Effect of Cytosolic pH on Inward Currents Reveals Structural Characteristics of the Proton Transport Cycle in the Influenza A Protein M2 in Cell-Free Membrane Patches of Xenopus oocytes

Transport activity through the mutant D44A of the M2 proton channel from influenza virus A was measured in excised inside-out macro-patches of Xenopus laevis oocytes at cytosolic pH values of 5.5, 7.5 and 8.2. The current-voltage relationships reveal some peculiarities: 1. “Transinhibition”, i.e., instead of an increase of unidirectional outward current with increasing cytosolic H⁺ concentration, a decrease of unidirectional inward current was found. 2. Strong inward rectification. 3. Exponential rise of current with negative potentials. In order to interpret these findings in molecular terms, different kinetic models have been tested. The transinhibition basically results from a strong binding of H⁺ to a site in the pore, presumably His37. This assumption alone already provides inward rectification and exponential rise of the IV curves. However, it results in poor global fits of the IV curves, i.e., good fits were only obtained for cytosolic pH of 8.2, but not for 7.5. Assuming an additional transport step as e.g. caused by a constriction zone at Val27 resulted in a negligible improvement. In contrast, good global fits for cytosolic pH of 7.5 and 8.2 were immediately obtained with a cyclic model. A “recycling step” implies that the protein undergoes conformational changes (assigned to Trp41 and Val27) during transport which have to be reset before the next proton can be transported. The global fit failed at the low currents at pH_cyt = 5.5, as expected from the interference of putative transport of other ions besides H⁺. Alternatively, a regulatory effect of acidic cytosolic pH may be assumed which strongly modifies the rate constants of the transport cycle.     

Structure and catalysis by carbonic anhydrase II: role of active-site tryptophan 5

The tryptophan residue Trp5, highly conserved in the α class of carbonic anhydrases including human carbonic anhydrase II (HCA II), is positioned at the entrance of the active site cavity and forms a π-stacking interaction with the imidazole ring of the proton shuttle His64 in its outward orientation. We have observed that replacement of Trp5 in HCA II caused significant structural changes, as determined by X-ray diffraction, in the conformation of 11 residues at the N-terminus and in the orientation of the proton shuttle residue His64. Most significantly, two variants W5H and W5E HCA II had His64 predominantly outward in orientation, while W5F and wild type showed the superposition of both outward and inward orientations in crystal structures. Although Trp5 influences the orientation of the proton shuttle His64, this orientation had no significant effect on the rate constant for proton transfer near 1 μs⁻¹, determined by exchange of ¹⁸O between CO₂ and water measured by mass spectrometry. The apparent values of the pK_a of the zinc-bound water and the proton shuttle residue suggest that different active-site conformations influence the two stages of catalysis, the proton transfer stage and the interconversion of CO₂ and bicarbonate.     

Functional analysis of amino acids of the Na+/H+ exchanger that are important for proton translocation

The Na⁺/H⁺ exchanger is an integral membrane protein found in the plasma membrane of eukaryotic and prokaryotic cells. In eukaryotes it functions to exchange one proton for a sodium ion. In mammals it removes intracellular protons while in plants and fungal cells the plasma membrane form removes intracellular sodium in exchange for extracellular protons. In this study we used the Na⁺/H⁺ exchanger of Schizosaccharomyces pombe (Sod2) as a model system to study amino acids critical for activity of the protein. Twelve mutant forms of the Na⁺/H⁺ exchanger were examined for their ability to translocate protons as assessed by a cytosensor microphysiometer. Mutation of the amino acid Histidine 367 resulted in defective proton translocation. The acidic residues Asp145, Asp178, Asp266 and Asp267 were important in the proton translocation activity of the Na⁺/H⁺ exchanger. Mutation of amino acids His98, His233 and Asp241 did not significantly impair proton translocation by the Na⁺/H⁺ exchanger. These results confirm that polar amino acids are important in proton flux activity of Na⁺/H⁺ exchangers.     

Inward and outward K+-selective currents in the plasma membrane of protoplasts from maize root cortex and stele

In an attempt to understand the processes mediating ion transport within the root, the patch clamp technique was applied to protoplasts isolated from the cortex and stele of maize roots and their plasma membrane conductances investigated. In the whole-cell configuration, membrane hyperpolarization induced a slowly activating inwardly rectifying conductance in most protoplasts isolated from the root cortex. In contrast, most protoplasts isolated from the stele contained a slowly activating outwardly rectifying conductance upon plasma membrane depolarization. The reversal potential of the inward current indicated that it was primarily due to the movement of K⁺; the outwardly rectifying conductance was comparatively less selective for K⁺. Membrane hyperpolarization beyond a threshold of about ?70 mV induced inward currents. When E_K was set negative of this threshold, inward currents activated negative of E_K and no outward currents were observed positive of E_K. Outward currents in the stelar protoplasts activated at potentials positive of ?85 mV. However, when E_K was set positive of ?85 mV a small inward current was also observed at potentials negative (and slightly positive) of the equilibrium potential for K⁺. Inwardly and outwardly rectifying K⁺ channels were observed in outside-out patches from the plasma membrane of cortical and stelar cells, respectively. Characterization of these channels showed that they were likely to be responsible for the macroscopic ‘whole-cell’ currents. Inward and outward currents were affected differently by various K⁺ channel blockers (TEA⁺, Ba²⁺ and Cs⁺). In addition, Ca²⁺ above 1 mM partially blocked the inward current in a voltage-dependent manner but had little effect on the outward current. It is suggested that the inwardly rectifying conductance identified in protoplasts isolated from the cortex probably represents an important component of the low-affinity K⁺ uptake mechanism (mechanism II) identified in intact roots. The outwardly rectifying conductance identified in protoplasts isolated from the stele could play a role in the release of cations into the xylem vessels for transport to the shoot.     

Whole-cell K+ current activation in response to voltages and carbachol in gastric parietal cells isolated from guinea pig

Summary Patch-clamp studies of whole-cell ionic currents were carried out in parietal cells obtained by collagenase digestion of the gastric fundus of the guinea pig stomach. Applications of positive command pulses induced outward currents. The conductance became progressively augmented with increasing command voltages, exhibiting an outwardly rectifying current-voltage relation. The current displayed a slow time course for activation. In contrast, inward currents were activated upon hyperpolarizing voltage applications at more negative potentials than the equilibrium potential to K⁺ (E _K). The inward currents showed time-dependent inactivation and an inwardly rectifying current-voltage relation. Tail currents elicited by voltage steps which had activated either outward or inward currents reversed at nearE _K, indicating that both time-dependent and voltagegated currents were due to K⁺ conductances. Both outward and inward K⁺ currents were suppressed by extracellular application of Ba²⁺, but little affected by quinine. Tetraethylammonium inhibited the outward current without impairing the inward current, whereas Cs⁺ blocked the inward current but not the outward current. The conductance of inward K⁺ currents, but not outward K⁺ currents, became larger with increasing extracellular K⁺ concentration. A Ca²⁺-mobilizing acid secretagogue, carbachol, and a Ca²⁺ ionophore, ionomycin, brought about activation of another type of outward K⁺ currents and voltage-independent cation currents. Both currents were abolished by cytosolic Ca²⁺ chelation. Quinine preferentially inhibited this K⁺ current. It is concluded that resting parietal cells of the guinea pig have two distinct types of voltage-dependent K⁺ channels, inward rectifier and outward rectifier, and that the cells have Ca²⁺-activated K⁺ channels which might be involved in acid secretion under stimulation by Ca²⁺-mobilizing secretagogues.     

A Cellular Automata Model of Proton Hopping Down a Channel

Proton hopping is the process where a H‐atom on a hydronium ion forms a H‐bond with the O‐atom of a neighboring H₂O molecule. There is then an exchange of bonding forces when that covalent bond of the H‐atom in the hydronium ion changes to a H‐bond, and the previous H‐bond changes to a covalent bond with the neighboring O‐atom. The neighboring molecule now becomes a hydronium (H₃O⁺) ion. This process repeats itself very rapidly among neighboring hydronium and H₂O molecules. There is a flow of protonic character through bulk H₂O, referred to as proton hopping. This process carries information through living systems where H₂O is present. A cellular automata model of proton hopping down a channel has been created and studied. Variations in the rate of proton entry into the channel and the effects of the polar character of the channel walls was studied using the model. The behavior of the models corresponds to experimental results.     

Proton transfer in catalysis and the role of proton shuttles in carbonic anhydrase

The undisputed role of His64 in proton transfer during catalysis by carbonic anhydrases in the α class has raised questions concerning the details of its mechanism. The highly conserved residues Tyr7, Asn62, and Asn67 in the active-site cavity function to fine tune the properties of proton transfer by human carbonic anhydrase II (HCA II). For example, hydrophobic residues at these positions favor an inward orientation of His64 and a low pK_a for its imidazole side chain. It appears that the predominant manner in which this fine tuning is achieved in rate constants for proton transfer is through the difference in pK_a between His64 and the zinc-bound solvent molecule. Other properties of the active-site cavity, such as inward and outward conformers of His64, appear associated with the change in ΔpK_a; however, there is no strong evidence to date that the inward and outward orientations of His64 are in themselves requirements for facile proton transfer in carbonic anhydrase.     

Route,mechanism, and implications of proton import during Na+/K+ exchange by native Na+/K+-ATPase pumps

A single Na⁺/K⁺-ATPase pumps three Na⁺ outwards and two K⁺ inwards by alternately exposing ion-binding sites to opposite sides of the membrane in a conformational sequence coupled to pump autophosphorylation from ATP and auto-dephosphorylation. The larger flow of Na⁺ than K⁺ generates outward current across the cell membrane. Less well understood is the ability of Na⁺/K⁺ pumps to generate an inward current of protons. Originally noted in pumps deprived of external K⁺ and Na⁺ ions, as inward current at negative membrane potentials that becomes amplified when external pH is lowered, this proton current is generally viewed as an artifact of those unnatural conditions. We demonstrate here that this inward current also flows at physiological K⁺ and Na⁺ concentrations. We show that protons exploit ready reversibility of conformational changes associated with extracellular Na⁺ release from phosphorylated Na⁺/K⁺ pumps. Reversal of a subset of these transitions allows an extracellular proton to bind an acidic side chain and to be subsequently released to the cytoplasm. This back-step of phosphorylated Na⁺/K⁺ pumps that enables proton import is not required for completion of the 3 Na⁺/2 K⁺ transport cycle. However, the back-step occurs readily during Na⁺/K⁺ transport when external K⁺ ion binding and occlusion are delayed, and it occurs more frequently when lowered extracellular pH raises the probability of protonation of the externally accessible carboxylate side chain. The proton route passes through the Na⁺-selective binding site III and is distinct from the principal pathway traversed by the majority of transported Na⁺ and K⁺ ions that passes through binding site II. The inferred occurrence of Na⁺/K⁺ exchange and H⁺ import during the same conformational cycle of a single molecule identifies the Na⁺/K⁺ pump as a hybrid transporter. Whether Na⁺/K⁺ pump–mediated proton inflow may have any physiological or pathophysiological significance remains to be clarified.     

Proton transport by gastric membrane vesicles

A highly purified membrane fraction was derived from hog gastric mucosa by a combination of differential and density gradient centrifugation and free flow electrophoresis. This final fraction was 35-fold enriched with respect to cation activated ouabain-insensitive ATPase. Antibody against this fraction was shown to be bound to the luminal surface of the gastric glands. The addition of ATP to this fraction or the density gradient fraction resulted in H⁺ uptake into an osmotically sensitive space. The apparent K_m for ATP was 1.7 · 10^?4 M in the absence of a K⁺ gradient similar to that found for ATPase activity. The reaction is specific for ATP and requires cation in the sequence K⁺ > Rb⁺ > Cs⁺ > Na⁺ > Li⁺ and is inhibited by ATPase inhibitors such as N,N′-dicylclohexylcarbodiimide. Maximal H⁺ uptake occurs with an outward K⁺ gradient but the minimal apparent K_A is found in the absence of a K⁺ gradient. The pH optimum for H⁺ uptake is between 5.8 and 6.2 which corresponds to the pH range for phosphorylation of the enzyme, but is considerably less than the pH maximum of the K⁺ dependent dephosphorylation. In the presence of an inward K^? gradient, protonophores such as tetrachlorsalicylanilide only partially abolish the H⁺ gradient but valinomycin dissipates 75% of the gradient, and nigericin abolishes the gradient. The vesicles therefore have a low K⁺ conductance but a measurable H⁺ conductance, hence a K⁺ gradient can produce an H⁺ gradient in the presence of valinomycin. The uptake and spontaneous leak of H⁺ are temperature sensitive skin with a similar transition temperature. Ultraviolet irradiation inactivates ATPase and proton transport at the same rate, approximately at twice the rate of p-nitrophenylphosphatase inactivation. It is concluded that H⁺ uptake by these vesicles is probably due to a dimeric (H⁺ + K⁺)-ATPase and is probably non-electrogenic.     

Macroscopic Ca2+ -Na+ and K+ currents in single heart and aortic cells

Summary The whole-cell voltage clamp technique was used to study the slow inward currents and K⁺ outward currents in single heart cells of embryonic chick and in rabbit aortic cells. In single heart cells of 3-day-old chick embryo three types of slow inward Na⁺ currents were found. The kinetics and the pharmacology of the slow I_Na, were different from those of the slow Ica in older embryos. Two types of slow inward currents were found in aortic single cells of rabbit; angiotensin 11 increased the sustained type and d-cAMP and d-cGMP decreased the slow transient component. Two types of outward K⁺ currents were found in both aortic and heart cells. Single channel analysis demonstrated the presence of a high single K⁺ channel conductance in aortic cells. In cardiac and vascular smooth muscles, slow inward currents do share some pharmacological properties, although the regulation of these channels by cyclic nucleotides and several drugs seems to be different.     

K Channels Are Responsible for an Inwardly Rectifying Current in the Plasma Membrane of Mesophyll Protoplasts of Avena sativa

In whole-cell recording, the conductance of the plasma membrane of protoplasts isolated from mesophyll cells of leaves of oat (Avena sativa) was greater for inward than outward current. The inward current in both the whole-cell mode and with isolated patches was dependent on [K⁺]_o. When the membrane voltage was more positive than −50 millivolts, the membrane conductance in the whole-cell mode was low, and K⁺ channels in cell-attached or outside-out patches had a low probability of being open. At a membrane voltage more negative than −50 millivolts, the membrane conductance increased by sevenfold in the whole-cell mode, and the probability of the channels being open increased. The inward current was highly selective for K⁺ compared with Cs⁺, Na⁺, choline or Cl⁻. Low concentrations of [Cs⁺]_o or [Na⁺]_o blocked the inward current in a strongly voltage-dependent fashion. Comparison of single-channel with the macroscopic current yields an estimate of about 200 inwardly rectifying K⁺ channels per cell at a density of 0.035 per square micrometer. At physiological membrane voltages and [K⁺]_o about 10 millimolar, the influx through these channels is sufficient to increase the internal [K⁺] by 2 millimolar per minute. These K⁺ channels are activated by membrane voltages in the normal physiological range and could contribute to K⁺ uptake whenever the membrane is more negative than the K⁺ equilibrium potential.     

The membrane potential of Arabidopsis thaliana guard cells; depolarizations induced by apoplastic acidification

The apoplastic pH of guard cells probably acidifies in response to light, since light induces proton extrusion by both guard cells and epidermal leaf cells. From the data presented here, it is concluded that these apoplastic pH changes will affect K⁺ fluxes in guard cells of Arabidopsis thaliana (L.) Heynh. Guard cells of this species were impaled with double-barrelled microelectrodes, to measure the membrane potential (E_m) and the plasma-membrane conductance. Guard cells were found to exhibit two states with respect to their E_m, a depolarized and a hyperpolarized state. Apoplastic acidification depolarized E_m in both states, though the origin of the depolarization differed for each state. In the depolarized state, the change in E_m was the result of a combined pH effect on instantaneously activating conductances and on the slow outward rectifying K⁺ channel (s-ORC). At a more acidic apoplastic pH, the current through instantaneously activated conductances became more inwardly directed, while the maximum conductance of s-ORC decreased. The effect on s-ORC was accompanied by an acceleration of activation and deactivation of the channel. Experiments with acid loading of guard cells indicated that the effect on s-ORC was due to a lowered intracellular pH, caused by apoplastic acidification. In the hyperpolarized state, the pH-induced depolarization was due to a direct effect of the apoplastic pH on the inward rectifying K⁺ channel. Acidification shifted the threshold potential of the channel to more positive values. This effect was accompanied by a decrease in activation times and an increase of deactivation times, of the channel. From the changes in E_m and membrane conductance, the expected effect of acidification on K⁺ fluxes was calculated. It was concluded that apoplastic acidification will increase the K⁺-efflux in the depolarized state and reduce the K⁺-influx in the hyperpolarized state. Received: 28 April 1997 / Accepted: 10 November 1997     

Evaluating how rimantadines control the proton gating of the influenza A M2-proton port via allosteric binding outside of the M2-channel: MD simulations

In order to understand how rimantadine (RMT) inhibits the proton conductance in the influenza A M2 channel via the recently proposed “allosteric mechanism”, molecular dynamics simulations were applied to the M2-tetrameric protein with four RMTs bound outside the channel at the three protonation states: the 0H-closed, 1H-intermediate and 3H-open situations. In the 0H-closed state, a narrow channel with the RMT-Asp44-Trp41 H-bond network was formed, therefore the water penetration through the channel was completely blocked. The Trp41-Asp44 interaction was absent in the 1H-intermediate state, whilst the binding of RMT to Asp44 remained, which resulted in a weakened helix-helix packing, therefore the channel was partially prevented. In the 3H-open state it was found that the electrostatic repulsion from the three charged His37 residues allowed the Trp41 gate to open, permitting water to penetrate through the channel. This agreed well with the potential of the means force which is in the following order: 0H > 1H > 3H.     

Molecular Modeling of the NPY Binding Site on the Y1 Receptor

A three-dimensional model of the neuropeptide Y (NPY) - rat Y₁ (rY₁) receptor complex and of the NPY _13-36 - rY₁ receptor complex was constructed by molecular modeling based on the electron density projection map of rhodopsin and on site-directed mutagenesis studies of neuropeptide receptors. In order to further guide the modeling, the nucleotide sequences encoding Trp287, Cys295 and His297 in the third extracellular loop of the rY₁ receptor, were altered by site-directed mutagenesis experiments. Single-point mutated receptors were expressed in COS-7 cells, and tested for their ability to bind radio labelled NPY (³H-NPY). Mutations of Trp287 and His297 completely abolished binding of ³H-NPY. The Cys295Ser mutation only slightly decreased the binding of ³H-NPY, suggesting that the involvement of Cys295 in a disulphide bond is not essential for maintaining the correct three-dimensional structure of the binding site for NPY. Molecular dynamics simulations of NPY-rY₁ receptor interactions suggested that Asp199, Asp103 and Asp286 in the receptor interact, respectively, with Lys4, Arg33 and Arg35 of NPY. The simulations also suggested that His297 acts as a hydrogen acceptor from Arg35 in NPY, and that Tyr1 of NPY interacts with a binding pocket on the receptor formed by Asn115, Asp286, Trp287 and His297. Tyr36 in NPY interacted both with Thr41 and Tyr99 via hydrogen bonds, and also with Asn296, His297 and Phe301. The present study suggests that amino acid residues at the extracellular end of the transmembrane helices and in the extracellular loops are strongly involved in binding to NPY and NPY_13-36.Electronic Supplementary Material available.     

Voltage-dependent and Src-mediated regulation of NMDA receptor single channel outward currents in cortical neurons

A voltage-dependent but Ca²⁺-independent regulation of N-methyl-D-aspartate (NMDA) receptor outward activity was studied at the single channel level using outside-out patches of cultured mouse cortical neurons. Unlike the inward activity associated with Ca²⁺ and Na⁺ influx, the NMDA receptor outward K⁺ conductance was unaffected by changes in Ca²⁺ concentration. Following a depolarizing pre-pulse, the single channel open probability (NP _o), amplitude, and open duration of the NMDA inward current decreased, whereas the same pre-depolarization increased those parameters of the NMDA outward current (pre-pulse facilitation). The outward NP _o was increased by the pre-pulse facilitation, disregarding Ca²⁺ changes. The voltage–current relationships of the inward and outward currents were shifted by the pre-depolarization toward opposite directions. The Src family kinase inhibitor, PP1, and the Src kinase antibody, but not the anti-Fyn antibody, blocked the pre-pulse facilitation of the NMDA outward activity. On the other hand, a hyperpolarizing pre-pulse showed no effect on NMDA inward currents but inhibited outward currents (pre-pulse depression). Application of Src kinase, but not Fyn kinase, prevented the pre-pulse depression. We additionally showed that a depolarization pre-pulse potentiated miniature excitatory synaptic currents (mEPSCs). The effect was blocked by application of the NMDA receptor antagonist AP-5 during depolarization. These data suggest a voltage-sensitive regulation of NMDA receptor channels mediated by Src kinase. The selective changes in the NMDA receptor-mediated K⁺ efflux may represent a physiological and pathophysiological plasticity at the receptor level in response to dynamic changes in the membrane potential of central neurons.     

Easily polarizable proton transfer hydrogen bonds between the side chains of histidine and the carboxylic acid groups of glutamic and aspartic acid residues in proteins

The nature of the hydrogen bonds formed between glutamic acid and histidine residues between aspartic acid and histidine residues is studied by i.r. spectroscopy. These studies were performed with $\text{(}\text{l}\text{-Glu}\text{)}{}_{\mathrm{n}}\text{+(}\text{l}\text{-His}\text{)}{}_{\mathrm{n}}$ and with associates of monomeric Glu + His and Asp + His systems in solutions whereby these amino acids had protected α-amino and α-carboxylic groups. It is shown that the OH …N?^?O?H⁺N bonds are easily polarizable proton transfer hydrogen bonds. The residence time of the proton at the His is a little larger in the case of the Asp + His than in the case of the Glu + His systems. Polar environments shift this proton transfer equilibrium in favour of the proton limiting structure ^?O?H⁺N, and less polar ones in favour of the structure OH?N. These results demonstrate that the large proton polarizability of the hydrogen bonded system in the active centre of chymotrypsin is responsible for the charge shift caused by the substrate, and thus for the increase in reactivity of the serine residue and the catalytic activity of the enzyme.     

Different Inward and Outward Conduction Mechanisms in NaVMs Suggested by Molecular Dynamics Simulations

Rapid and selective ion transport is essential for the generation and regulation of electrical signaling pathways in living organisms. Here, we use molecular dynamics (MD) simulations with an applied membrane potential to investigate the ion flux of bacterial sodium channel Na_VMs. 5.9 µs simulations with 500 mM NaCl suggest different mechanisms for inward and outward flux. The predicted inward conductance rate of ∼27±3 pS, agrees with experiment. The estimated outward conductance rate is 15±3 pS, which is considerably lower. Comparing inward and outward flux, the mean ion dwell time in the selectivity filter (SF) is prolonged from 13.5±0.6 ns to 20.1±1.1 ns. Analysis of the Na⁺ distribution revealed distinct patterns for influx and efflux events. In 32.0±5.9% of the simulation time, the E53 side chains adopted a flipped conformation during outward conduction, whereas this conformational change was rarely observed (2.7±0.5%) during influx. Further, simulations with dihedral restraints revealed that influx is less affected by the E53 conformational flexibility. In contrast, during outward conduction, our simulations indicate that the flipped E53 conformation provides direct coordination for Na⁺. The free energy profile (potential of mean force calculations) indicates that this conformational change lowers the putative barriers between sites S_CEN and S_HFS during outward conduction. We hypothesize that during an action potential, the increased Na⁺ outward transition propensities at depolarizing potentials might increase the probability of E53 conformational changes in the SF. Subsequently, this might be a first step towards initiating slow inactivation.     

Blockade of potassium channels in the plasmalemma ofChara corallina by tetraethylammonium,Ba2+, Na+ and Cs+

Summary The outer membranes of plant cells contain channels which are highly selective for K⁺. In the giant-celled green algaChara corallina, K⁺ currents in the plasmalemma were measured during the action potential and when the cell was depolarized to the K⁺ equilibrium potential in high external K⁺ concentrations. Currents in both conditions were reduced by externally added tetraethylammonium (TEA⁺), Ba²⁺, Na⁺ and Cs⁺. In contrast to inhibition by TEA⁺, the latter three ions inhibited inward K⁺ current in a voltage-dependent manner, and reduced inward current more than outward. Ba²⁺ and Na⁺ also appeared to inhibit outward current in a strongly voltage-dependent manner. The blockade by Cs⁺ is studied in more detail in the following paper. TEA⁺ inhibited both inward and outward currents in a largely voltage-independent manner, with an apparentK _D of about 0.7 to 1.1mm, increasing with increasing external K⁺. All inhibitors reduced current towards a similar linear leak, suggesting an insensitivity of the background leak inChara to these various K⁺ channel inhibitors. The selectivity of the channel to various monovalent cations varied depending on the method of measurement, suggesting that ion movement through the K⁺-selective channel may not be independent.     

The Hydronium Tetrafluoroborate Dimer in Nonpolar Media and its Proton NMR Spectrum

Hydronium tetrafluoroborate ion pairs, H₃O⁺·BF₄ ^- have been shown computationally to be unstable toward decomposition, in the absence of solvation or electrostatic interactions existing in crystals. As the proton NMR spectrum of a hydronium salt with the octanesulfonate-antimony pentachloride complex anion was reported in freon solution, we investigated the hypothesis that larger ionic clusters were present in the nonpolar solvent. It was found that the dimer (H₃O⁺·BF₄ ^-)₂ was stable at the MP2/6-31G* level. GIAO-B3LYP chemical shift calculations with the same basis set and also with the 6-31G**, 6-31++G**, 6-311++G**, dzvp, tzp, tz2p, and qz2p basis sets conducted on the hydronium fluoroborate dimer reproduce the main features of the experimental spectrum: the existence of two signals with a two-to-one intensity ratio and the more intense resonance at higher frequency (more deshielded). The alternative structures, of hydronium tetrafluoroborate ion pairs with one and with two hydrogen bonds between anion and cation, give calculated chemical shifts which are farther from the experimental values.     

Proton conductance of cell membranes

Several workers have suggested that cell membranes have a high proton conductance. Our interest in this concept arose from the possibility that the nutrient (submucosal-facing) membrane of the gastric mucosa may have a high proton or hydroxyl ion conductance which would play a role in the regulation of the acid-base balance of the cell. We found that wide changes in the H⁺ concentration of the fluid bathing the nutrient side of the in vitro frog gastric mucosa did not result in significant changes in p.d. However, a maintained change of the H⁺ concentration of the bathing fluid would be expected to produce only a temporary change in p.d. Since a diffusion barrier is present on the nutrient side the temporary change in p.d. might be masked. An analysis of this possibility was made on the basis of a conceptual model and as a result of the analysis it is concluded that the proton (and/or OH^?) conductance of the nutrient membrane of the frog gastric mucosa is not a significant fraction of its total conductance. The present status of the proton conductance hypothesis with respect to striated muscle and to the secretory membrane of the gastric mucosa is reviewed.