitivities of the sodium and the asymmetrical displacement currents is 2.3+/- 0.2. This result suggests two independent identical gating particles per sodium channel in the membrane of myelinated nerve.

Animals↗