Ion conduction and selectivity in acid-sensing ion channel 1

The ability of acid-sensing ion channels (ASICs) to discriminate among cations was assessed based on changes in conductance and reversal potential with ion substitution. Human ASIC1a was expressed in Xenopus laevis oocytes, and acid-induced currents were measured using two-electrode voltage clamp. Replacement of extracellular Na⁺ with Li⁺, K⁺, Rb⁺, or Cs⁺ altered inward conductance and shifted the reversal potentials consistent with a selectivity sequence of Li ∼ Na > K > Rb > Cs. Permeability decreased more rapidly than conductance as a function of atomic size, with P_K/P_Na = 0.1 and G_K/G_Na = 0.7 and P_Rb/P_Na = 0.03 and G_Rb/G_Na = 0.3. Stimulation of Cl⁻ currents when Na⁺ was replaced with Ca²⁺, Sr²⁺, or Ba²⁺ indicated a finite permeability to divalent cations. Inward conductance increased with extracellular Na⁺ in a hyperbolic manner, consistent with an apparent affinity (K_m) for Na⁺ conduction of 25 mM. Nitrogen-containing cations, including NH₄⁺, NH₃OH⁺, and guanidinium, were also permeant. In addition to passing through the channels, guanidinium blocked Na⁺ currents, implying competition for a site within the pore. The role of negative charges in an external vestibule of the pore was evaluated using the point mutation D434N. The mutant channel had a decreased single-channel conductance, measured in excised outside-out patches, and a macroscopic slope conductance that increased with hyperpolarization. It had a weakened interaction with Na⁺ (K_m = 72 mM) and a selectivity that was shifted toward larger atomic sizes. We conclude that the selectivity of ASIC1 is based at least in part on interactions with binding sites both within and internal to the outer vestibule.     

Selectivity of the Ca2+-activated and Light-dependent K+ Channels for Monovalent Cations

The ionic selectivity of the Ca²⁺-activated K⁺ channel of Aplysia neurons and of the light-dependent K⁺ channel of Pecten photoreceptors to metal and organic cations was studied. The selectivity sequence determined from reversal potential measurements is T1⁺ K⁺ > Rb⁺ > NH⁺₄ > Cs⁺ > Na⁺, Li⁺ and is identical to the sequence determined previously for voltage-dependent K⁺ channels in a variety of tissues. Our results suggest that some physical aspect of the K⁺ channel is conserved in phyllogenetically different tissues and cells.     

Altered Ionic Selectivity of the Sodium Channel Revealed by Cysteine Mutations within the Pore

To explore the role of pore-lining amino acids in Na⁺ channel ion-selectivity, pore residues were  replaced serially with cysteine in cloned rat skeletal muscle Na⁺ channels. Ionic selectivity was determined by measuring permeability and ionic current ratios of whole-cell currents in Xenopus oocytes. The rSkM1 channels displayed an ionic selectivity sequence Na⁺>Li⁺>NH₄ ⁺>>K⁺>>Cs⁺ and were impermeable to divalent cations.  Replacement of residues in domain IV showed significantly enhanced current and permeability ratios of NH₄ ⁺ and K⁺, and negative shifts in the reversal potentials recorded in the presence of external Na⁺ solutions when compared to cysteine mutants in domains I, II, and III (except K1237C). Mutants in domain IV showed altered selectivity sequences: W1531C (NH₄ ⁺>K⁺>Na⁺≥Li⁺≈Cs⁺), D1532C, and G1533C (Na⁺>Li⁺≥NH₄ ⁺>K⁺>Cs⁺). Conservative replacement of the aromatic residue in domain IV (W1531) with phenylalanine or tyrosine retained Na⁺ selectivity of the channel while the alanine mutant (W1531A) reduced ion selectivity. A single mutation within the third pore forming region (K1237C) dramatically altered the selectivity sequence of the rSkM1 channel (NH₄ ⁺>K⁺>Na⁺≥Li⁺≈Cs⁺) and was permeable to divalent cations having the selectivity sequence Ca²⁺≥Sr²⁺>Mg²⁺>Ba²⁺. Sulfhydryl modification of K1237C, W1531C or D1532C with methanethiosulfonate derivatives that introduce a positively charged ammonium group, large trimethylammonium moiety, or a negatively charged sulfonate group within the pore was ineffective in restoring Na⁺ selectivity to these channels. Selectivity of D1532C mutants could be largely restored by increasing extracellular pH suggesting altering the ionized state at this position influences selectivity. These data suggest that K1237 in domain III and W1531, D1532, and G1533 in domain IV play a critical role in determining the ionic selectivity of the Na⁺ channel.     

Hydrogen currents through aconitine-modified sodium channels in the nerve fiber membrane

Ionic currents through aconitine-modified sodium channels of the Ranvier node membrane were measured by a voltage clamp method in an external medium free from sodium ions. A shift of pH of the solution below 4.6 led to the appearance of inward ionic currents, whose kinetics and activation region were characteristic of aconitine-modified sodium channels at low pH. These currents were blocked by the local anesthetic benzocaine in a concentration of 2 mM. Experiments with variation of the concentration of Ca⁺⁺, Tris⁺, TEA⁺, and choline⁺ in acid sodium-free solutions showed that these cations make no appreciable contribution to the inward current. It is concluded that the inward currents observed under these conditions are carried by H⁺ (or H₃O⁺) through aconitine-modified sodium channels. From the shifts of reversal potentials of the ionic currents the relative permeability (P_H/P_Na) for H⁺ was determined: 1059 ± 88. The results agree with the view that the aconitine-modified sodium channel is a relatively wide water pore, and that movement of H⁺ through it is limited by its binding with an acid group.Institute of Cytology, Academy of Sciences of the USSR, Leningrad. Translated from Neirofiziologiya, Vol. 14, No. 5, pp. 508–516, September–October, 1982.     

Monovalent Cation Permeation through the Connexin40 Gap Junction Channel : Cs,Rb, K,Na, Li,TEA, TMA,TBA, and Effects of Anions Br,Cl, F,Acetate, Aspartate,Glutamate, and NO3

The unitary conductances and permeability sequences of the rat connexin40 (rCx40) gap junction channels to seven monovalent cations and anions were studied in rCx40-transfected neuroblastoma 2A (N2A) cell pairs using the dual whole cell recording technique. Chloride salt cation substitutions (115 mM principal salt) resulted in the following junctional maximal single channel current-voltage relationship slope conductances (γ_j in pS): CsCl (153), RbCl (148), KCl (142), NaCl (115), LiCl (86), TMACl (71), TEACl (63). Reversible block of the rCx40 channel was observed with TBA. Potassium anion salt γ_j are: Kglutamate (160), Kacetate (160), Kaspartate (158), KNO₃ (157), KF (148), KCl (142), and KBr (132). Ion selectivity was verified by measuring reversal potentials for current in rCx40 gap junction channels with asymmetric salt solutions in the two electrodes and using the Goldman-Hodgkin-Katz equation to calculate relative permeabilities. The permeabilities relative to Li⁺ are: Cs⁺ (1.38), Rb⁺ (1.32), K⁺ (1.31), Na⁺ (1.16), TMA⁺ (0.53), TEA⁺ (0.45), TBA⁺ (0.03), Cl⁻ (0.19), glutamate⁻ (0.04), and NO₃− (0.14), assuming that the monovalent anions permeate the channel by forming ion pairs with permeant monovalent cations within the pore thereby causing proportionate decreases in the channel conductance. This hypothesis can account for why the predicted increasing conductances with increasing ion mobilities in an essentially aqueous channel were not observed for anions in the rCx40 channel. The rCx40 effective channel radius is estimated to be 6.6 Å from a theoretical fit of the relationship of relative permeability and cation radius.     

Potassium Channels in Myelinated Nerve : Selective permeability to small cations

The permeability of K channels to various cations is studied in myelinated nerve. Ionic currents under voltage clamp are measured in Ringer solution containing tetrodotoxin and a high concentration of the test ion. Reversal potentials for current in K channels are determined and used with the Goldman-Hodgkin-Katz equation to calculate relative permeabilities. The ratios P_Tl:P_K:P_Rb:P_NHNH4 are 2.3:1.00:0.92:0.13. No other ions are found to be measurably permeant including Li⁺, Na⁺, Cs⁺, methylamine, guanidine, hydrazine, or hydroxylamine. The ratio P_Na/P_K is less than 0.01. Potassium conductance is depressed at pH values below 5.0. Leakage conductance is higher in K, Rb, Cs, NH₄, and Tl Ringer than in Na Ringer, but the selectivity sequence probably is not the same as for K channels. The hypothesis is offered that the narrowest part of the K channel is a circle of oxygen atoms about 3 Å in diameter with low electrostatic field strength.     

Further Studies on the Roles of Sodium and Potassium in the Generation of the Electro-Olfactogram : Effects of mono-, di-, and trivalent cations

In the negative EOG-generating process a cation which can substitute for Na⁺ was sought among the monovalent ions, Li⁺, Rb⁺, Cs⁺, NH₄⁺, and TEA⁺, the divalent ions, Mg⁺⁺, Ca⁺⁺, Sr⁺⁺, Ba⁺⁺, Zn⁺⁺, Cd⁺⁺, Mn⁺⁺, Co⁺⁺, and Ni⁺⁺, and the trivalent ions, Al⁺⁺⁺ and Fe⁺⁺⁺. In Ringer solutions in which Na⁺ was replaced by one of these cations the negative EOG's decreased in amplitude and could not maintain the original amplitudes. In K⁺-Ringer solution in which Na⁺ was replaced by K⁺, the negative EOG's reversed their polarity. Recovery of these reversed potentials was examined in modified Ringer solutions in which Na⁺ was replaced by one of the above cations. Complete recovery was found only in the normal Ringer solution. Thus, it was clarified that Na⁺ plays an irreplaceable role in the generation of the negative EOG's. The sieve hypothesis which was valid for the positive EOG-generating membrane or IPSP was not found applicable in any form to the negative EOG-generating membrane. The reversal of the negative EOG's found in K⁺- , Rb⁺- , and Ba⁺⁺-Ringer solutions was attributed to the exit of the internal K⁺. It is, however, not known whether or not Cl^- permeability increases in these Na⁺-free solutions and contributes to the generation of the reversed EOG's.     

Daptomycin forms cation- and size-selective pores in model membranes

Daptomycin is a lipopeptide antibiotic that is used clinically to treat severe infections caused by Gram-positive bacteria. Its bactericidal action involves the calcium-dependent binding to membranes containing phosphatidylglycerol, followed by the formation of membrane-associated oligomers. Bacterial cells exposed to daptomycin undergo membrane depolarization, suggesting the formation of channels or pores in the target membranes. We here used a liposome model to detect and characterize the permeability properties of the daptomycin pores. The pores are selective for cations, with permeabilities being highest for Na⁺, K⁺, and other alkali metal ions. The permeability is approximately twice lower for Mg⁺⁺, and lower again for the organic cations choline and hexamethonium. Anions are excluded, as is the zwitterion cysteine. These observations account for the observed depolarization of bacterial cells by daptomycin and suggest that under typical in vivo conditions depolarization is mainly due to sodium influx.     

Molecular basis of ion permeability in a voltage‐gated sodium channel

Voltage‐gated sodium channels are essential for electrical signalling across cell membranes. They exhibit strong selectivities for sodium ions over other cations, enabling the finely tuned cascade of events associated with action potentials. This paper describes the ion permeability characteristics and the crystal structure of a prokaryotic sodium channel, showing for the first time the detailed locations of sodium ions in the selectivity filter of a sodium channel. Electrostatic calculations based on the structure are consistent with the relative cation permeability ratios (Na⁺ ≈ Li⁺ ≫ K⁺, Ca²⁺, Mg²⁺) measured for these channels. In an E178D selectivity filter mutant constructed to have altered ion selectivities, the sodium ion binding site nearest the extracellular side is missing. Unlike potassium ions in potassium channels, the sodium ions in these channels appear to be hydrated and are associated with side chains of the selectivity filter residues, rather than polypeptide backbones.     

The k/na selectivity of a cation channel in the plasma membrane of root cells does not differ in salt-tolerant and salt-sensitive wheat species

The characteristics of cation outward rectifier channels were studied in protoplasts from wheat root (Triticum aestivum L. and Triticum turgidum L.) cells using the patch clamp technique. The cation outward rectifier channels were voltage-dependent with a single channel conductance of 32 ± 1 picosiemens in 100 millimolar KCl. Whole-cell currents were dominated by the activity of the cation outward rectifiers. The time- and voltage-dependence of these currents was accounted for by the summed behavior of individual channels recorded from outside-out detached patches. The K⁺/Na⁺ permeability ratio of these channels was measured in a salt-sensitive and salt-tolerant genotype of wheat that differ in rates of Na⁺ accumulation, using a voltage ramp protocol on protoplasts in the whole-cell configuration. Permeability ratios were calculated from shifts in reversal potentials following ion substitutions. There were no significant differences in the K⁺/Na⁺ permeability ratios of these channels in root cells from either of the two genotypes tested. The permeability ratio for K⁺/Cl⁻ was greater than 50:1. The K⁺/Na⁺ permeability ratio averaged 30:1, which is two to four times more selective than the same type of channel in guard cells and suspension culture cells. Lowering the Ca²⁺ concentration in the bath solution to 0.1 millimolar in the presence of 100 millimolar Na⁺ had no significant effect on the K⁺/Na⁺ permeability ratios of the channel. It seems unlikely that the mechanism of salt tolerance in wheat is based on differences in the K⁺/Na⁺ selectivity of these channels.     

Cation Penetration through Isolated Leaf Cuticles

The rates of penetration of various cations through isolated apricot Prunus armeniaca L. leaf cuticles were determined. Steady state rates were measured by using a specially constructed flow-through diffusion cell. The penetration rates of the monovalent cations in group IA followed a normal lyotropic series, i.e., CS⁺ ≥ Rb⁺ > K⁺ > Na⁺ > Li⁺. The divalent cations all penetrated through the cuticle more slowly than the monovalent cations. Comparison of the relative values of k (permeability coefficient) and D (diffusion coefficient) indicates that the penetration of ions through isolated cuticles took place by diffusion and was impeded by charge interactions between the solute and charge sites in the penetration pathway. Cuticular penetration rates of K⁺ and H₂O at pH above 9 were of similar magnitude. At pH 5.5 H₂O penetration was not affected but that of K⁺ was greatly reduced. From this observation and from data on cuticle titration and ion adsorption studies, we hypothesize that cuticular pores are lined with a substance (perhaps a protein) which has exposed positively charged sites.     

Role of external calcium in sodium and chloride transport in the gills of seawater-adaptedMugil capito

Summary When the mulletMugil capito is transferred to medium lacking Ca⁺⁺ (either Ca⁺⁺-free seawater or distilled water) the passive permeability of the gill to Na⁺ and Cl^– is increased and the activating effect of external K⁺ on the Na⁺ and Cl^– effluxes in hyposaline media is inhibited. The permeability of the gill increases progressively in proportion to the time of Ca⁺⁺ deprivation; it declines when Ca⁺⁺ is added again to the external medium. The active mechanisms for ion excretion are not reversible. At external Ca⁺⁺ concentrations from 0.1 to 10 mM the Na⁺ permeability is constant but the activation of Na⁺ efflux by K⁺ shows a maximum at a Ca⁺⁺ concentration of about 1 mM. For activation of Cl^– efflux external bicarbonate must be present, in addition to Ca⁺⁺, suggesting the existence of a Cl^–/HCO ₃ ^– exchange. The mechanism by which Ca⁺⁺ controls the passive branchial permeability is thus probably different from that involved in K⁺ activation of ion excretion. The Ca⁺⁺ effect on the K⁺ sensitive ionic excretory mechanisms seems to be related to intracellular Ca⁺⁺ movements. Thus, on the one hand, substances such as Ruthenium Red and La⁺⁺⁺ which both inhibit Ca⁺⁺ exchange, in media containing Ca⁺⁺ and HCO ₃ ^– also inhibit K⁺ activation of Na⁺ and Cl^– effluxes; on the other hand, the ionophore A 23187, a stimulator of Ca⁺⁺ exchange, when added to these media, activates the Na⁺ and Cl^– effluxes; its maximal effect on the Na⁺ flux occurs at 2 mM Ca⁺⁺.Abbreviations ASW-Ca artificial seawater minus calcium - DW deionised water - DWCa deionised water with 1 mM Ca⁺⁺ added - DWCaHCO ₃ DW with calcium plus bicarbonate - DWHCO ₃ DW with 1 mM sodium bicarbonate added - FW freshwater (tap water) - FWK freshwater with K⁺ added - P. D. potential difference - SW seawater The experiments reported in this paper were done with Jean Maetz who tragically died in August 1977. It is the last report about several years of friendly collaboration     

Selectivity and sensitivity to hydrogen ion blocking of batrachotoxin-modified sodium channels in nerve fiber membrane

Currents through batrachotoxin-modified sodium channels were measured by the voltage clamp method on the Ranvier node membrane. In experiments with replacement of Na⁺ in the external solution by K⁺ or NH ₄ ⁺ the following series of permeabilities, determined as reversal potentials according to the equation of a static field, was obtained — P_Na: \(P_{NH_4 }\) :P_K=1:0.47:0.19. The relative permeability for H⁺ was determined by measuring currents after replacement of Na⁺ in the external solution by nonpenetrating choline ions and lowering pH to 3.7–3.8. The ratio p_H/p_Na for sodium channels modified by batrachotoxin averaged 528±46. Modified channels were less sensitive to the blocking action of H⁺ than normal sodium channels. The difference in the effective values of pK of the acid group of normal and modified channels was 0.40–0.45.     

Diversity of Channels Generated by Different Combinations of Epithelial Sodium Channel Subunits

The epithelial sodium channel is a multimeric protein formed by three homologous subunits: α, β, and γ; each subunit contains only two transmembrane domains. The level of expression of each of the subunits is markedly different in various Na⁺ absorbing epithelia raising the possibility that channels with different subunit composition can function in vivo. We have examined the functional properties of channels formed by the association of α with β and of α with γ in the Xenopus oocyte expression system using two-microelectrode voltage clamp and patch-clamp techniques. We found that αβ channels differ from αγ channels in the following functional properties: (a) αβ channels expressed larger Na⁺ than Li⁺ currents (I_Na+/I_Li+ 1.2) whereas αγ channels expressed smaller Na⁺ than Li⁺ currents (I_Na+/I_Li+ 0.55); (b) the Michaelis Menten constants (K _m) of activation of current by increasing concentrations of external Na⁺ and Li⁺ of αβ channels were larger (K _m > 180 mM) than those of αγ channels (K _m of 35 and 50 mM, respectively); (c) single channel conductances of αβ channels (5.1 pS for Na⁺ and 4.2 pS for Li⁺) were smaller than those of αγ channels (6.5 pS for Na⁺ and 10.8 pS for Li⁺); (d) the half-inhibition constant (K _i) of amiloride was 20-fold larger for αβ channels than for αγ channels whereas the K _i of guanidinium was equal for both αβ and αγ. To identify the domains in the channel subunits involved in amiloride binding, we constructed several chimeras that contained the amino terminus of the γ subunit and the carboxy terminus of the β subunit. A stretch of 15 amino acids, immediately before the second transmembrane domain of the β subunit, was identified as the domain conferring lower amiloride affinity to the αβ channels. We provide evidence for the existence of two distinct binding sites for the amiloride molecule: one for the guanidium moiety and another for the pyrazine ring. At least two subunits α with β or γ contribute to these binding sites. Finally, we show that the most likely stoichiometry of αβ and αγ channels is 1α:1β and 1α:1γ, respectively.     

Activation of Slo2.1 channels by niflumic acid

Slo2.1 channels conduct an outwardly rectifying K⁺ current when activated by high [Na⁺]_i. Here, we show that gating of these channels can also be activated by fenamates such as niflumic acid (NFA), even in the absence of intracellular Na⁺. In Xenopus oocytes injected with <10 ng cRNA, heterologously expressed human Slo2.1 current was negligible, but rapidly activated by extracellular application of NFA (EC₅₀ = 2.1 mM) or flufenamic acid (EC₅₀ = 1.4 mM). Slo2.1 channels activated by 1 mM NFA exhibited weak voltage dependence. In high [K⁺]_e, the conductance–voltage (G-V) relationship had a V_1/2 of +95 mV and an effective valence, z, of 0.48 e. Higher concentrations of NFA shifted V_1/2 to more negative potentials (EC₅₀ = 2.1 mM) and increased the minimum value of G/G_max (EC₅₀ = 2.4 mM); at 6 mM NFA, Slo2.1 channel activation was voltage independent. In contrast, V_1/2 of the G-V relationship was shifted to more positive potentials when [K⁺]_e was elevated from 1 to 300 mM (EC₅₀ = 21.2 mM). The slope conductance measured at the reversal potential exhibited the same [K⁺]_e dependency (EC₅₀ = 23.5 mM). Conductance was also [Na⁺]_e dependent. Outward currents were reduced when Na⁺ was replaced with choline or mannitol, but unaffected by substitution with Rb⁺ or Li⁺. Neutralization of charged residues in the S1–S4 domains did not appreciably alter the voltage dependence of Slo2.1 activation. Thus, the weak voltage dependence of Slo2.1 channel activation is independent of charged residues in the S1–S4 segments. In contrast, mutation of R190 located in the adjacent S4–S5 linker to a neutral (Ala or Gln) or acidic (Glu) residue induced constitutive channel activity that was reduced by high [K⁺]_e. Collectively, these findings indicate that Slo2.1 channel gating is modulated by [K⁺]_e and [Na⁺]_e, and that NFA uncouples channel activation from its modulation by transmembrane voltage and intracellular Na⁺.     

Properties and the Cytoskeletal Control of Ca++-independent Large Conductance K+ Channels in Neonatal Rat Hippocampal Neurons

A member of the family of Ca⁺⁺-independent large conductance K⁺ channels (termed BK channels) was identified in patch clamp experiments with cultured neonatal rat hippocampal neurons. Permeation was characterized (at 5 mmol/l external, 140 mmol/l internal K⁺; 135 mmol/l external Na⁺) by a conductance of 107 pS, a ratio P_Na/P_K∼ 0.01, and outward rectification near the reversal potential. Channel activity was not voltage-dependent, could not be reduced by internal TEA or by a shift of internal pH from 7.4 to 6.8, i.e., discriminating features within the Ca⁺⁺-independent BK channel family. Cytosolic proteolysis abolished the functional state of hippocampal Ca⁺⁺-independent BK channels, in contrast to the pronase resistance of hippocampal Ca⁺⁺-activated BK channels which suggests structural dissimilarities between these related channels. Cytoskeletal alterations had an activating influence on Ca⁺⁺-independent BK channels and caused a 3–4-fold rise in P _o , but patch excision and channel isolation from the natural environment provoked the strongest increase in P _o , from 0.07 ± 0.03 to 0.73 ± 0.04. This activation process operated slowly, on a minute time scale and can be most easily explained with the loss of a membrane-associated inhibitory particle. Once activated, Ca⁺⁺-independent BK channels reacted sensitively to a Mg-ATP supplemented brain tissue extract with a P _o decline, from 0.60 ± 0.06 to 0.10 ± 0.05. Heated extracts failed to induce significant channel inhibition, providing evidence for a heat-unstable molecule with reassociates with the internal channel surface to reestablish channel inhibition. A dualistic channel control, by this membrane-associated molecule and by the cytoskeleton seems possible. Received: 16 July 1997/Revised: 3 November 1997     

Stimulation of plasmalemma adenosine triphosphatase from oat roots by inorganic and organic cations: concentration-dependence, selectivity, and sites

K⁺-stimulated ATPase activity of a plasmalemma-enriched fraction from excised roots of oat was triphasic in the range 5 to 80 millimolar KCl. The phases obeyed Michaelis-Menten kinetics and were separated from each other by jumps or sharp breaks at about 10 and 20 millimolar. Stimulation by alkali cations was in the order K⁺ > Rb⁺ > Na⁺ > Cs⁺ > Li⁺ or in a closely related sequence. The specificity reflected differences in V_max, not in affinity (K_m⁻¹). Stimulation by the organic cations ethanolamine and choline in the interval 11 to 80 millimolar appeared monophasic rather than biphasic. Substitution on the quaternary nitrogen of the amino alcohols decreased their effectiveness, as did extension and branching of the chain. Stimulation was maximal at about pH 7 both for K⁺ and choline.     

Ca2+-Activated K+ channel from human erythrocyte membranes: Single channel recification and selectivity

Summary The Ca²⁺-activated K⁺ channel of the human red cell membranes was characterized with respect to rectification and selectivity using the patch-clamp technique. In inside-out patches exposed to symmetric solutions of K⁺, Rb⁺, and NH ₄ ⁺ , respectively, inward rectifyingi-V curves were obtained. The zero current conductances were: K⁺ (23.5 pS±3.2)>NH ₄ ⁺ (14.2 pS±1.2)>Rb⁺ (11.4 pS±1.8). With low extracellular K⁺ concentrations (substitution with Na⁺) the current fluctuations reversed close to the Nernst potential for the K ion and the rectification as well as thei-V slopes decreased. With mixed intracellular solutions of K⁺ and Na⁺ enhanced rectification were observed due to a Na⁺ block of outward currents. From bi-ionic reversal potentials the following permeability sequence (P _K/P _X) was calculated: K⁺ (1.0)>Rb⁺ (1.4±0.1)>NH ₄ ⁺ (8.5±1.3)>Li⁺(>50); Na⁺ (>110); Cs⁺ (5). Li⁺, Na⁺, and Cs⁺ were not found to carry any current, and only minimum values of the permeability ratios were estimated. Tl⁺ was permeant, but the permeability and conductance were difficult to quantify, since with this ion the single channel activity was extremely low and the channels seemed to inactivate. The inward rectification in symmetric solutions indicate an asymmetric open channel structure, and the different selectivity sequences based on conductances and permeabilities reflect interionic interactions in the permeation process.     

The Permeability of the Sodium Channel to Organic Cations in Myelinated Nerve

The relative permeability of sodium channels to 21 organic cations was studied in myelinated nerve fibers. Ionic currents under voltage-clamp conditions were measured in sodium-free solutions containing the test cation. The measured reversal potential and the Goldman equation were used to calculate relative permeabilities. The permeability sequence was: sodium ≈ hydroxylamine > hydrazine > ammonium ≈ formamidine ≈ guanidine ≈ hydroxyguanidine > aminoguanididine >> methylamine. The cations of the following compounds were not measurably permeant: N-methylhydroxylamine, methylhydrazine, methylamine, methylguanidine, acetamidine, dimethylamine, tetramethylammonium, tetraethylammonium, ethanolamine, choline, tris(hydroxymethyl)amino methane, imidazole, biguanide, and triaminoguanidine. Thus methyl and methylene groups render cations impermeant. The results can be explained on geometrical grounds by assuming that the sodium channel is an oxygen-lined pore about 3 A by 5 A in cross-section. One pair of oxygens is assumed to be an ionized carboxylic acid. Methyl and amino groups are wider than the 3 A width of the channel. Nevertheless, cations containing amino groups can slide through the channel by making hydrogen bonds to the oxygens. However, methyl groups, being unable to form hydrogen bonds, are too wide to pass through.     

Characterization of a k-stimulated adenosine triphosphatase associated with the plasma membrane of red beet

A membrane fraction enriched with a magnesium-dependent, monovalent cation-stimulated ATPase was isolated from red beet (Beta vulgaris L.) storage roots by a combination of differential centrifugation, extraction with KI, and sucrose density gradient centrifugation. This fraction was distinct from endoplasmic reticulum, Golgi, mitochondrial, and possibly tonoplast membranes as determined from an analysis of marker enzymes. The ATPase activity associated with this fraction was further characterized and found to have a pH optimum of 6.5 in the presence of both Mg²⁺ and K⁺. The activity was substrate specific for ATP and had a temperature optimum near 40°C. Kinetics with Mg:ATP followed a simple Michaelis-Menten relationship. However the kinetics of K⁺-stimulation were complex and suggestive of negative cooperativity. When monovalent cations were present at 2.5 millimolarity, ATPase was stimulated in the sequence K⁺ > Rb⁺ > Na⁺ > Li⁺ but when the concentration was raised to 50 millimolarity, the sequence changed to K⁺ ≥ Na⁺ ≥ Rb⁺ > Li. The activity was not synergistically stimulated by combinations of Na⁺ and K⁺. The enzyme was insensitive to NaN₃, oligomycin, ouabain, and sodium molybdate but sensitive to N,N′-dicyclohexylcarbodiimide, diethylstilbestrol, and sodium vanadate. Based on the similarity between the properties of this ATPase activity and those from other well characterized plant tissues, it has been concluded that this membrane fraction is enriched with plasma membrane vesicles.