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Shroom is a family of related proteins linked to the actin cytoskeleton, and one of them, xShroom1, is constitutively expressed in Xenopus laevis oocytes which is required for the expression of the epithelial sodium channel (ENaC). On the other hand, ENaC and the cystic fibrosis transmembrane regulator (CFTR) are co-expressed in many types of cells with a negative or positive interaction depending on the studied tissues. Here, we measured the amiloride-sensitive ENaC currents (INa amil ) and CFTR currents (I CFTR ) with voltage clamp techniques in oocytes co-injected with ENaC and/or CFTR and xShroom1 antisense oligonucleotides. The objective was to study the mechanism of regulation of ENaC by CFTR when xShroom1 was suppressed and the endocytic traffic of CFTR was blocked. CFTR activation had a measurable negative effect on ENaC and this activation resulted in a greater inhibition of INa amil than with xShroom1 antisense alone. Our results with Dynasore, a drug that acts as an inhibitor of endocytic pathways, suggest that the changes in INa amil by xShroom1 downregulation were probably due to an increment in channel endocytosis. An opposite effect was observed when I CFTR was measured. Thus, when xShroom1 was downregulated, the I CFTR was larger than in the control experiments and this effect is not observed with Dynasore. A speculative explanation could be that xShroom1 exerts a dual effect on the endocytic traffic of ENaC and CFTR and these actions were canceled with Dynasore. In the presence of Dynasore, no difference in either INa amil or I CFTR was observed when xShroom1 was downregulated., (© 2022. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

The regulation of the epithelial Na⁺ channel (ENaC) during cell swelling is relevant in cellular processes in which cell volume changes occur, i.e., migration, proliferation and cell absorption. Its sensitivity to hypotonically induced swelling was investigated in the Xenopus oocyte expression system with the injection of the three subunits of mouse ENaC. We used voltage-clamp techniques to study the amiloride-sensitive Na⁺ currents (INa(amil)) and video microscopic methodologies to assess oocyte volume changes. Under conditions of mild swelling (25 % reduced hypotonicity) inward current amplitude decreased rapidly over 1.5 min. In contrast, there was no change in current amplitude of H₂O-injected oocytes to the osmotic insult. INa(amil) kinetics analysis revealed a decrease in the slower inactivation time constant during the hypotonic stimuli. Currents from ENaC-injected oocytes were not sensitive to external Cl⁻ reduction. Neither short- nor long-term cytochalasin D treatment affected the observed response. Oocytes expressing a DEG mutant β-ENaC subunit (β-S518K) with an open probability of 1 had reduced INa(amil) hypotonic response compared to oocytes injected with wild-type ENaC subunits. Finally, during the hypotonic response ENaC-injected oocytes did not show a cell volume difference compared with water-injected oocytes. On this basis we suggest that hypotonicity-dependent ENaC inhibition is principally mediated through an effect on open probability of channels in the membrane.

Aldosterone is a key regulator of the epithelial sodium channel (ENaC) and stimulates protein methylation on the β-subunit of the ENaC. We found that aldosterone (100 nM) promotes cellular migration in a wound-healing model in trophoblastic BeWo cells. Here, we tested if the positive influence of aldosterone on wound healing is related to methylation reactions. Cell migration and proliferation were measured in BeWo cells at 6 h, when mitosis is still scarce. Cell migration covered 12.4, 25.3, 19.6 and 45.1 % of the wound when cultivated under control, aldosterone (12 h), 8Br-cAMP and aldosterone plus 8Br-cAMP, respectively. Amiloride blocked the effects of aldosterone alone or in the presence of 8Br-cAMP on wound healing. Wound healing decreased in aldosterone (plus 8Br-cAMP) coexposed with the methylation inhibitor 3-deaza-adenosine (3-DZA, 12.9 % reinvasion of the wound). There was an increase in wound healing in aldosterone-, 8Br-cAMP- and 3-DZA-treated cells in the presence of AdoMet, a methyl donor, compared to cells in the absence of AdoMet (27.3 and 12.9 % reinvasion of the wound, respectively). Cell proliferation assessed with the reagent MTT was not changed in any of these treatments, suggesting that cellular migration is the main factor for reinvasion of wound healing. Electrophysiological studies showed an increase in ENaC current in the presence of aldosterone. This effect was higher with 8Br-cAMP, and there was a decrease when 3-DZA was present. AdoMet treatment partially reversed this phenomenon. We suggest that aldosterone positively influences wound healing in BeWo cells, at least in part through methylation of the ENaC.

Cell migration/proliferation processes associated with wound healing were measured in BeWo cells at 6 h, when mitosis is still scarce. Cells were cultured in medium with 1% fetal bovine serum to minimize proliferation. BeWo cell migration covered 20.6 +/- 7.0%, 38.0 +/- 5.4%, 16.6 +/- 4.8% and 13.7 +/- 3.6% of the wound when cultivated under control, aldosterone (100 nM, 12 h), aldosterone plus amiloride (10 muM) and amiloride treatments, respectively. When BeWo cells were treated with aldosterone, there was an increase in wound healing (P < 0.05), which was prevented by adding the ENaC blocker amiloride (P < 0.05, n = 16). Immunocytochemistry studies showed that the three ENaC subunits showed greater expression at the leading edge of the wound 3 h after injury, supporting the notion that these proteins participate in a postinjury signal. Antisense oligonucleotides directed against the alpha-ENaC subunit decreased the migratory response of the cells compared to the sense treated cells or the cells without oligonucleotides (P < 0.001, n = 16): 30.2 +/- 3.7%, 17.6 +/- 1.3%, 27.5 +/- 1.5% and 20.2 +/- 1.5% reinvasion of the wound with aldosterone, aldosterone plus antisense, aldosterone plus sense treatments and control conditions, respectively. Aldosterone and amiloride influence wound healing in BeWo cells, probably by their effects upon ENaCs, transmitting a signal to the cell cytoplasm for the release of several agents that promote cell migration.

The present study was performed to assay sodium currents in BeWo cells. These cells comprise a human trophoblast cell line which displays many of the biochemical and morphological properties similar to those reported for the in uterus proliferative cytotrophoblast. For whole-cell patch-clamp experiments, BeWo cells treated for 12 h with 100 nM aldosterone were exposed to 8Br-cAMP, a membrane-permeable cAMP analogue, to induce channel activity. Cells showed an amiloride-sensitive ion current (IC50 of 5.77 microM). Ion substitution experiments showed that the amiloride-sensitive current carried cations with a permeability rank order of Li+ > Na+ > K+ > NMDG (PLi/PNa = 1.3, PK/PNa = 0.6, PNMDG/PNa = 0.2). In cells pretreated with aldosterone, we observed that nearly half of successful patches had sodium channels with a linear conductance of 6.4 +/- 1.8 pS, a low voltage-independent Po and a PK/PNa of 0.19. Using RT-PCR, we determined that control cells express the alpha-, but not beta- and gamma-, epithelial sodium channel (ENaC) mRNA. When cells were treated with aldosterone (100 nM, 12 h), all alpha-, beta- and gamma-ENaC mRNAs were detected. The presence of ENaC subunit proteins in these cells was confirmed by Western blot analysis and immunolocalization with specific ENaC primary antibodies. In summary, our results suggest that BeWo cells express ENaC subunits and that aldosterone was able to modulate a selective response by generating amiloride-sensitive sodium currents similar to those observed in other human tissues.

The human ether-a-go-go related gene (HERG1) K+ channel is expressed in neoplastic cells, in which it was proposed to play a role in proliferation, differentiation and/or apoptosis. K562 cells (a chronic myeloid leukemic human cell line) express both the full-length (herg1a) and the N-terminally truncated (herg1b) isoforms of the gene, and this was confirmed with Western blots and coimmunoprecipitation experiments. Whole-cell currents were studied with a tail protocol. Seventy-eight percent of cells showed a HERG1-like current: repolarization to voltages negative to -40 mV produced a transient peak inward tail current, characteristic of HERG1 channels. Cells were exposed to a HERG-specific channel blocker, E4031. Half-maximal inhibitory concentration (IC50) of the blocker was 4.69 nM: The kinetics of the HERG1 current in K562 cells resembled the rapid component of the native cardiac delayed rectifier current, known to be conducted by heterotetrameric HERG1 channels. Fast and slow deactivation time constants at -120 mV were 27.5 and 239.5 ms, respectively. Our results in K562 cells suggest the assembling of heterotetrameric channels, with some parameters being dominated by one of the isoforms and other parameters being intermediate. Hydrogen peroxide was shown to increase HERG1a K+ current in heterologous expression systems, which constitutes an apoptotic signal. However, we found that K562 HERG1 whole-cell currents were not activated by H2O2.

The cystic fibrosis transmembrane regulator (CFTR) is a Cl(-) channel known to influence other channels, including connexin (Cx) channels. To study the functional interaction between CFTR and gap junction channels, we coexpressed in Xenopus oocytes CFTR and either Cx45, Cx40, Cx32 or Cx50 and monitored junctional conductance (G (j)) and its sensitivity to transjunctional voltage (V (j)) by the dual voltage-clamp method. Application of forskolin induced a Cl(-) current; increased G (j) approximately 750%, 560%, 64% and 8% in Cx45, Cx40, Cx32 and Cx50, respectively; and decreased sensitivity to V (j ) gating, monitored by a change in the ratio between G (j) steady state and G (j) peak (G (j)SS/G (j)PK) at the pulse. In oocyte pairs expressing just Cx45 in one oocyte (#1) and both Cx45 and CFTR in the other (#2), with negative pulses applied to oocyte #1 forskolin application still increased G (j) and decreased the sensitivity to V (j) gating, indicating that CFTR activation is effective even when it affects only one of the two hemichannels and that the G (j) and V (j) changes are not artifacts of decreased membrane resistance in the pulsed oocyte. COOH-terminus truncation reduced the forskolin effect on Cx40 (Cx40TR) but not on Cx32 (Cx32TR) channels. The data suggest a cross-talk between CFTR and a variety of gap junction channels. Cytoskeletal scaffolding proteins and/or other intermediate cytoplasmic proteins are likely to play a role in CFTR-Cx interaction.

The cystic fibrosis transmembrane conductance regulator (CFTR) is a chloride (Cl(-)) channel known to influence the function of other channels, including connexin channels. To further study potential functional interactions between CFTR and gap junction channels, we have co-expressed CFTR and connexin45 (Cx45) in Xenopus oocytes and monitored junctional conductance and voltage sensitivity by dual voltage clamp electrophysiology. In single oocytes expressing CFTR, an increase in cAMP caused by forskolin application induced a Cl(-) current and increased membrane conductance; application of diphenylamine carboxylic acid (CFTR blocker) readily blocked the Cl(-) current. With co-expression of CFTR and Cx45, application of forskolin to paired oocytes induced a typical outward current and increased junctional conductance (G(j)). In addition, the presence of CFTR reduced the transjunctional voltage sensitivity of Cx45 channels without affecting the kinetics of junctional current inactivation. The drop in voltage sensitivity was further enhanced by forskolin application. The data indicate that CFTR influences cell-to-cell coupling mediated by Cx45 channels.

C1- currents (ICl) were measured in short fibers (1-2 mm) from the lumbricalis muscle of toads (Bufo arenarum) with two microelectrodes (15 degrees C). Initially the fibers were equilibrated in a high (K+)-containing solution: (mM) K2SO4 68; Na2SO4 20; KCl 60; CaSO4 8; MgSO4 1; HEPES 2.5. Constant pulses were applied when all the external K+ was replaced by Cs+: Cs2SO4 68; Na2SO4 20; CsCl 60; CaSO4 8; HEPES 2.5 (pH 7.5). Under these conditions about 80-90% of the current is carried by Cl-. The current-voltage relation is almost linear implying constant conductance and hence voltage-independent permeability. The voltage dependence of the net C1- current could be fitted by constant field equation with a PCl of 3.3 x 10-6 cm/sec. In a separate group of experiments a two-pulse technique was used to estimate the availability and the inactivation of the initial ICl during a test pulse. After returning the potential to the holding potential for various times, test pulses of the same amplitude and duration of the prepulses were applied. The initial current during the test pulse was 70% of the initial current during the prepulse and the recovery was complete in less than 300 msec with a linear relationship between the current during the test pulse and the amplitude of the preceding prepulse. When the test pulses were preceded by a positive prepulse, the initial current for any given test pulse was larger than with a negative prepulse. If we assumed that the initial current during the test pulse is a measure of the number of channels open at the end of the prepulse, these results suggest that hyperpolarizing pulses inactivate and depolarizing prepulses activate the ICl.

Hydroxide, bicarbonate and buffer anion permeabilities in semitendinosus muscle fibers of Rana pipiens were measured. In all experiments, the fibers were initially equilibrated in isotonic, high K2SO4 solutions at pHo = 7.2 buffered with phosphate. Two different methods were used to estimate permeabilities: (i) membrane potential changes were recorded in response to changes in external ion concentrations, and (ii) intracellular pH changes were recorded in response to changes in external concentrations of ions that alter intracellular pH. Constant field equations were used to calculate relative or absolute permeabilities. In the first method, to increase the size of the membrane potential change produced by a sudden change in anion entry, external K+ was replaced by Cs+ prior to changes of the anion under study. At constant external Cs+ activity, a hyperpolarization results from increasing external pH from 7.2 to 10.0 or higher, using either CAPS (3-[cyclohexylamino]-1-propanesulfonic acid) or CHES (2-[N-cyclohexylamino]-ethanesulfonic acid) as buffer. For each buffer, the protonated form is a zwitterion of zero net charge and the nonprotonated form is an anion. Using reported values of H+ permeability, calculations show that the reduction in [H+]o cannot account for the hyperpolarizations produced by alkaline solutions. Membrane hyperpolarization increases with increasing total external buffer concentration at constant external pH, and with increasing external pH at constant external buffer anion concentration. Taken together, these observations indicate that both OH- and buffer anions permeate the surface membrane. The following relative permeabilities were obtained at pHo = 10.0 +/- 0.3: (POH/PK) = 890 +/- 150, (PCAPS/PK) = 12 +/- 2, (PCHES/PK) = 5.3 +/- 0.9, and (PNO3/PK) = 4.7 +/- 0.5. PNO3/PK was independent of pHo up to 10.75. At pHo = 9.6, (PHCO3/PK) = 0.49 +/- 0.03; at pHo = 8.9, (PCl/PK) = 18 +/- 2 and at pHo = 7.1, (PHEPES/PK) = 20 +/- 2. In the second method, on increasing external pH from 7.2 to 10.0, using 2.5 mM CAPS (total buffer concentration), the internal pH increases linearly with time over the next 10 min. This alkalinization is due to the entry of OH- and the absorption of internal H+ by entering CAPS- anion. The rate of CAPS- entry was determined in experiments in which the external CAPS concentration was increased at constant external pH. Such increases invariably produced an increase in the rate of internal alkalinization, which was reversed when the CAPS concentration was reduced to its initial value.(ABSTRACT TRUNCATED AT 400 WORDS)

The effects of pH on the permeability and conductance of the membranes to nitrate and to chloride of semitendinosus and lumbricalis muscle fibers were examined. Membrane potential responses to quick solution changes were recorded in semitendinosus fibers initially equilibrated in isotonic, high K2SO4 solutions. External solutions were first changed to ones in which either Rb+ or Cs+ replaced K+ and then to solutions containing either NO3- or Cl- to replace SO4(2-). The hyperpolarizations produced by Cl- depend on external pH, being smaller in acid than in alkaline solutions. By contrast, hyperpolarizations produced by NO3- were independent of external pH over a pH range from 5.5 to 9.0. In addition, voltage-clamp measurements were made on short lumbricalis muscle fibers. Initially they were equilibrated in isotonic solutions containing mainly K2SO4 plus Na2SO4. KCl or KNO3 were added to the sulfate solutions and the fibers were equilibrated in these new solutions. When finally equilibrated the fibers had the same volume they had in the sulfate solutions before the additions. Constant hyperpolarizing voltage pulses of 0.6-sec duration were applied when all external K+ was replaced by TEA+. For these conditions, inward currents flowing during the voltage pulses were largely carried by Cl- or NO3- depending on the final equilibrating solution. Cl- currents during voltage pulses were both external pH and time dependent. By contrast, NO3- currents were independent of both external pH and time. The voltage dependence of NO3- currents could be fit by constant field equations with a PNO3 of 3.7.10(-6) cm/sec. The voltage dependence of the initial or "instantaneous" Cl- currents at pH 7.5 and 9.0 could also be fit by constant field equations with PCl of 5.8 x 10(-6) and 7.9 x 10(-6) cm/sec, respectively. At pH 5.0, no measurable "instantaneous" Cl- currents were found. From these results we conclude that NO3- does not pass through the pH, time-dependent Cl- channels but rather passes through a distinct set of channels. Furthermore, Cl- ions do not appear to pass through the channels which allow NO3- through. Consequently, the measured ratio of PCl/PNO3 based on membrane potential changes to ionic changes made on intact skeletal muscle fibres is not a measure of the selectivity of a single anion channel but rather is a measure of the relative amounts of different channel types.(ABSTRACT TRUNCATED AT 400 WORDS)