Basolateral Membrane Cl Research Articles

Taking intestinal anion secretion to TASK: a role for K2P channels in cyclic AMP-regulated anion secretion.

Intestinal anion secretion has been the focus of numerous investigations ever since the discovery of its essential role in diarrheal disease. A study in this issue of The Journal of Physiology by Julio-Kalajzić et al. (2018) provides new insights into this process via the molecular identification and functional characterization of TASK-2 potassium channels as components of what appears to be a network of K+ channels that support cyclic AMP-activated anion secretion. The standard model of anion secretion involves activation of apical CFTR following protein kinase A phosphorylation and ATP binding to the channel. Earlier studies using rodent intestine and colon have shown that CFTR is capable of conducting either Cl− or HCO3− ions and therefore supports transepithelial Cl− or HCO3− secretion in response to elevations in intracellular cAMP (Tang et al. 2015). CFTR-mediated anion efflux would be expected to produce membrane depolarization that would reduce the driving force for further anion movement out of the cell if not for the activation of basolateral, cAMP-activated KCNQ1/KCNE3 K+ channels, which serve as a mechanism for charge compensation. These channels also support the functions of the Na+–K+-ATPase and NKCC1 cotransporters by providing a pathway for K+ recycling across the basolateral membrane that maintains concentration gradients for Na+ and K+. In the study by Julio-Kalajzić et al. (2018), tissue-specific expression of a dominant-negative mutation in KCNE3 (KCNE3-D90N) effectively reduced forskolin/3-isobutyl-1-methylxanthine (IBMX)-stimulated anion secretion in the colon relative to WT mice. However, KCNE3-D90N mice still exhibited residual cAMP-dependent anion secretion amounting to ∼40% of that observed in WT colon. Interestingly, attempts to inhibit the remaining anion secretion by employing KCa3.1-deficient mice expressing the KCNE3-D90N mutation were unsuccessful, although Ca2+-activated anion secretion evoked by carbachol was abolished. This result indicated that crosstalk between cAMP and Ca2+ signalling pathways, which could potentially activate KCa3.1 channels, does not account for residual anion secretion observed following suppression of KCNQ1/KCNE3 channel activity. However, when basolateral tetrapentylammonium [TPeA (a membrane-permeant, quaternary ammonium compound known to inhibit K2P channels, among other subtypes)] was tested on the colonic epithelium of KCNE3-D90N mice, most of the residual anion secretion was inhibited, but no effect was observed on carbachol-stimulated anion secretion. These results suggested that a K+ channel distinct from KCNQ1/KCNE3 and KCa3.1 was involved in sustaining the remaining anion secretion. Previous RT-PCR and presently reported immunocytochemistry experiments revealed the presence of TASK-2 K+ channels expressed in portions of the crypt and villous cells of the ileum and in the crypts and surface cells of the colon. Furthermore, when TASK-2-deficient mice were used to investigate the effects of forskolin/IBMX on anion secretion, no effect of TPeA was observed, indicating that TPeA-sensitive anion secretion was dependent on TASK-2 expression. To test whether a combination of TASK-2 and KCNQ1/KCNE3 channel activities completely accounted for forskolin/IBMX-stimulated anion secretion, the authors generated Kcne3−/−/Kcnk5−/− double knockout mice. Ussing chamber experiments with colons from these mice revealed a small amount of forskolin/IBMX-stimulated anion secretion that was not affected by basolateral treatment with C293B, although basolateral TPeA produced a further decrease in secretion. In contrast, carbachol elicited robust anion secretion in colons from Kcne3−/−/Kcnk5−/− mice. These results confirmed a role for both KCNQ1/KCNE3 and TASK-2 K+ channels in facilitating cAMP-dependent anion secretion and suggested that when both of these channels are absent, another TPeA-sensitive K+ channel is present that supports the remaining level of cAMP-dependent anion secretion. An issue that was not specifically addressed in the study by Julio-Kalajzić et al. (2018) concerns the relative amounts of Cl− and HCO3− secretion that constitute the Isc response following forskolin/IBMX stimulation. It is possible that TASK-2 channels do not contribute equally to these two components of the forskolin/IBMX response. Earlier studies have shown that forskolin-stimulated Cl− secretion in rodent colon depends on basolateral, bumetanide-sensitive NKCC cotransport activity (∼40–70% depending on the region of the colon) and 4-acetamido-4'-isothiocyano-2,2'-stilbene disulfonate (SITS)-sensitive, HCO3−-dependent Cl− uptake that appears to involve AE2 Cl−/HCO3− exchangers (Schultheiss et al. 1998; Gawenis et al. 2010). Cl− uptake by AE2 results in HCO3− efflux, likely causing localized alkalinization within the unstirred fluid layer adjacent to the basolateral membrane. In a previous study of HCO3− absorption in murine proximal tubules, basolateral alkalinization was proposed to activate TASK-2 channels resulting in membrane hyperpolarization necessary for sustaining electrogenic Na+–(HCO3−)3 cotransport activity (Warth et al. 2004). In the colon, increases in AE2 activity may provide a mechanism for coupling activation of TASK-2 K+ channels with Cl− uptake across the basolateral membrane and apical Cl− efflux through CFTR (Fig. 1). The author has no conflicts of interest to report. National Institute of Allergy and Infectious Disease. Grant Number: R01 AI128729-01

Loss of kAE1 expression in collecting ducts of end-stage kidneys from a family with SLC4A1 G609R-associated distal renal tubular acidosis

Distal renal tubular acidosis caused by missense mutations in kidney isoform of anion exchanger 1 (kAE1/SLC4A1), the basolateral membrane Cl−/HCO3− exchanger of renal alpha-intercalated cells, has been extensively investigated in heterologous expression systems but rarely in human kidneys. The preferential apical localization of distal renal tubular acidosis (dRTA)-associated kAE1 mutants R901X, G609R and M909T in cultured epithelial monolayers has not been examined in human kidney. Here, we present kidney tissues from dRTA-affected siblings heterozygous for kAE1 G609R, characterized by predominant absence rather than mistargeting of kAE1 in intercalated cells. Thus, studies of heterologous recombinant expression of mutant proteins should be, whenever possible, interpreted in comparison to affected patient tissues.

Mechanisms of bicarbonate secretion: lessons from the airways.

Early studies showed that airway cells secrete HCO(3)(-) in response to cAMP-mediated agonists and HCO(3)(-) secretion was impaired in cystic fibrosis (CF). Studies with Calu-3 cells, an airway serous model with high expression of CFTR, also show the secretion of HCO(3)(-) when cells are stimulated with cAMP-mediated agonists. Activation of basolateral membrane hIK-1 K(+) channels inhibits HCO(3)(-) secretion and stimulates Cl(-) secretion. CFTR mediates the exit of both HCO(3)(-) and Cl(-) across the apical membrane. Entry of HCO(3)(-) on a basolateral membrane NBC or Cl(-) on the NKCC determines which anion is secreted. Switching between these two secreted anions is determined by the activity of hIK-1 K(+) channels.

Basolateral membrane Cl conductance in guinea pig distal colon activates during electrogenic K secretion and Cl secretion

Epinephrine (epi) activates transient Cl secretion (sec) and sustained K sec across isolated distal colonic mucosa of guinea pig. The Ca-activated Cl channel inhibitor CaCCinh-A01 [30μM] reduced electrogenic K sec, detected as short-circuit current (Isc), consistent with basolateral Cl channels providing an exit pathway for Cl entering via Na/K/2Cl-cotransporters. CaCCinh-A01 inhibited both Isc and transepithelial conductance in a concentration dependent manner, IC50 = 6.3μM. GlyH-101, another Cl channel inhibitor, also reduced K secretory Isc (IC50 = 9.4μM). Epi activated whole-cell Cl current in isolated intact colonic crypts. This epi-activated Cl current also was inhibited by CaCCinh-A01 or GlyH-101. In contrast to K sec, CaCCinh-A01 augmented epi-activated Cl secretory Isc as well as PGE2-activated Cl sec. Synergistic Cl sec activated by cholinergic/PGE2 stimulation was insensitive to CaCCinh-A01. Colonic expression of Tmem16A, a Ca-activated Cl channel, was supported by RT-PCR of Tmem16A-mRNA, immuno-blot with Tmem16A-antibodies, and immuno-reactivity in lateral membranes of epithelial cells. Alternative splices of Tmem16A were detected for exons involved in channel activation. Inhibition of K sec and augmentation of Cl sec by CaCCinh-A01 supports a common colonic cell model for these two ion secretory processes with basolateral membrane Cl channels that contribute to production of electrogenic K sec and limiting Cl sec. Maximal Cl sec occurs only for synergistic activation mechanisms that inactivate these basolateral membrane Cl channels. [NIH DK65845]

Activation of the basolateral membrane Cl−conductance essential for electrogenic K+secretion suppresses electrogenic Cl−secretion

Adrenaline activates transient Cl(-) secretion and sustained K(+) secretion across isolated distal colonic mucosa of guinea-pigs. The Ca(2+)-activated Cl(-) channel inhibitor CaCCinh-A01 (30 μm) significantly reduced electrogenic K(+) secretion, detected as short-circuit current (I(sc)). This inhibition supported the cell model for K(+) secretion in which basolateral membrane Cl(-) channels provide an exit pathway for Cl(-) entering the cell via Na(+)-K(+)-2Cl(-) cotransporters. CaCCinh-A01 inhibited both I(sc) and transepithelial conductance in a concentration-dependent manner (IC(50) = 6.3 μm). Another Cl(-) channel inhibitor, GlyH-101, also reduced sustained adrenaline-activated I(sc) (IC(50) = 9.4 μm). Adrenaline activated whole-cell Cl(-) current in isolated intact colonic crypts, confirmed by ion substitution. This adrenaline-activated whole-cell Cl(-) current was also inhibited by CaCCinh-A01 or GlyH-101. In contrast to K(+) secretion, CaCCinh-A01 augmented the electrogenic Cl(-) secretion activated by adrenaline as well as that activated by prostaglandin E(2). Synergistic Cl(-) secretion activated by cholinergic/prostaglandin E(2) stimulation was insensitive to CaCCinh-A01. Colonic expression of the Ca(2+)-activated Cl(-) channel protein Tmem16A was supported by RT-PCR detection of Tmem16A mRNA, by immunoblot with a Tmem16A antibody, and by detection of immunofluorescence in lateral membranes of epithelial cells. Alternative splices of Tmem16A were detected for exons that are involved in channel activation. Inhibition of K(+) secretion and augmentation of Cl(-) secretion by CaCCinh-A01 support a common colonic cell model for these two ion secretory processes, such that activation of basolateral membrane Cl(-) channels contributes to the production of electrogenic K(+) secretion and limits the rate of Cl(-) secretion. Maximal physiological Cl(-) secretion occurs only for synergistic activation mechanisms that close these basolateral membrane Cl(-) channels.

Secretory activation of basolateral membrane Cl−channels in guinea pig distal colonic crypts

Cell-attached recordings revealed Cl(-) channel activity in basolateral membrane of guinea pig distal colonic crypts isolated from basement membrane. Outwardly rectified currents ((gp)Cl(or)) were apparent with a single-channel conductance (gamma) of 29 pS at resting membrane electrical potential; another outward rectifier with gamma of 24 pS was also observed ( approximately 25% of (gp)Cl(or)). At a holding potential of -80 mV gamma was 18 pS for both (gp)Cl(or) currents, and at +80 mV gamma was 67 and 40 pS, respectively. Identity as Cl(-) channels was confirmed in excised patches by changing bath ion composition. From reversal potentials, relative permeability of K(+) over Cl(-) (P(K)/P(Cl)) was 0.07 +/- 0.03, with relative permeability of Na(+) over Cl(-) (P(Na)/P(Cl)) = 0.08 +/- 0.04. A second type of Cl(-) channel was seen with linear current-voltage (I-V) relations ((gp)Cl(L)), having subtypes with gamma of 21, 13, and 8 pS. Epinephrine or forskolin increased the number of open (gp)Cl(or) and (gp)Cl(L). Open probabilities (P(o)) of (gp)Cl(or), (gp)Cl(L21), and (gp)Cl(L13) were voltage dependent in cell-attached patches, higher at more positive potentials. Kinetics of (gp)Cl(or) were more rapid with epinephrine activation than with forskolin activation. Epinephrine increased P(o) at the resting membrane potential for (gp)Cl(L13). Secretagogue activation of these Cl(-) channels may contribute to stimulation of electrogenic K(+) secretion across colonic epithelium by increasing basolateral membrane Cl(-) conductance that permits Cl(-) exit after uptake via Na(+)-K(+)-2Cl(-) cotransport.

Basolateral membrane Cl(-)-, Na(+)-, and K(+)-coupled base transport mechanisms in rat MTALH.

Mechanisms involved in basolateral HCO transport were examined in the in vitro microperfused rat medullary thick ascending limb of Henle (MTALH) by microfluorometric monitoring of cell pH. Removing peritubular Cl(-) induced a cellular alkalinization that was inhibited in the presence of peritubular 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (DIDS) and blunted in the absence of external CO(2)/HCO. The alkalinization elicited by removing peritubular Cl(-) persisted in the bilateral absence of Na(+), together with a voltage clamp. When studied in Cl(-)-free solutions, lowering peritubular pH induced a base efflux that was inhibited by peritubular DIDS or by the absence of external CO(2)/HCO. Removing peritubular Na(+) elicited a cellular acidification that was accounted for by stimulation of a DIDS- and ethylisopropylamiloride (EIPA)-insensitive Na(+)-HCO cotransport and inhibition of a basolateral Na(+)/H(+) exchange. Increasing bath K(+) induced an intracellular alkalinization that was inhibited in the absence of external CO(2)/HCO. At 2 mM, peritubular Ba(2+), which inhibits the K(+)-Cl(-) cotransport, did not induce any change in transepithelial voltage but elicited a cellular alkalinization and inhibited K(+)-induced cellular alkalinization, consistent with the presence of a basolateral, electroneutral Ba(2+)-sensitive K(+)-Cl(-) cotransport that may operate as a K(+)-HCO cotransport. This cotransport was inhibited in the peritubular presence of furosemide, [(dihydroindenyl)oxy]alkanoic acid, 5-nitro-2-(3-phenylpropylamino)benzoate, or DIDS. At least three distinct basolateral HCO transport mechanisms are functional under physiological conditions: electroneutral Cl(-)/HCO exchange, DIDS- and EIPA-insensitive Na(+)-HCO cotransport, and Ba(2+)-sensitive electroneutral K(+)-Cl(-)(HCO) cotransport.

Basolateral potassium channels and epithelial ion transport.

Basolateral potassium channels and epithelial ion transport.

Cl- channels in basolateral renal medullary membranes. XII. Anti-rbClC-Ka antibody blocks MTAL Cl- channels.

Cl- channels in the medullary thick ascending limb (MTAL) studied by either patch-clamp technique or reconstitution into lipid bilayers are activated by increases in intracellular Cl- concentrations. rbClC-Ka, a ClC Cl- channel, may represent this channel. We therefore evaluated the role of rbClC-Ka in transcellular MTAL Cl- transport in two separate ways. First, an antibody was raised against a fusion protein containing a 153-amino acid fragment of rbClC-Ka. Immunostaining of rabbit kidney sections with the antibody was localized to basolateral regions of MTAL and cortical thick ascending limb (CTAL) segments and also to the cytoplasm of intercalated cells in the cortical collecting duct. Second, Cl- uptake and efflux were measured in suspensions of mouse MTAL segments. Cl- uptake was bumetanide sensitive and was stimulated by treatment with a combination of vasopressin + forskolin + dibutyryl adenosine 3',5-cyclic monophosphate (DBcAMP). Cl- efflux was also increased significantly by vasopressin + forskolin + DBcAMP from 114 +/- 20 to 196 +/- 36 nmol.mg protein-1.45 s-1 (P = 0.003). Cl- efflux was inhibited by the Cl- channel blocker diphenylamine-2-carboxylate (154 +/- 26 vs. 70 +/- 21 nmol.mg protein-1.45 s-1, P = 0.003). An anti-rbClC-Ka antibody, which inhibits the activity of MTAL Cl- channels in lipid bilayers, reduced Cl- efflux from intact MTAL segments (154 +/- 28 vs. 53 +/- 14 nmol.mg protein-1.45 s-1, P = 0.02). These results support the view that rbClC-Ka is the basolateral membrane Cl- channel that mediates vasopressin-stimulated net Cl- transport in the MTAL segment.

Extracellular ATP Activates Calcium Signaling, Ion, and Fluid Transport in Retinal Pigment Epithelium

The presence of receptors for ATP has not been established in any native preparation of retinal neurons or glia. In the present study, we used conventional electrophysiological and [Ca2+]in fluorescence imaging techniques to investigate the effects of ATP added to Ringer's solution perfusing the retinal-facing (apical) membrane of freshly isolated monolayers of bovine retinal pigment epithelium (RPE). ATP (or UTP) produced large, biphasic voltage and resistance changes with a Kd of approximately 5 microM for ATP and approximately 1 microM for UTP. Electrical and pharmacological evidence indicates that the first and second phases of the response are attributable to an increase in basolateral membrane Cl conductance and a decrease in apical membrane K conductance, respectively. The ATP-induced responses were not affected by adenosine, but were reduced by the P2-purinoceptor blocker suramin. ATP also produced a large, transient increase in [Ca2+]in that was blocked by cyclopiazonic acid, an inhibitor of endoplasmic reticulum Ca2+-ATPases. The calcium buffer BAPTA attenuated the voltage effects of ATP. We also found that apical DIDS significantly inhibited the ATP-evoked [Ca2+]in and electrical responses, suggesting that DIDS blocked the purinoceptor. Measurements of fluid movement across the RPE using the capacitance probe technique demonstrated a significant increase in fluid absorption by apical UTP. These data indicate the presence of metabotropic P2Y/P2U-purinoceptors at the RPE apical membrane and implicate extracellular ATP in vivo as a retinal signaling molecule that could help regulate the hydration and chemical composition of the subretinal space.

Cl- channels in basolateral renal medullary vesicles XI. rbClC-Ka cDNA encodes basolateral MTAL Cl- channels.

The present experiments examined whether rbClC-Ka, a CIC family Cl-channel cDNA from rabbit outer medulla, encodes a basolateral membrane Cl- channel mediating net medullary thick ascending limb (MTAL) Cl- absorption. MTAL cells contain a Cl- channel having certain properties that make it a plausible candidate for the basolateral Cl- channel in that segment. Especially pertinent among properties is the fact that cytosolic Cl- increases in the range 2-25 mM activated these Cl- channels. Cultured mouse MTAL cells were grown in the presence of an antisense oligonucleotide specific for rbCIC-Ka or a random oligonucleotide with no complementarity to rbCIC-Ka. The abundance of Cl- channels was assessed by the frequency of incorporation of Cl- channels from membrane vesicles prepared from these cells into lipid bilayers and by Western blot analysis using an antiserum to the COOH terminus of the rbClC-ka protein. With the use of vesicles from untreated cells or cells treated with the random oligonucleotide, Cl- channels were incorporated into bilayers in 17% and 16% of trials, respectively. However, when vesicles were prepared from cells pretreated with antisense oligonucleotide, there was a virtual abolition of Cl- channel incorporation into bilayers but no effect on the frequency of K+ channel incorporation. In parallel with the reduction in Cl- channel incorporation, the abundance of rbClC-Ka protein was reduced approximately 50% on Western blots. Finally, exposure of Cl- channels in lipid bilayers to the rbClC-Ka antiserum resulted in a block in channel activity. These results support the contention that the basolateral Cl- channel mediating net Cl- absorption in the MTAL is encoded by rbClC-Ka.

Nonsteroidal anti-inflammatory drugs alter chloride and fluid transport in bovine retinal pigment epithelium.

Nonsteroidal anti-inflammatory drugs (NSAIDs) were added to the solutions bathing the apical membrane of bovine retinal pigment epithelium (RPE)-choroid explants. For example, niflumic acid (100 microM) depolarized the basolateral membrane voltage (VB) by approximately 12 mV, increased transepithelial potential by 4.5 mV, decreased intracellular Cl activity by 13 mM, decreased transepithelial resistance by 17 omega.cm2, and increased the ratio of apical to basolateral membrane resistance nearly threefold. All of these changes are consistent with an increase in basolateral membrane Cl conductance. In addition, niflumic acid caused intracellular Ca concentration to decrease by 16 nM and fluid transport rate to increase by 1.5 microliters.cm-2.h-1. Flufenamic acid, which is structurally very similar to niflumic acid, had the opposite effects on membrane voltage and resistance. Basal application of the Cl channel blocker 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid or current clamping VB to the reversal potential for Cl practically abolished the niflumic acid response. The niflumic acid results suggest that certain NSAIDs can directly alter Cl conductance in the bovine RPE, apparently independently of cyclooxygenase inhibition.

Cl- channels in basolateral renal medullary vesicles X. Cloning of a Cl- channel from rabbit outer medulla

These experiments were intended to identify candidate cDNAs which might encode basolateral membrane Cl- channels of the mTAL using a homology-based cloning strategy. We prepared a cDNA library using a 1.8 to 3.2 kb mRNA fraction from rabbit outer medulla that induces a Cl- conductance in cellular membranes of Xenopus laevis oocytes. The cDNA library was screened with two 32P-oligonucleotide probes corresponding to highly conserved sequences in other Cl- channels. We isolated two cDNAs: rbClC-Ka and rbClC-Kb. The protein sequences deduced from these two cDNAs had 99% homology. Using RT-PCR technology, cultured mouse mTAL cells were found to contain mRNA corresponding to those two cDNAs. Expression of the mRNAs corresponding to these two cDNAs was kidney-specific and was greater in rabbit renal medulla than rabbit renal cortex. Finally, by using RT-PCR technology in combination with microdissected glomeruli or tubule segments, we found mRNA for rbClC-Ka in glomeruli, proximal convoluted tubules, mTAL and cortical collecting tubules.

Cl- channels in basolateral renal medullary vesicles. IX. Channels from mouse MTAL cell patches and medullary vesicles.

The experiments reported herein compared Cl- channels fused into bilayers from rabbit outer medullary vesicles with Cl- channels in excised patches of basolateral membranes from cultured mouse medullary thick ascending limb (MTAL) cells and evaluated whether the latter were plausible candidates for the Cl- channels mediating net NaCl absorption in microperfused mouse MTAL segments. The unique signature characteristics of Cl- channels incorporated into lipid bilayers from outer medullary vesicles include activation of open probability (Po) by increases in the Cl- concentrations bathing intracellular faces; activation of Po by protein kinase A (PKA) + ATP, when the Cl- concentrations bathing intracellular faces are low; and no effect of PKA + ATP on Po with high cytoplasmic-face Cl- concentrations. These same properties were observed in Cl- channels studied using excised patches of basolateral membranes from mouse MTAL cells. Moreover, in both bilayers and in excised patches, the sharpest fractional increase in Cl- channel Po occurred with cytosolic-face Cl- concentration increases to values similar to the antidiuretic hormone (ADH)-dependent values of intracellular Cl- activity in microperfused mouse MTAL segments, and these fractional Po increases were adequate to account quantitatively for the ADH-dependent increase in basolateral membrane Cl- conductance in microperfused mouse MTAL segments. Thus the excised-patch basolateral Cl- channels reported here are reasonable candidates for those mediating net Cl- absorption in the MTAL.

The delayed basolateral membrane hyperpolarization of the bovine retinal pigment epithelium: mechanism of generation.

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 apical bumetanide (100 microM); this response was completely restored after bumetanide removal. 4. Reducing apical [K+]o, adding apical bumetanide (500 mM), or removing apical Cl- decreased aCli by 25 +/- 6 (n = 8), 28 +/- 1 (n = 2) and 26 +/- 5 mM (n = 3), respectively; adding 100 microM apical bumetanide decreased aCli by 12 +/- 2 mM (n = 3). Adding apical bumetanide or removing apical bath Cl- hyperpolarized the basolateral membrane and decreased the apparent basolateral membrane conductance (GB). 5. DIDS (4,4'-diisothiocyanostilbene-2,2'-disulphonic acid) blocked the RPE basolateral membrane Cl- conductance and inhibited the DBMH and the basolateral membrane hyperpolarization produced by apical bumetanide addition or by removal of apical Cl-o. The present results show that the DBMH is caused by delta[K]o-induced inhibition of the apical membrane Na(+)-K(+)-2Cl- cotransporter; the subsequent decrease in aCli generated a hyperpolarization at the basolateral membrane Cl- channel.

Cryoprotection of Aplysia gut basolateral membranes by trehalose

Cl −-ATPase activity resides in the basolateral membrane of gut cells. The sugar alcohols sorbitol, mannitol and glycerol could not maintain any cryoprotection of the basolateral membrane fraction for at least 12hr. The sugar, mannose, could not maintain any cryoprotection of basolateral membrane for at least 12 hr, while sucrose could only maintain 20% of basolateral membrane Cl −-ATPase activity after 12 hr. Trehalose cryoprotected basolateral membrane Cl −-ATPase activity by 98% of control up to 48 hr.

K+ and Cl- transport mechanisms in bovine pigment epithelium that could modulate subretinal space volume and composition.

1. Conventional and ion-selective double-barrelled microelectrodes were used in an in vitro bovine retinal pigment epithelium (RPE)-choroid preparation to measure the changes in membrane voltage, resistance and intracellular K+ and Cl- activities produced by small, physiological changes in extracellular potassium ([K+]o). 2. In the intact eye, light-induced changes in [K+]o occur in the extracellular (or subretinal) space that separates the neural retina and the RPE apical membrane. These [K+]o changes can be approximated in vitro by decreasing apical bath [K+]o from 5 to 2 mM. 3. This in vitro change in [K+]o simultaneously decreased intracellular Cl- and K+ activities (aCli and aKi) by 25 +/- 6 mM (n = 8) and 19 +/- 7 mM (n = 4) (mean +/- S.D.), respectively. In control Ringer solution (5 mM [K+]o) aCli and aKi were 65 +/- 10 mM (n = 28) and 65 +/- 8 mM (n = 6), respectively. 4. The [K+]o-induced decreases in aCli and aKi were both significantly inhibited, either by blocking the apical membrane K+ conductance with Ba2+ or the basolateral membrane Cl- conductance with DIDS (4,4'-diisothiocyano-stilbene-2,2'-disulphonic acid). 5. Transepithelial current pulses were used to determine the relative basolateral membrane Cl- conductance, TClBAS, was approximately 0.6 (n = 3), and the relative apical membrane K+ conductance, TKAP, was approximately 0.7 (n = 2). Step changes in basal bath [K+]o were used to estimate the relative basolateral membrane K+ conductance, TKBAS, was approximately 0.34 (n = 3). 6. These data show that the apical membrane K+ conductance and the basolateral membrane Cl- conductance are electrically coupled. In vivo, this coupling could have significant functional importance by modulating the relative hydration of the subretinal space, regulating RPE cell volume, and buffering the chemical composition of the subretinal space.

Basolateral membrane Cl- and K+ conductances of the dark-adapted chick retinal pigment epithelium.

1. We characterized the basolateral membrane Cl- and K+ conductances of the dark-adapted chick neural retina-retinal pigment epithelium (RPE)-choroid preparation. Conventional microelectrodes were used to measure apical (V(ap)) and basolateral (Vba) membrane voltage, and double-barreled Cl- and K+ selective microelectrodes were used to follow the time course and magnitude of ion concentration changes outside the basolateral (basal) membrane. 2. In response to a fivefold decrease in basal [Cl-]o, Vba rapidly depolarized by 6.4 +/- 0.7 (SE) mV, and the apparent resistance of the basolateral membrane (Rba) increased. The Cl- channel blocker 4,4'-diisothiocyanostilbene-2,2'-disulfonate (DIDS) suppressed the Vba depolarization by 40% and blocked the Rba increase. Estimates of the relative Cl- conductance (transference number, TCl) from the DIDS-sensitive component of the Cl- diffusion potential gave an average value for TCl of 0.22 +/- 0.03. 3. Further evidence for a Cl- conductance was obtained by measuring changes in intracellular Cl- activity (aCli) induced by transtissue current. Depolarizing Vba elevated aiCl, whereas hyperpolarizing Vba had the opposite effect, consistent with conductive Cl- movement across the basal membrane. TCl estimated from these data averaged 0.23 +/- 0.02. 4. In response to a sixfold increase in basal [K+]o, Vba depolarized 6.1 +/- 0.8 mV. The amplitude of this K+ diffusion potential was inhibited 44 and 67% by 5 and 10 mM Ba2+, respectively. TK was estimated to be 0.61 +/- 0.05. 5. The rapid c-wave membrane hyperpolarizations in response to the light-evoked decrease in subretinal [K+]o were used to calculate the equivalent resistances of the apical membrane (R(ap)), basolateral membrane (Rba), and the paracellular shunt pathway (Rs). They were 152 +/- 10, 615 +/- 38, and 138 +/- 7 omega.cm2 (n = 11 tissues), respectively. From these data the equivalent electromotive force for the basal (Eba) and apical (Eap) membranes were estimated to be -45 +/- 2 and -77 +/- 1 mV, respectively. This estimate of Eba is in the range of that predicted from our estimates of TCl and TK, indicating that, in the dark-adapted chick retina, the resting conductance of the basal membrane can largely be accounted for by the Cl- and K+ conductances described here.

Light-evoked modulation of basolateral membrane Cl- conductance in chick retinal pigment epithelium: the light peak and fast oscillation

1. We studied the ionic mechanism of the light-peak voltage of the DC electroretinogram (DC ERG) in an in vitro preparation of chick neural retina-retinal pigment epithelium (RPE)-choroid. The light peak originates from a depolarization of the RPE basolateral (basal) membrane, associated with an increase in its conductance. Using conventional and Cl(-)-selective microelectrodes, we tested the hypothesis that the light-peak voltage is generated by an increase in Cl- conductance (gCl) of the basolateral (basal) membrane. 2. Perfusion of the RPE basal membrane with 4,4'-diisothiocyanostilbene-2,2'-disulfonate (DIDS), a known blocker of gCl in chick RPE, suppressed both the light-peak depolarization and the accompanying conductance increase of the basal membrane. 3. Using sustained transepithelial current to clamp the basal membrane potential at different levels, we estimated the reversal potential of the light peak. At membrane potentials above the equilibrium potential for Cl- (ECl = -40 +/- 10 mV mean +/- SE), light-peak polarity was reversed. Current-voltage (I-V) curves measured in the dark and at the peak of the light peak also gave a reversal potential in the same range as ECl. In addition, shifting ECl by changing intracellular Cl- (aCli) via passage of transepithelial current or perfusing the apical side of the RPE with the Cl- uptake blocker, furosemide, shifted the light-peak reversal potential in the same direction as the change in ECl. 4. The transference number for Cl-, TCl, was estimated from step decreases in basal Cl- and increased from 0.20 +/- 0.01 in the dark to 0.31 +/- 0.01 during the light peak. These results indicate an average increase of 55% in the relative conductance of the basal membrane for Cl-. 5. Light-evoked changes in aCli, measured with Cl(-)-selective microelectrodes, were too small to account for the change in basal membrane potential during the light peak. These data strongly support the hypothesis that the light peak originates from an increase in RPE basal membrane permeability to Cl-. 6. We also obtained support for the model of Joseph and Miller that the fast-oscillation trough of the DC ERG, generated by a delayed basal membrane hyperpolarization of the RPE, originates from light-evoked modulation of the Cl- transport pathway. Perfusing either the apical side of the RPE with furosemide or the basal side with DIDS suppressed the fast oscillation. The delayed basal hyperpolarization reversed polarity at membrane potentials positive to ECl.(ABSTRACT TRUNCATED AT 400 WORDS)

Cl? channels in basolateral renal medullary membranes: VII. Characterization of the intracellular anion binding sites

A unique property of basolateral membrane Cl- channels from the mTAL is that the Cl- concentration facing the intracellular aspects of these channels is a determinant of channel open time probability (Po). The K1/2 for maximal activation of Po by Cl- facing intracellular domains of these channels is 10 mM Cl-. The present experiments evaluated the nature of these Cl(-)-interactive sites. First, we found that the impermeant anion isethionate, when exposed to intracellular Cl- channel faces, could augment Po with a K1/2 in the range of 10 mM isethionate without affecting conductance (gCl, pS). Second, pretreatment of the solutions facing the intracellular aspects of the channels with either 1 mM phenylglyoxal (PGO), an arginine-specific reagent, or the lysine/terminal amine reagent trinitrobenzene sulfonic acid (TNBS, 1 mM), prevented the activation of Po usually seen when the Cl- concentration of solutions facing intracellular channel domains was raised from 2 to 50 mM. However, when the Cl- channel activity was increased by first raising the Cl- concentration bathing intracellular channel faces from 2 to 50 mM, subsequent addition of either PGO or TNBS to solutions bathing intracellular Cl- channel faces had no effect on Po. We conclude that the intracellular aspects of these Cl- channels contain Cl(-)-interactive loci (termed [Cl]i) which are accessible to impermeant anions in intracellular fluids and which contain arginine- and lysine-rich domains which can be inactivated, at low ambient Cl- or isethionate concentrations, by interactions with PGO or TNBS.