Zero-current Conductance Research Articles

Permeation properties of Na+ and Ca2+ ions through the mouse epsilon2/zeta1 NMDA receptor channel expressed in Xenopus oocytes.

Ion permeation properties of the mouse e2/zeta1 NMDA receptor channel expressed in Xenopus oocytes were studied using the outside-out patch-clamp technique. In symmetrical Na+ solutions, the single-channel I-V relations were almost linear at low electrolyte concentrations, but rectified inwardly for Na+ concentrations above 50 mm. In symmetrical Na+ solutions, the "zero-current conductance" increased with Na+ concentration and saturated according to a hyperbolic curve, the half-maximal saturating activity, KM(Na), being 14.2 mm and the maximal conductance, Gmax(Na), 53.9 pS. When Ca2+ was present with Na+ in the external solution, the single-channel current was lower than in pure Na+, although the reversal potential indicated a higher permeability for Ca2+ than for Na+. Using ion activities, PCa/PNa was found to be about 17. The I-V data were fitted with a model based on the Eyring's rate theory, assuming a one-ion pore with three energy barriers and two sites. The KM(Ca) and Gmax (Ca) were 76.5 microm and 21.2 pS, respectively. According to the estimated rate constants, KM for Ca2+ is mainly determined by the binding strength of a site located 80% away from the channel opening at the external membrane-solution interface, a position similar to that postulated previously for the Mg2+ blocking site.

Valinomycin acts as a channel in ultrathin lipid membranes

When the thickness of monolayer membranes formed by bolaform archaeal lipids is reduced to the approximate length of two valinomycin molecules, the zero-current conductance does not show any more a linear dependence on valinomycin concentration; instead, a quadratic behaviour is observed. This suggests that a dimer permeation pore is formed and therefore the conduction mechanism changes from carrier to channel.

The conductance of single cardiac sodium channels from guinea pig depends on the intracellular sodium concentration

Currents through DPI 201-106 modified single sodium channels have been measured in cell-free inside-out patches from guinea-pig ventricular myocytes. Single-channel conductance and reversal potential of the sodium channel have been calculated at different intracellular sodium concentrations ([Na +] 1) from microscopic I− V curves, which were obtained by application of linear voltage ramps. The relation between the reversal potential and [Na +] 1 could be fitted with a modified Goldman-Hodgkin-Katz equation with a relative permeability for K + over Na + ions of 0.054. The zero-current conductance of the Na channel as a function of [Na +] 1 shows a plateau value at low Na concentrations, and increases in a sigmoidal manner at higher concentrations. It is concluded that the Na channel can carry outward currents and that its conductance depends on [Na +] 1.

The properties and function of inward rectification in rod photoreceptors of the tiger salamander.

1. Rod photoreceptors were isolated from the retinae of tiger salamanders and voltage clamped using the whole-cell patch-clamp technique. 2. Hyperpolarizing the cell to potentials more negative than -50 mV evoked an inward current termed Ih. 3. Ih did not turn on immediately following a hyperpolarizing step but showed a marked delay. The activation time course of Ih could be described by the sum of two exponential components of opposite polarity. 4. The steady-state chord-conductance was half activated at -67 mV. 5. The reversal potential of Ih was close to -30 mV in normal standard salt solution. Increasing the external potassium concentration tenfold shifted the reversal potential by +17 mV. 6. The conductance-voltage relation and the kinetic parameters were not affected by changes in the external potassium concentration. 7. When fully activated, the zero-current conductance underlying Ih depended on the square root of the concentration of external potassium. 8. The permeability ratio PNa/PK depended on the external potassium concentration. It was 0.2 at an external potassium concentration of 2.0 mM and 0.3 at an external potassium concentration of 10.0 mM. The interaction of potassium with Ih suggests that Ih is a multi-ion pore. 9. It is concluded that Ih differs from the inward rectifier that is found in egg cells, frog muscle and heart muscle. 10. The kinetics and voltage sensitivity of Ih suggest that it does not play a role in the dark resting state or in the response to dim flashes of light. Its properties indicate that it may have a major role in the response to bright flashes.

Voltage dependence of the Ca2+-activated K+ conductance of human red cell membranes is strongly dependent on the extracellular K+ concentration.

The conductance of the Ca2+-activated K+ channel (gK(Ca)) of the human red cell membrane was studied as a function of membrane potential (Vm) and extracellular K+ concentration ([K+]ex). ATP-depleted cells, with fixed values of cellular K+ (145 mM) and pH (approximately 7.1), and preloaded with approximately 27 microM ionized Ca were transferred, with open K+ channels, to buffer-free salt solutions with given K+ concentrations. Outward-current conductances were calculated from initial net effluxes of K+, corresponding Vm, monitored by CCCP-mediated electrochemical equilibration of protons between a buffer-free extracellular and the heavily buffered cellular phases, and Nernst equilibrium potentials of K ions (EK) determined at the peak of hyperpolarization. Zero-current conductances were calculated from unidirectional effluxes of 42K at (Vm-EK) approximately equal to 0, using a single-file flux ratio exponent of 2.7. Within a [K+]ex range of 5.5 to 60 mM and at (Vm-EK) greater than or equal to 20 mV a basic conductance, which was independent of [K+]ex, was found. It had a small voltage dependence, varying linearly from 45 to 70 microS/cm2 between 0 and -100 mV. As (Vm-EK) decreased from 20 towards zero mV gK(Ca) increased hyperbolically from the basic value towards a zero-current value of 165 microS/cm2. The zero-current conductance was not significantly dependent on [K+]ex (30 to 156 mM) corresponding to Vm (-50 mV to 0). A further increase in gK(Ca) symmetrically around EK is suggested as (Vm-EK) becomes positive. Increasing the extracellular K+ concentration from zero and up to approximately 3 mM resulted in an increase in gK(Ca) from approximately 50 to approximately 70 microS/cm2. Since the driving force (Vm-EK) was larger than 20 mV within this range of [K+]ex this was probably a specific K+ activation of gK(Ca). The Ca2+-activated K+ channel of the human red cell membrane is an inward rectifier showing the characteristic voltage dependence of this type of channel.

Vibrating probe analysis of teleost opercular epithelium: Correlation between active transport and leak pathways of individual chloride cells

We have utilized the vibrating probe technique to examine transport by individual chloride cells in the short-circuited fish opercular epithelium. Variability in the steady state and in response to rapid perturbations, including fast-acting hormones and ion replacement, was analyzed. Negative short-circuit currents, corresponding to chloride secretion, were associated with the apical crypts of all but five of 386 chloride cells sampled. Average chloride cell short-circuit current and conductance were 2.7 +/- 0.1 nA and 87.7 +/- 3.8 nS, respectively, or 19 mA cm-2 and 620 mS cm-2 (resistance = 1.6 omega cm2) when normalized to apical crypt surface area. Exposure to 1 microM epinephrine rapidly inhibited the tissue short-circuit current by inhibiting the current pumped by all chloride cells, i.e. all chloride cells have adrenergic receptors. The time course of inhibition for each cell mirrored that of the whole tissue. Reversal of epinephrine inhibition of the tissue short-circuit current by glucagon and phosphodiesterase inhibition was by reversal of epinephrine's inhibition of individual chloride cells, and not by turning on cells which were previously inactive or uninhibited, or by stimulating nonchloride cells. A great amount of variability existed among chloride cells in the ability of these agents to reverse epinephrine-inhibited current. Likewise, considerable variability in the response of chloride cell conductance to these perturbations was observed, and in many instances a clear dissociation between current and conductance was noted. In the steady state, variability among cells in a single tissue always defined a linear relationship between chloride cell current and conductance with zero-current conductance intercept at zero. Equivalent circuit modeling indicates that the leak conductance of chloride cells within a single tissue always contributes the same proportion to the total individual chloride cell conductance, such that the ratio between the conductances of the active and leak pathways of chloride cells is constant. The leak pathway is almost certainly dominated by a sodium-selective paracellular pathway. The results suggest that these cells control the permeability of their paracellular pathway. A possible mechanism for this control is discussed.

The Current-voltage Behavior of Ion Channels: Important Features of the Energy Profile of the Gramicidin Channel Deduced from the Conductance-voltage Characteristic in the Limit of Low Ion Concentration

The conductance-voltage (G-V) characteristic of a single-filing, multi-barrier, multi-occupancy channel depends in the limit of low ion concentration upon only two parameters: the voltage dependence of the entry step and the ratio of the rate constant for leaving the channel to that for crossing its middle (14,17,20). We show that the G-V shape in this low concentration limit can be measured accurately using a triangular wave, many-channel technique and demonstrate that the observed shape is incompatible with that expected if the only important rate limiting barrier at low concentration were at the channel mouth. Instead the central barrier turns out, surprisingly, in view of the markedly sublinear I-V shape at low concentration, to be even slightly larger than the exit barrier. Additionally, we find that it is not possible to fit both the G-V shape and the concentration dependence of the zero-current conductance simultaneously with a 3-barrier 2-site model. However, by adding additional sites to yield a 3-barrier 4-site model either of the type 3B4S" where the extra site in each channel half is external to the mouth of the channel or of the type 3B4S' where the extra site is internal to the mouth of the channel, we obtain good agreement. Additionally, using the flux ratio data of Procopio and Andersen (19) to discriminate between 3B4S and 3B4S" models, we find the 3B4S" model to be the only satisfactory one.

Effects of unstirred layers on the steady-state zero-current conductance of bilayer membranes mediated by neutral carriers of ions.

Some effects of diffusion polarization and chemical reactions on the steady-state zero-current conductance of lipid bilayers mediated by neutral carriers of ions have been studied theoretically and experimentally. Assuming that ion permeation across the interfaces occurs via a heterogeneous reaction between ions in the solution and carriers in the membrane, the relationship between the conductance and the aqueous concentration of carriers is shown to be linear only in a limited range of sufficiently low concentrations. At higher carrier concentrations, which for the most strongly bound cations are within the range of the experimentally accessible values, the conductance is expected to become limited by diffusion of the carried ion in the unstirred layers and therefore reach an upper limiting value independent of the membrane properties. This expectation has been successfully verified for glyceryl-monooleate membranes in the presence of the ions K+, Rb+ and NH+4 and carriers such as valinomycin and trinactin. The experimental results support, at least for the present system, the generally accepted view that complexation between ions and the macrocyclic antibiotics occurs at the membrane surface; it is shown, in fact, that for a different mechanism, such as that by which the complexes would form in the aqueous solutions and cross the interfaces as lipid-soluble ions, the same type of saturation would be expected to be observable only for unrealistically high values of the rate constants of the ion-carrier association. A previously proposed criterion to distinguish between these two mechanisms, based on the dependence of the conductance on the ion concentration, is discussed from the viewpoint of this more comprehensive model.

Theory for carrier-mediated zero-current conductance of bilayers extended to allow for nonequilibrium of interfacial reactions, spatially dependent mobilities and barrier shape

A generalized form of the electrodiffusion equation, allowing for any shape of symmetrical energy barrier and any spatial dependence of the diffusion coefficient, is used to deduce theoretically the carrier-mediated conductance for thin (e.g., bilayer) membranes in the limit of low applied current. Both the Nernst-Planck and the Eyring single-barrier treatments are special cases of this more general approach, which allows for the effect of non-uniform properties of the lipid and non-uniform profiles of the forces acting within the membrane interior. Two independent mechanisms for ions to cross the membrane-solution interfaces are considered; namely, (1) the reaction at the interface between ions from solution and carriers from the membrane, and (2) the partition across the interfaces of complexes already formed in the solution. The rates of these reactions are taken into account using the rate equations of chemical kinetics; and the Poisson-Boltzmann equation is integrated in the aqueous solutions to evaluate the effect of charged polar head groups of the lipid. The analysis leads to an expression for the conductance, which, in the approximation of constant field, is an explicit function of such experimentally variable parameters as the concentrations and types of permeant ions and carriers in the aqueous phases, the total ionic strength and the nature of the polar head groups of the lipid. The functional relationship observable in an unknown membrane can, in principle, enable one to deduce such information as the mechanism of ion permeation across the interfaces, the magnitude of the surface charge, and the degree of ion-carrier complexation in the aqueous solutions.