RESUMEN

Human red cells were prepared with various cellular Na+ and K+ concentrations at a constant sum of 156 mM. At maximal activation of the K+ conductance. gK(Ca). the net efflux of K+ was determined as a function of the cellular Na+ and K+ concentrations and the membrane potential. Vm, at a fixed [K+]ex of approximately 3.5 mM. Vm was only varied from (Vm-EK) approximately equal to 25 mV and upwards, that is, outside the range of potentials with a steep inward rectifying voltage dependence (Stampe & Vestergaard-Bogind, 1988). gK(Ca) as a function of cellular Na+ and K+ concentrations at Vm = -40.0 and 40 mV indicated a competitive, voltage-dependent block of the outward current conductance by cellular Na+. Since the present Ca2+-activated K+ channels have been shown to be of the multi-ion type, the experimental data from each set of Na+ and K+ concentrations were fitted separately to a Boltzmann-type equation, assuming that the outward current conductance in the absence of cellular Na+ is independent of voltage. The equivalent valence determined in this way was a function of the cellular Na+ concentration increasing from 0.5 to 1.5 as this concentration increased from 11 to 101 mM. Data from a previous study of voltage dependence as a function of the degree of Ca2+ activation of the channel could be accounted for in this way as well. It is therefore suggested that the voltage dependence of gK(Ca) for outward currents at (Vm-Ek) greater than 25 mV reflects a voltage-dependent Na+ block of the Ca2+-activated K+ channels.

Asunto(s)

RESUMEN

The voltage dependence for outward-going current of the Ca-activated K+ conductance (gK(Ca] of the human red cell membrane has been examined over a wide range of membrane potentials (Vm at constant values of [K+]ex, [K+]c and pHc, the intact cells being preloaded to different concentrations of ionized calcium. Outward-current conductances were calculated from initial net effluxes of K+ and the corresponding (Vm - EK) values. The basic conductance, defined as the outward-current conductance at (Vm - EK) greater than or equal to 20 mV and [K+]ex greater than or equal to 3 mM (B. Vestergaard-Bogind, P. Stampe and P. Christophersen, J. Membrane Biol. 95:121-130, 1987) was found to be a function of cellular ionized Ca. At all degrees of Ca activation gK(Ca) was an apparently linear function of voltage (Vm range -40 to +70 mV), the absolute level as well as the slope decreasing with decreasing activation. In a simple two-state model the constant voltage dependence can, at the different degrees of Ca activation, be accounted for by a Boltzmann-type equilibrium function with an equivalent valence of approximately 0.4, assuming chemical equilibrium at Vm = 0 mV. Alternatively, the phenomenon might be explained by a voltage-dependent block of the outward current by an intracellular ion. Superimposed upon the basic conductance is the apparently independent inward-rectifying steep voltage function with an equivalent valence of approximately 5 and chemical equilibrium at the given EK value.

Asunto(s)

RESUMEN

UNLABELLED: 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). IN CONCLUSION: 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.

Asunto(s)

RESUMEN

The conductance of the Ca2+-sensitive K+-channels in human red cell membranes has been determined as a function of the intracellular pH. A sudden increase in the intracellular concentration of ionized calcium was established by addition of ionophore A23187 to a suspension of cells in buffer-free, Ca2+-containing salt solution. At the various cellular pH-values cellular concentrations of ionized Ca, saturating with respect to activation of the Ca2+-sensitive K+-conductance, were obtained by the use of varied concentrations of extracellular Ca2+ and added ionophore A23187. Changes in membrane potential was monitored as CCCP-mediated changes in extracellular pH. Initial net effluxes of K+, cellular K+ contents and the K+ Nernst equilibrium potentials were calculated from flame photometric measurements. Cellular Ca-contents were determined by aid of 45Ca. With cellular Ca2+ at the saturating level with respect to activation of the K+-channel the K+-conductance calculated from these data was independent of extracellular pH and a steep function of cellular pH with a half maximal conductance of 31 microSeconds/cm2 at a cellular pH of 6.1. The K+-conductance is not a simple function of cellular pH (pHc). From pHc = 6.5 and down to pHc = 6.0 a Hill-coefficient of 2.5 was found, indicating cooperativity between at least two sites regulating the conductance. Below pHc = 6.0 an extremely high Hill-coefficient of 11 was found, probably indicating that the additional titration of the channel protein leads to an increased cooperativity. The importance, as a physiological regulatory mechanism, of a K+-conductance increasing from zero to maximal conductance within less than one unit of pH, is discussed.

Asunto(s)

RESUMEN

The ratio between the unidirectional fluxes through the Ca2+-activated K+-specific ion channel of the human red cell membrane has been determined as a function of the driving force (Vm-EK). Net effluxes and 42K influxes were determined during an initial period of approximately 90 sec on cells which had been depleted of ATP and loaded with Ca. The cells were suspended in buffer-free salt solutions in the presence of 20 microM of the protonophore CCCP, monitoring in this way changes in membrane potential as changes in extracellular pH. (Vm-EK) was varied at constant EK by varying the Nernst potential and the conductance of the anion and the conductance of the potassium ion. In another series of experiments EK was varied by suspending cells in salt solutions with different K+ concentrations. At high extracellular K+ concentrations both of the unidirectional fluxes were determined as 42K in- and effluxes in pairs of parallel experiments. Within a range of (Vm-EK) of -6 to 90 mV the ratio between the unidirectional fluxes deviated strongly from the values predicted by Ussing's flux ratio equation. The Ca2+-activated K+ channel of the human red cell membrane showed single-file diffusion with a flux ratio exponent n of 2.7. The magnitude of n was independent of the driving force (Vm-EK), independent of Vm and independent of the conductance gK.

Asunto(s)

RESUMEN

Ionophore A23187-mediated net influx of Ca2+ in ATP-depleted human red cells was studied as a function of the pH and the proton concentration gradient across the membranes. Utilizing the Ca2+-induced increase in K+ conductance of the cell membranes, various CCCP-mediated proton gradients were raised across the membranes of cells suspended in unbuffered salt solutions with different K+ concentrations. In ionophore-mediated equilibrium the concentration ratios of ionized Ca between ATP-depleted, DIDS-treated cells and their suspension medium were equal to the concentration ratios of protons raised to the second power. With no proton concentration gradient across the membranes the net influxes of Ca2+ as a function of pH resembled a titration curve of a weak acid, with half maximal net influx at pH 7.3, at 100 microM extracellular Ca2+. With cellular pH fixed at various values, the net influx of Ca2+ was determined as a function of the proton concentration gradient. A linear relationship between the logarithm of net influx and the difference between extracellular and cellular pH was found at all cellular pH values tested, but the proton concentration gradient acceleration was a function of the cellular pH. Accelerations between 10- and 40- times per unit delta pH were found and net effluxes were correspondingly decreased. The results are discussed in relation to present models of the mechanism of ionophore A23187-mediated Ca2+ transport. The importance of the proton concentration gradient dependency is discussed in relation to the induced oscillations in K+-conductance of human red cell membranes previously reported (Vestergaard-Bogind and Bennekou (1982) Biochim. Biophys. Acta 688, 37-44).

Asunto(s)

RESUMEN

Ionophore A23187-mediated Ca2+-induced oscillations in the conductance of the Ca2+-sensitive K+ channels of human red cells were monitored with ion specific electrodes. The membrane potential was continuously reflected in CCCP-mediated pH changes in the buffer-free medium, changes in extracellular K+ activity were followed with a K+-selective electrode, and changes in the intracellular concentration of ionized calcium were calculated on the basis of cellular 45Ca content. An increased cellular 45Ca content at the successive minima of the oscillations where the K+ channels are closed indicates that the activation of the channels might be a (dCa2+/dt)-sensitive process and that accommodation to enhanced levels of intracellular free calcium may occur. An incipient inactivation of the K+ channels at intracellular ionized calcium levels of about 10 microM and a concurrent membrane potential of about -65 mV was observed. At a membrane potential of about -70 mV and an intracellular concentration of about 2 X 10(-4) M no inactivation of K+ channels took place. Inactivation of the K+ channels is suggested to be a compound function of the intracellular level of free calcium and the membrane potential. The observed sharp peak values in cellular 45Ca content support the notion that a necessary component of the oscillatory system is a Ca2+ pump operating with a significant delay in the activation/inactivation process in response to changes in cellular concentration of ionized calcium.

Asunto(s)

RESUMEN

The time-dependence of ionophore A23187-induced changes in the conductance of the Ca2+-sensitive K+ channels of the human red cell has been monitored with ion-specific electrodes. The membrane potential was reflected in CCCP-mediated pH changes in a buffer-free extracellular medium, and changes in extracellular K+ activity and electrode potential of an extracellular Ca2+-electrode were recorded. Within a narrow range of ionophore-mediated Ca2+ influx, the above-mentioned parameters were found to oscillate when ionophore was added to a suspension of glucose-fed cells. The period of oscillation was about 2 min/cycle depending on ionophore concentration, and the amplitude of hyperpolarization was about 60 mV, corresponding to a maximal gK+ of the same magnitude as gCl-. Without CCCP present no oscillation in K+ conductance was observed. The Ca2+ affinity for the opening process was in the micromolar range. The closing of the K+ channels was a spontaneous process in that the depolarization was well under way before the Ca2+-ATPase-mediated Ca2+ net efflux started. Below the Ca2+ influx range for oscillations, no response was observed for up to 20 min after the addition of ionophore. Above the upper limit, a permanent hyperpolarization resulted with an extracellular K+ activity increasing monotonically as a function of time. In experiments with ATP-depleted cells, responses of the latter type ensued at all ionophore concentrations above the lower limit. Addition of surplus EGTA to suspensions of hyperpolarized cells restores the normal membrane potential in the case of glucose-fed cells, whereas the K+-channels in ATP-depleted cells remained open.

Asunto(s)

Asunto(s)

RESUMEN

Like most other red cells, the giant erythrocytes of Amphiuma means possess a system for rapid exchange of chloride across the membrane. Also, there are indications that the net transport of chloride in these cells is slow. The size of Amphiuma erythrocytes allows direct measurements of membrane potential with microelectrodes. The present work exploits the possibility that such measurements can be used to give a quantitative estimate of the chloride conductance (GCl) of the Amphiuma red cell membrane. The membrane potential was measured as a function of extracellular chloride concentration (5-120mM), using an impermeant anion (Para-amino-hippurate) as a substitute. Furthermore, the effect of different pH values (6.0-7.2) was studied. For each extracellular chloride concentration the membrane potential was determined at a pH at which hydroxyl, hydrogen, and bicarbonate ions were in electrochemical equilibrium. From these membrane potentials and the corresponding chloride concentrations in the medium (at constant intracellular ion concentrations), the GCl of the membrane was calculated to be 3.9 x 10-7 omega-1 cm-2. This value is some six orders of magnitude smaller than that calculated from the rate of tracer exchange under equilibrium conditions. The experimental strategy used gives the values for a "partial transference number" which takes into account only ions which are not in electrochemical equilibrium. Whereas this approach gives a value for GCl, it does not permit calculation of the overall membrane conductance. From the calculated value of GCl it is possible to estimate that the maximal value of the combined conductances of hydroxyl (or proton) and bicarbonate ions is 0.6 x 10-7 omega-1 cm-2. The large discrepancy between the rate of exchange of chloride and its conductance is in agreement with measurements on human and sheep red cells employing the ionophore valinomycin to increase the potassium conductance of the membrane. The results in the present study were, however, obtained without valinomycin and an accompaning assumption of a constant field in the membrane. Therefore, the present measurements give independent support to the above mentioned conclusions.

Asunto(s)

RESUMEN

A number of instantaneous changes occurred when picrate was added to a suspension of human red cells in steady state with respect to glycolysis and ion distribution across the membrane at pH 7.40. The rate of glycolysis increased, without change in glycolytic quotient, to a new steady-state value, the effect reaching a maximum of 1.75 times the rate of the control at 0.5 mM picrate. Inorganic phosphate (P(i)) was released at a relatively constant rate, increasing with picrate concentration to 1.0 mmol P(i)/liter cells x h at 5-6 mM picrate. The steady- state concentrations of ATP and 1,3-diphosphoglycerate (1,3-DPG) decreased to new stable values within 15-45 min after the addition of picrate. The ATP level was affected only at picrate concentrations of 1 mM or more, and the level of ATP stabilized at 75 percent of the control values at 4 mM of picrate. In contrast, 1,3-DPG concentrations decreased to 40 percent of the control value of 0.5 mM picrate. Higher concentrations of picrate resulted in only a small additional decrease in the stationary concentration of 1,3-DGP. A net efflux of cellular potassium at constant rate took place. This net efflux was an almost linear function of picrate concentration in the range of 0.1-3 mM. At the latter concentration the net efflux amounted to about 2.7 meq/liter cells x h and a further increase in picrate concentration caused only a minor increase in the potassium efflux. Possible mechanisms for the effects of picrate on human red cell glycolysis are discussed.

Asunto(s)

RESUMEN

An increase in extracellular Ca concentration causes the membrane of giant red cells of the salamander, Amphiuma means, to undergo a marked, transient hyperpolarization. This hyperpolarization is caused by an increase in K permeability of the membrane as judged from the K sensitivity of the membrane potential and from the rate of K loss under influence of raised extracellular Ca concentration. At constant external pH, the induction of hyperpolarization by increased extracellular Ca has a relatively well-defined threshold concentration. Furthermore the phenomenon is of an "all or none" type with most of the cells having membrane potential values either in the normal range (about -15 mV) or in the range -40 to -70mV. Shortly after suspension in Ringer's with 15 mm Ca, most if not all of the individual cells are hyperpolarized. Upon continued exposure (5-20 min) to the higher Ca concentration the membrane potential returns to the normal value in a fashion compatible with an "all or none" response. The observed Ca effect is sensitive to the pH of the suspending medium. At pH 6.2 the response is absent whereas the hyperpolarization is markedly stronger at pH 8.2 than at PH 7.2. It is argued that a reliable transport number for K under influence of Ca cannot be estimated from the slope of membrane potential vs. log (extracellular K concentration). This is probably related to the fact that the membrane potentials of the cells in the population do not stay constant in time. The above phenomenon is compared with the Ca-induced K permeability in poisoned human red cells or red cell ghosts. It is important to note that the cells employed in the present study are neither poisoned nor mechanically disrupted. This study emphasizes that the role of Ca in regulating cell membrane permeability to K seems to be a general feature.

Asunto(s)

Asunto(s)

Asunto(s)