Search

Cultured L cells were found previously to have an oscillating membrane potential. Current-voltage ( I-V) curves were measured during these oscillations. Two I-V curves were recorded, one at the maximum and one at the minimum of oscillations. Each curve is nonlinear, and when they are subtracted from one another, the result gives the I-V curve for the K current producing oscillations. This I- V curve is zero for −85 to −90mV and saturates for positive and high negative membrane potentials. When the external K is increased the I-V curve is shifted and its zero current potential is reduced. The K zero current potential follows a Nernst relation when plotted against the external K concentration. The I- V curves all have a similar shape at different K concentrations, showing a saturation on each side of the zero current potential. The results can be explained satisfactorily in term of a carrier model for K ions. [ABSTRACT FROM AUTHOR]

The membrane potentials and resistances of L cells were measured using a standard electrophysiological technique. The values obtained in physiological media were around −15 mV and 37 MΩ, respectively. Almost all the large nondividing L cells (giant L cells) showed spontaneous oscillations of the membrane potential between around −15 and −40 mV. Application of an appropriate electrical or mechanical stimulus was also capable of eliciting responses but such were usually induced only once. The total membrane conductance increased significantly and in parallel with such a hyperpolarizing response. Cooling of the cells and application of metabolic inhibitors to the cells completely blocked the spontaneous oscillation despite the fact that the electrically induced hyperpolarizing response remained. Intracellular K, Na and Cl concentrations were measured by means of a flame photometer and a chloridometer, and the equilibrium potential for each ion was estimated. [ABSTRACT FROM AUTHOR]

Current-voltage relations have been measured across lecithin bilayers doped with alamethicin molecules. The results show that there are two aspects of the induced conductances, a voltage-dependent and a voltage-independent conductance. Both have been characterized as a function of alamethicin and KCl concentration. The two aspects of the conductances do not show the same changes with those two variables. The voltage-independent conductance is affected very little by changes in KCl concentration, and its dependance on alamethicin concentration reveals that it is produced by two or three alamethicin molecules. The voltage-dependent conductance is shifted by the changes in KCl concentration only when the concentrations are ≧ 100 mm; below 100 mm KCl the slope of the log conductance-voltage curve is also reduced. The effect of changing alamethicin concentration reveals that nine or ten molecules are involved for KCl concentrations larger than 100 mm; if the KCl concentration is less than 100 mm, the effect of changing the alamethicin concentration is reduced. Time-dependent measurements have also been performed; only one time constant was found and it is strongly voltage-dependent. Also a very slow voltage-dependent absorption process is found. These results can be explained if it is assumed that pores are formed of a mixture of charged and uncharged alamethicin molecules when a voltage is applied and that uncharged alamethicin can also form pores without applying a voltage, once the absorption process has been started by previously applied voltages. The voltage dependence of the time constant seems to indicate that the voltage-dependent pore formation is produced by aggregates of charged alamethicin rather than independent molecules. [ABSTRACT FROM AUTHOR]

Single-channel patch-clamp experiments were performed on MDCK cells in order to characterize the ionic channels participating in regulatory volume decrease (RVD). Subconfluent layers of cultured cells were exposed to a hypotonic medium (150 mOsm), and the membrane currents at the single-channel level were measured in cell-attached experiments. The results indicate that MDCK cells respond to a hypotonic swelling by activating several different ionic conductances. In particular, a potassium and a chloride channel appeared in the recordings more frequently than other channels, and this allowed a more detailed study of their properties in the inside-out configuration of the patch-clamp technique. The potassium channel had a linear I/V curve with a unitary conductance of 24±4 pS in symmetrical K concentrations (145 m m). It was highly selective for K ions vs. Na ions: P/P<0.04. The time course of its open probability ( P)showed that the cells responded to the hypotonic shock with a rapid activation of this channel. This state of high activity was maintained during the first minute of hypotonicity. The chloride channel participating in RVD was an outward-rectifying channel: outward slope conductance of 63.3+ 4.7 pS and inward slope conductance of 26.1±4.9 pS. It was permeable to both Cl and NOand its maximal activation after the hypotonic shock was reached after several seconds (between 30 and 100 sec). The activity of this anionic channel did not depend on cytoplasmic calcium concentration. Quinine acted as a rapid blocker of both channels when applied to the cytoplasmic side of the membrane. In both cases, 1 m m quinine reversibly reduced single-channel current amplitudes by 20 to 30%. These results indicate that MDCK cells responded to a hypotonic swelling by an early activation of highly selective potassium conductances and a delayed activation of anionic conductances. These data are in good agreement with the changes of membrane potential measured during RVD. [ABSTRACT FROM AUTHOR]

Kinetic studies of cotransport mechanisms have so far been limited to the conventional steady-state approach which does not allow in general to resolve either isomerization or ratelimiting steps and to determine the values of the individual rate constants for the elementary reactions involved along a given transport pathway. Such questions can only be answered using presteady-state or relaxation experiments which, for technical reasons, have not yet been introduced into the field of cotransport kinetics. However, since two recent reports seem compatible with the observation of such transient kinetics, it would appear that theoretical studies are needed to evaluate the validity of such claims and to critically evaluate the expectations from a presteady-state approach. We thus report such a study which was performed on a simple four-state mechanism of carrier-mediated transport. The time-dependent equation for zero- trans substrate uptake was thus derived and then extended to models with p intermediary steps. It is concluded that ( p-1) exponential terms will describe the approach to the steady state but that such equations have low analytical value since the parameters of the flux equation cannot be expressed in terms of the individual rate constants of the elementary reactions for models with p>5. We thus propose realistic simplifications based on the time-scale separation hypothesis which allows replacement of the rate constants of the rapid steps by their equilibrium constants, thereby reducing the complexity of the kinetic system. Assuming that only one relaxation can be observed, this treatment generates approximate models for which analytical expressions can easily be derived and simulated through computer modeling. When performed on the four-state mechanism of carrier-mediated transport, the simulations demonstrate the validity of the approximate solutions derived according to this hypothesis. Moreover, our approach clearly shows that presteady-state kinetics, should they become applicable to (co)transport kinetics, could be invaluable in determining more precise transport mechanisms. [ABSTRACT FROM AUTHOR]

A fundamental approach to calculate the diffusion of ions through membranes is introduced. The membran eis considered as a heterogeneous structure with molecules that can have a selective affinity for a certain class of diffusing ions. To diffuse through a membrane an ion must become associated with, or dissolved into, at least one component of that membrane. Diffusion is produced by thermal jumps from one molecular site to another. It is assumed that the electric field can change the binding properties between ions and membrane molecules. The kinetics of the conductances are calculated from the chemical kinetic theory. The calculations are compared to the squid axon data and the unknown parameters are adjusted to fit the data curves. The results are very satisfactory. The calculated activation energies correspond to the measuredQ 10 in the squid axon. The calculated and measured action potentials are quite similar. [ABSTRACT FROM AUTHOR]