The mitochondria-rich (mr) cell of amphibian skin epithelium is differentiated as a highly specialised pathway for passive transepithelial transport of chloride. The apical membrane of mr cells ...expresses several types of Cl
− channels, of which the function of only two types has been studied in detail. (i) One type of channel is gated by voltage and external chloride concentration. This intriguing type of regulation leads to opening of channels only if Cl
−
o is in the millimolar range and if the electrical potential is of a polarity that secures an inwardly directed net flux of this ion. Reversible voltage activations of the conductance proceed with long time constants, which depend on
V in such a way that the rate of conductance activation increases when
V is clamped at more negative values (serosal bath grounded). The gating seems to involve processes that are dependent on F-actin localised in the submembrane domain in the neck region of the flask-shaped mr cell. (ii) The other identified Cl
− pathway of mr cells is mediated by small-conductance apical CFTR chloride channels as concluded from its activation via β-adrenergic receptors, ion selectivity, genistein stimulation and inhibition by glibenclamide.
bbCFTR has been cloned, and immunostaining has shown that the gene product is selectively expressed in mr cells. There is cross-talk between the two pathways in the sense that activation of the conductance of the mr cell by voltage clamping excludes activation via receptor occupation, and vice versa. The mechanism of this cross-talk is unknown.
The isolated epithelium of toad skin was disintegrated into single cells by treatment with collagenase and trypsine. Chloride channels of cell-attached and excised inside-out apical membrane-patches ...of mitochondria-rich cells were studied by the patch-clamp technique. The major population of Cl- channels constituted small 7-pS linear channels in symmetrical solutions (125 mM Cl-). In cell-attached and inside-out patches the single channel i/V-relationship could be described by electrodiffusion of Cl- with a Goldmann-Hodgkin-Katz permeability of, PCl = 1.2 x 10(-14) - 2.6 x 10(-14) cm3. s-1. The channel exhibited voltage-independent activity and could be activated by cAMP. This channel is a likely candidate for mediating the well known cAMP-induced transepithelial Cl- conductance of the amphibian skin epithelium. Another population of Cl- channels exhibited large, highly variable conductances (upper limit conductances, 150-550 pS) and could be activated by membrane depolarization. A group of intermediate-sized Cl(-)-channels included: (a) channels (mean conductance, 30 pS) with linear or slightly outwardly rectifying i/V-relationships and activity occurring in distinct "bursts," (b) channels (conductance-range, 10-27 pS) with marked depolarization-induced activity, and (c) channels with unresolvable kinetics. The variance of current fluctuations of such "noisy" patches exhibited a minimum close to the equilibrium-potential for Cl-. With channels occurring in only 38% of sealed patches and an even lower frequency of voltage-activated channels, the chloride conductance of the apical membrane of mitochondria-rich cells did not match quantitatively that previously estimated from macroscopic Ussing-chamber experiments. From a qualitative point of view, however, we have succeeded in demonstrating the existence of Cl-channels in the apical membrane with features comparable to macroscopic predictions, i.e., activation of channel gating by cAMP and, in a few patches, also by membrane depolarization.
We have hypothesized that a major role of the apical H(+)-pump in mitochondria-rich (MR) cells of amphibian skin is to energize active uptake of Cl- via an apical Cl-/HCO3(-)-exchanger. The activity ...of the H+ pump was studied by monitoring mucosal H+-profiles with a pH-sensitive microelectrode. With gluconate as mucosal anion, pH adjacent to the cornified cell layer was 0.98 +/- 0.07 (mean +/- SEM) pH-units below that of the lightly buffered bulk solution (pH = 7.40). The average distance at which the pH-gradient is dissipated was 382 +/- 18 microns, corresponding to an estimated "unstirred layer" thickness of 329 +/- 29 microns. Mucosal acidification was dependent on serosal pCO2, and abolished after depression of cellular energy metabolism, confirming that mucosal acidification results from active transport of H+. The H+ was practically similar adjacent to all cells and independent of whether the microelectrode tip was positioned near an MR-cell or a principal cell. To evaluate H+-profiles created by a multitude of MR-cells, a mathematical model is proposed which assumes that the H+ distribution is governed by steady diffusion from a number of point sources defining a set of particular solutions to Laplace's equation. Model calculations predicted that with a physiological density of MR cells, the H+ profile would be governed by so many sources that their individual contributions could not be experimentally resolved. The flux equation was integrated to provide a general mathematical expression for an external standing H+-gradient in the unstirred layer. This case was treated as free diffusion of protons and proton-loaded buffer molecules carrying away the protons extruded by the pump into the unstirred layer; the expression derived was used for estimating stationary proton-fluxes. The external H+-gradient depended on the mucosal anion such as to indicate that base (HCO3-) is excreted in exchange not only for Cl-, but also for Br- and I-, indicating that the active fluxes of these anions can be attributed to mitochondria-rich cells.
1. The inward facing membranes of in vitro frog skin epithelium were depolarized with solutions of high K concentration. The electrical properties of the epithelium are then expected to be governed ...by the outward facing, Na‐selective membrane.
2. In this state, the transepithelial voltage (V) was clamped to zero and step‐changes of Na activity in the outer solution ((Na)o) were performed with a fast‐flow chamber at constant ionic strength, while the short‐circuit current was recorded.
3. At pre‐selected times after a step‐change of (Na)o the current response (I) to a fast voltage staircase was recorded. This procedure was repeated after blocking the Na channels with amiloride to obtain the current—voltage curve of transmembrane and paracellular shunt pathways. The current—voltage curve of the Na channels was computed by subtracting the shunt current from the total current.
4. The instantaneous INa—V curve thus obtained at a given (Na)o could easily be fitted with the constant field equation in the range between ‐50 and zero mV. This fit yielded approximate estimates of PNa, the Na— permeability of the Na‐selective membrane (at this (Na)o) and the cellular Na activity, (Na)c. As residual properties of the serosal membrane were ignored the computed values are expected to underestimate the true ones.
5. At constant (Na)c, the steady‐state value of 1/PNa increases linearly with (Na)o. Error analysis and the effect of drugs show that the dependence is not due to the residual properties of the inward facing membranes but reflects the true behaviour of PNa.
6. The steady‐state PNa at a given (Na)o is smaller than the transient PNa observed right after a stepwise increase of (Na)o to this value. The time constant of PNa‐relaxation is in the order of seconds.
7. In conclusion, Na transport through open Na‐selective channels of the outward facing membrane of the stratum granulosum cells can be described as an electrodiffusion process which as such does not saturate with increasing (Na)o. However, when added to the outer border of the membrane Na causes a decrease of PNa within several seconds. It is considered that binding of Na results in closure of Na channels.
Proton Pump Activity of Mitochondria-rich Cells Jensen, Lars J.; Sørensen, Jens N.; Larsen, E. Hviid ...
The Journal of general physiology,
01/1997, Letnik:
109, Številka:
1
Journal Article
Recenzirano
Odprti dostop
We have hypothesized that a major role of the apical H+-pump in mitochondria-rich (MR) cells of amphibian skin is to energize active uptake of Cl− via an apical Cl−/HCO3−-exchanger. The activity of ...the H+ pump was studied by monitoring mucosal H+-profiles with a pH-sensitive microelectrode. With gluconate as mucosal anion, pH adjacent to the cornified cell layer was 0.98 ± 0.07 (mean ± SEM) pH-units below that of the lightly buffered bulk solution (pH = 7.40). The average distance at which the pH-gradient is dissipated was 382 ± 18 μm, corresponding to an estimated “unstirred layer” thickness of 329 ± 29 μm. Mucosal acidification was dependent on serosal pCO2, and abolished after depression of cellular energy metabolism, confirming that mucosal acidification results from active transport of H+. The H+ was practically similar adjacent to all cells and independent of whether the microelectrode tip was positioned near an MR-cell or a principal cell. To evaluate H+-profiles created by a multitude of MR-cells, a mathematical model is proposed which assumes that the H+ distribution is governed by steady diffusion from a number of point sources defining a set of particular solutions to Laplace's equation. Model calculations predicted that with a physiological density of MR cells, the H+ profile would be governed by so many sources that their individual contributions could not be experimentally resolved. The flux equation was integrated to provide a general mathematical expression for an external standing H+–gradient in the unstirred layer. This case was treated as free diffusion of protons and proton-loaded buffer molecules carrying away the protons extruded by the pump into the unstirred layer; the expression derived was used for estimating stationary proton-fluxes. The external H+-gradient depended on the mucosal anion such as to indicate that base (HCO3−) is excreted in exchange not only for Cl −, but also for Br− and I−, indicating that the active fluxes of these anions can be attributed to mitochondria-rich cells.
Chloride Channels in Toad Skin Larsen, E. Hviid; Rasmussen, B. E.
Philosophical transactions of the Royal Society of London. Series B, Biological sciences,
12/1982, Letnik:
299, Številka:
1097
Journal Article
Recenzirano
A study of the voltage and time dependence of a transepithelial Cl-current in toad skin (Bufo bufo) by the voltage-clamp method leads to the conclusion that potential has a dual role for ...Cl-transport. One is to control the permeability of an apical membrane Cl-pathway, the other is to drive Cl-ions through this pathway. Experimental analysis of the gating kinetics is rendered difficult owing to a contamination of the gated currents by cellular ion redistribution currents. To obtain insight into the effects of accumulation-depletion currents on voltage clamp currents of epithelial membranes, a mathematical model of the epithelium has been developed for computer analysis. By assuming that the apical membrane Cl-permeability is governed by a single gating variable (Hodgkin-Huxley kinetics), the model predicts fairly well steady-state current-voltage curves, the time course of current activations from a closed state, and the dependence of unidirectional fluxes on potential. Other predictions of the model do not agree with experimental findings, and it is suggested that the gating kinetics are governed by rate coefficients that also depend on the holding potential. Evidence is presented that Cl-transport through open channels does not obey the constant-field equation.
The optical sectioning video imaging technique was used for measurements of the volume of mitochondria-rich (m.r.) cells of the isolated epithelium of toad skin. Under short-circuit conditions, cell ...volume decreased by about 14% in response to bilateral exposure to Cl-free (gluconate substitution) solutions, apical exposure to a sodium-free solution, or to amiloride. Serosal exposure to ouabain resulted in a large increase in volume, which could be prevented either by the simultaneous application of amiloride in the apical solution or by the exposure of the epithelium to bilateral Cl-free solutions. Unilateral exposure to a Cl-free solution did not prevent ouabain-induced cell swelling. It is concluded that m.r. cells have an amiloride-blockable Na conductance in the apical membrane, a ouabain-sensitive Na pump in the basolateral membrane, and a passive Cl permeability in both membranes. From the initial rate of ouabain-induced cell volume increase the active Na current carried by a single m.r. cell was estimated to be 9.9 +/- 1.3 pA. Voltage clamping of the preparation in the physiological range of potentials (0 to -100 mV, serosa grounded) resulted in a cell volume increase with a time course similar to that of the stimulation of the voltage-dependent Cl conductance. Volume increase and conductance activation were prevented by exposure of the tissue to a Cl-free apical solution. The steady-state volume of the m.r. cells increased with the clamping voltage, and at -100 mV the volume was about 1.15 times that under short-circuit conditions. The rate of volume increase during current passage was significantly decreased by lowering the serosal K concentration (Ki) to 0.5 mM, but was independent of whether Ki was 2.4, 5, or 10 mM. This indicates that the K conductance of the serosal membrane becomes rate limiting for the uptake of KCl when Ki is significantly lower than its physiological value. It is concluded that the voltage-activated Cl currents flow through the m.r. cells and that swelling is caused by an uptake of Cl ions from the apical bath and K ions from the serosal bath. Bilateral exposure of the tissue to hypo- or hypertonic bathing solutions changed cell volume without detectable changes in the Cl conductance. The volume response to external osmotic perturbations followed that of an osmometer with an osmotically inactive volume of 21%.
A special configuration of the data acquisition software package Spike2 (CED) has been developed to allow interactive computer control of a current-clamp intracellular recording system. Using the ...1401plus dedicated computer (CED) as an interface between the electrophysiological apparatus and a personal computer it was possible to have keyboard-control of intracellular current injection, single and repetitive pulse nerve stimulation, pressure ejection as well as on-going data acquisition. An analysis program was designed using the Spike2 programming language for the investigation of resting membrane properties, spike characteristics and synaptic input profiles of enteric neurones. The hardware configurations and associated software of our set-up may be of interest to electrophysiologists wishing to implement or extend a computer-based experimental system.
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