Effects of Ba2+ and Cs+ on apical membrane K+ conductance in toad retinal pigment epithelium (original) (raw)
Related papers
The electrogenic sodium pump of the frog retinal pigment epithelium
The Journal of Membrane Biology, 1978
It was previously shown that ouabain decreases the potential difference across an in vitro preparation of bullfrog retinal pigment epithelium (RPE) when applied to the apical, but not the basal, membrane and that the net basal-to-apical Na + transport is also inhibited by apical ouabain. This suggested the presence of a Na+-K § pump on the apical membrane of the RPE. In the present experiments, intracellular recordings from RPE cells show that this pump is electrogenic and contributes approximately-10 mV to the apical membrane potential (VAp). Apical ouabain depolarized Vae in two phases. The initial, fast phase was due to the removal of the direct, electrogenic component. In the first one minute of the response to ouabain, VAe depolarized at an average rate of 4.4 +0.42 mV/min (n= 10, mean • and VAe depolarized an average of 9.6_+0.5 mV during the entire fast phase. A slow phase of membrane depolarization, due to ionic gradients running down across both membranes, continued for hours at a much slower rate, 0.4 mV/min. Using a simple diffusion model and K+-specific microelectrodes, it was possible to infer that the onset of the ouabain-induced depolarization coincided with the arrival of ouabain molecules at the apical membrane. This result must occur if ouabain affects an electrogenic pump. Other metabolic inhibitors, such as DNP and cold, also produced a fast depolarization of the apical membrane. For a decrease in temperature of-~10~ the average depolarization of the apical membrane was 7.1 _+3.4mV (n =5) and the average decrease in transepithelial potential was 3.9 +_0.3 mV (n= 10). These changes in potential were much larger than could be explained by the effect of temperature on an RT/F electrodiffusion factor. Cooling the tissue inhibited the same mechanism as ouabain, since prior exposure to ouabain greatly reduced the magnitude of the cold effect. Bathing the tissue in 0 mM [K +] solution for 2 hr inhibited the electrogenic pump, and subsequent re-introduction of 2mM [K +] solution produced a rapid membrane hyperpolarization. We conclude that the electrogenic nature of this pump is important to retinal function, since its contribution to the apical membrane potential is likely to affect the transport of ions, metabolites, and fluid across the RPE.
Effect of intracellular potassium upon the electrogenic pump of frog retinal pigment epithelium
The Journal of Membrane Biology, 1978
We have studied the hyperpolarizing, electrogenic pump located on the apical membrane of the retinal pigment epithelium (RPE) in an in vitro preparation of bullfrog RPE-choroid. Changes in RPE [K+]i alter the current produced by this pump. Increasing [K+]o in the solution perfusing the basal membrane increases RPE [K+]~ (measured with a K+-specific microelectrode), and also depolarizes the apical membrane. This depolarization is due to a decrease in electrogenic pump current flowing across the apical membrane resistance, since it is abolished when the pump is inhibited by apical ouabain, by cooling the tissue, or by 0 mM [K+]o outside the apical membrane. Removal of C1-from the solution perfusing the basal membrane abolishes the K § apical depolarization by preventing the entry of K § (as KC1) into the cell. We conclude that the increase in [K+]~ causes the decrease in pump current. This result is consistent with the finding that [K+]~ is a competitive inhibitor of the Na §-K + pump in red blood cells. It is possible that the light-evoked changes in [K+]o in the distal retina could alter RPE [K+]i, and thus could affect the pump from both sides of the apical membrane. Any change in pump current is likely to influence retinal function, since this pump helps to determine the composition of the photoreceptor extracellular space.
Barium reverses the transretinal potassium gradient of the amphibian retina
Neuroscience Letters, 1987
Barium chloride (Ba 2+) was added to the bathing medium of the perfused retina-eyecup preparation of the tiger salamander. The electroretinogram (ERG) and intraretinal extracellular potassium activity ([K +]o) were analyzed using a double-barrel electrode, one of which was ion-selective for K +. The action of Ba 2+ on the ERG was to attenuate the c-wave/slow Pill complex. In addition, Ba 2+ dramatically decreased the [K ÷]o in the outer retina, enough to reverse the transretinal potassium gradient. These findings, together with the known properties of Miiller cell K + channels, form the basis of an explanation of why Ba 2+ blocks slow PIII and not the b-wave of the ERG. Recently there has been considerable interest in the actions that barium (Ba 2+) has on the electroretinogram (ERG). Studies [1, 6] have demonstrated that Ba 2+ blocks the c-wave/slow Pill complex of the ERG while leaving the other components relatively intact. Both the c-wave and the slow PIII are dependent upon the light evoked decrease in extracellular potassium activity ([K÷]o) that occurs in the outer (distal) retina, which largely reflects the activity of the photoreceptors [9]. Ba 2+ blocks K ÷ channels of pigment epithelium (PE) cells [4] and dissociated Miiller cells [5, 8]. Therefore, the elimination of the c-wave/slow PIII complex by Ba 2+ has been viewed as additional evidence for the association between the PE cells with c-wave generation and the Miiller cells with slow PIII generation. Substantial experimental evidence indicates that the b-wave of the ERG is also generated by M/iller cells [7], presumably through a light-evoked increase in [K+]o in the distal retina as a consequence of ON bipolar activation [3]. Because the b-wave persists in the presence of Ba 2+, the Miiller cell hypothesis of b-wave generation might seem doubtful. However, some clarification of this issue was derived from recent findings which demonstrate, that while Ba 2+ largely blocks K ÷ channels of dissociated M/iller cells, it has only small effects on K ÷ channels of intact Miiller cells [5] or those of Mfiller cells studied in the retinal tissue slice preparation [8].
Implications of an anomalous intracellular electrical response in bullfrog corneal epithelium
The Journal of Membrane Biology, 1985
The ionic dependencies of the transepithelial and intracellular electrical parameters were measured in the isolated frog cornea. In NaCI Ringer's the intracellular potential difference V~c measured under short-circuit conditions depolarized by nearly the same amount after either increasing the stromal-side KCI concentration from 2.5 to 25 mM or exposure to 2 mM BaC12 (K + channel blocker). With Ba 2+ the depolarization of the V~ by 25 mM K + was reduced to one-quarter of the control change. If the Ci-permselective apical membrane resistance Ro remained unchanged, the relative basolateral membrane resistance Ri, which includes the lateral intercellular space, increased at the most by less than twofold after Ba >. These effects in conjunction with the depolarization of the Vsc by 62 mV after increasing the stromal-side K + from 2.5 to 100 mM in Cl-free Ringer's as well as the increase of the apparent ratio of membrane resistances (a :: Ro/Ri) from 13 to 32 are all indicative of an appreciable basolateral membrane K + conductance. This ratio decreased significantly after exposure to either 25 mM K + or Ba 2+. The decline of R,,/Ri with 25 mM K + appears to be anomalous since this decrease is not consistent with just an increase of basolateral membrane conductance by 25 mM K +, but rather perhaps a larger decrease of Ro than Ri. Also an increase of lateral space resistance may offset the effect of decreasing R~ with 25 mM K +. In contrast, R,,/R~ did transiently increase during voltage clamping of the apical membrane potential difference Vo and exposure to 25 mM K'~ on the stromat side. This increase and subsequent decrease of R JR, supports the idea that increases in stromal K § concentration may produce secondary membrane resistance changes. These effects on Ro/Ri show that the presence of asymmetric ionic conductance properties in the apical and basolateral membranes can limit the interpretative value of this parameter. The complete substitution of Na § with n-methyl-glucamine in CIfree Ringer's on the stromal side hyperpolarized the V~o by 6 mV whereas 10-4 M ouabain depolarized the V~c by 7 inV. Thus the basolateral membrane contains K +, Na + and perhaps CI-pathways in parallel with the NalK pump component.
Passive ionic properties of frog retinal pigment epithelium
The Journal of Membrane Biology, 1977
The isolated pigment epithelium and choroid of frog was mounted in a chamber so that the apical surfaces of the epithelial cells and the choroid were exposed to separate solutions. The apical membrane of these cells was penetrated with microelectrodes and the mean apical membrane potential was-88inV. The basal membrane potential was depolarized by the amount of the transepithelial potential (8-20mV). Changes in apical and basal cell membrane voltage were produced by changing ion concentrations on one or both sides of the tissue. Although these voltage changes were altered by shunting and changes in membrane resistance, it was possible to estimate apical and basal cell membrane and shunt resistance, and the relative ionic conductance T~ of each membrane. For the apical membrane: TK~0.52, Tnco3_~0.39 and TNa-~0.05 , and its specific resistance was estimated to be 6000-7000f~ cm 2. For the basal membrane: TK~0.90 and its specific resistance was estimated to be 400-1200~ cm 2. From the basal potassium voltage responses the intracellular potassium concentration was estimated at ll0mM. The shunt resistance consisted of two pathways: a paracellular one, due to the junctional complexes and another, around the edge of the tissue, due to the imperfect nature of the mechanical seal. In well-sealed tissues, the specific resistance of the shunt was about ten times the apical plus basal membrane specific resistances. This epithelium, therefore, should be considered "tight". The shunt pathway did not distinguish between anions (HCOi, C1-, methylsulfate, isethionate) but did distinguish between Na + and K-. The pigment epithelial cells and the photoreceptors are closely associated in the vertebrate retina. Apical processes from the pigment epithelial cells lie alongside and, in some cases, surround the photoreceptor outer segments. The epithelium also separates the sensory retina from its choroidal blood supply and transports salts and metabolites to and from the retina (
The Journal of General Physiology, 1984
In the frog retinal pigment epithelium (RPE), the cellular levels of cyclic AMP (cAMP) were measured in control conditions and after treatment with substances that are known to inhibit phosphodiesterase (PDE) activity (isobutyl-1-methylxanthine, SQ65442) or stimulate adenylate cyclase activity (forskolin). The cAMP levels were elevated by a factor of 5-7 compared with the controls in PDE-treated tissues and by a factor of 18 in forskolin-treated tissues. The exogenous application of cAMP (1 mM), PDE inhibitors (0.5 mM), or forskolin (0.1 mM) all produced similar changes in epithelial electrical parameters, such as transepithelial potential (TEP) and resistance (Rt), as well as changes in active ion transport. Adding 1 mM cAMP to the solution bathing the apical membrane transiently increased the short-circuit current (SCC) and the TEP (apical side positive) and decreased Rt. Microelectrode experiments showed that the elevation in TEP is due mainly to a depolarization of the basal mem...
Current-voltage relations of Cs+-inhibited K+ currents through the apical membrane of frog skin
Pfl�gers Archiv European Journal of Physiology, 1988
The voltage-dependence of the inhibitory effect of mucosal Cs + on the inward K + current through the apical membrane of frog skin (Rana temporaria) was studied by recording transepithelial current-voltage relations. Experiments were performed with skins exposed to NaC1 and KC1 Ringer solutions on the serosal and mucosal side respectively (control skins), as well as with tissues incubated with K2SO4 Ringer solutions on both sides (depolarized skins). Studies of the dose-dependence of the Cs + block showed that under both experimental conditions the apparent affinity of Cs + increased as the transepithelial potential was clamped at higher mucosal positive voltages. Under control conditions, the concentration of Cs + required to block 50% of the K + current (Kc~) recorded while the transepithelial voltage was clamped at zero mV was 16 retool/1. Kcs decreased exponentially with mucosal positive voltages. The dependence of Kc~ on the membrane potential was analyzed with Eyring rate theory in which Cs + was assumed to block the K + transport by binding to a site within the channel. The analysis showed that this site is located at a relative electrical distance 6 = 0.32 of the voltage drop across the apical membrane, measured from the cytosolic side. The Hill coefficient obtained from this analysis was n = 3.1. Experiments with K +depolarized tissues showed that only inward K + currents recorded with positive transepithelial voltages were depressed by external Cs +. Also under these conditions Kcs showed an exponential dependence on the transepithelial potential. The analysis of these data with the rate theory revealed 6 = 0.09 and n = 1.7. The difference in 6 found in control and depolarized tissues can be explained by the influence of the basolateral membrane resistance on the I-V relations.
Biophysical Journal, 2000
The recently cloned retinal cone Na ϩ -Ca 2ϩ -K ϩ exchanger (NCKX) was expressed in cultured insect cells, and whole-cell patch clamp was used to measure transmembrane currents generated by this transcript and compare them with currents generated by retinal rod NCKX or by a deletion mutant rod NCKX from which the two large hydrophilic loops were removed. We have characterized the ionic currents generated by both the forward (Ca 2ϩ extrusion) and reverse (Ca 2ϩ influx) modes of all three NCKX proteins. Reverse NCKX exchange generated outward current that required the simultaneous presence of both external Ca 2ϩ and external K ϩ . Forward NCKX exchange carried inward current with Na ϩ , but not with Li ϩ in the bath solution. The cation dependencies of the three NCKX tested (external K ϩ , external Na ϩ , internal Ca 2ϩ ) were very similar to each other and to those reported previously for the in situ rod NCKX. These findings provide the first electrophysiological characterization of cone NCKX and the first electrophysiological characterization of potassium-dependent Na ϩ -Ca ϩ exchangers in heterologous systems. Our results demonstrate the feasibility of combining heterologous expression and biophysical measurements for detailed NCKX structure/function studies.
The Journal of Physiology, 1995
1. Conventional and ion‐selective double‐barrelled microelectrodes were used in an in vitro preparation of bovine retinal pigment epithelium (RPE)‐choroid to measure the changes in membrane voltage, resistance and intracellular Cl‐ activity (aCli) produced by small, physiological changes in extracellular potassium concentration ([K+]o). These apical [K+]o changes approximate those produced in the extracellular (subretinal) space between the photoreceptors and the RPE following transitions between light and dark. 2. Changing apical [K+]o from 5 to 2 mM in vitro elicited membrane voltage responses with three distinct phases. The first phase was generated by an apical membrane hyperpolarization, followed by a (delayed) basolateral membrane hyperpolarization (DBMH); the third phase was an apical membrane depolarization. The present experiments focus on the membrane and cellular mechanisms that generate phase 2 of the response, the DBMH. 3. The DBMH was abolished in the presence of apica...
J Gen Physiol, 1990
Changes in retinal pigment epithelial (RPE) cell volume were measured by monitoring changes in intracellular tetramethylammonium (TMA) using double-barreled K-resin microelectrodes. Hyperosmotic addition of 25 or 50 mM mannitol to the Ringer of the apical bath resulted in a rapid (-30 s) osmometric cell shrinkage. The initial cell shrinkage was followed by a much slower (minutes) secondary shrinkage that is probably due to loss of cell solute. When apical [K +] was elevated from 2 to 5 mM during or before a hyperosmotic pulse, the RPE cell regulated its volume by reswelling towards control within 3-10 min. This change in apical [K +] is very similar to the increase in subretinal [K+]o that occurs after a transition from light to dark in the intact vertebrate eye. The K-dependent regulatory volume increase (RVI) was inhibited by apical Na removal, CI reduction, or the presence of bumetanide. These results strongly suggest that a Na(K),CI cotransport mechanism at the apical membrane mediates RVI in the bullfrog RPE. A unique aspect of this cotransporter is that it also functions at a lower rate under steady-state conditions. The transport requirements for Na, K, and C1, the inhibition of RVI by bumetanide, and thermodynamic calculations indicate that this mechanism transports Na, K, and CI in the ratio of 1:1:2.