Swelling-activated Gd3+-sensitive Cation Current and Cell Volume Regulation in Rabbit Ventricular Myocytes

The role of swelling-activated currents in cell volume regulation is unclear. Currents elicited by swelling rabbit ventricular myocytes in solutions with 0.6–0.9× normal osmolarity were studied using amphotericin perforated patch clamp techniques, and cell volume was examined concurrently by digital video microscopy. Graded swelling caused graded activation of an inwardly rectifying, time-independent cation current (I_Cir,swell) that was reversibly blocked by Gd³⁺, but I_Cir,swell was not detected in isotonic or hypertonic media. This current was not related to I_K1 because it was insensitive to Ba²⁺. The P_K/P_Na ratio for I_Cir,swell was 5.9 ± 0.3, implying that inward current is largely Na⁺ under physiological conditions. Increasing bath K⁺ increased g_Cir,swell but decreased rectification. Gd³⁺ block was fitted with a K _0.5 of 1.7 ± 0.3 μM and Hill coefficient, n, of 1.7 ± 0.4. Exposure to Gd³⁺ also reduced hypotonic swelling by up to ∼30%, and block of current preceded the volume change by ∼1 min. Gd³⁺-induced cell shrinkage was proportional to I_Cir,swell when I_Cir,swell was varied by graded swelling or Gd³⁺ concentration and was voltage dependent, reflecting the voltage dependence of I_Cir,swell. Integrating the blocked ion flux and calculating the resulting change in osmolarity suggested that I_Cir,swell was sufficient to explain the majority of the volume change at –80 mV. In addition, swelling activated an outwardly rectifying Cl⁻ current, I_Cl,swell. This current was absent after Cl⁻ replacement, reversed at E_Cl, and was blocked by 1 mM 9-anthracene carboxylic acid. Block of I_Cl,swell provoked a 28% increase in swelling in hypotonic media. Thus, both cation and anion swelling-activated currents modulated the volume of ventricular myocytes. Besides its effects on cell volume, I_Cir,swell is expected to cause diastolic depolarization. Activation of I_Cir,swell also is likely to affect contraction and other physiological processes in myocytes.     

The Tyrosine Kinase p56lck Mediates Activation of Swelling-induced Chloride Channels in Lymphocytes

Osmotic cell swelling activates Cl⁻ channels to achieve anion efflux. In this study, we find that both the tyrosine kinase inhibitor herbimycin A and genetic knockout of p56^lck, a src-like tyrosine kinase, block regulatory volume decrease (RVD) in a human T cell line. Activation of a swelling-activated chloride current (I_Cl−swell) by osmotic swelling in whole-cell patch-clamp experiments is blocked by herbimycin A and lavendustin. Osmotic activation of I_Cl−swell is defective in p56^lck-deficient cells. Retransfection of p56^lck restores osmotic current activation. Furthermore, tyrosine kinase activity is sufficient for activation of I_Cl−swell. Addition of purified p56^lck to excised patches activates an outwardly rectifying chloride channel with 31 pS unitary conductance. Purified p56^lck washed into the cytoplasm activates I_Cl−swell in native and p56^lck-deficient cells even when hypotonic intracellular solutions lead to cell shrinkage. When whole-cell currents are activated either by swelling or by p56^lck, slow single-channel gating events can be observed revealing a unitary conductance of 25–28 pS. In accordance with our patch-clamp data, osmotic swelling increases activity of immunoprecipitated p56^lck. We conclude that osmotic swelling activates I_Cl−swell in lymphocytes via the tyrosine kinase p56^lck.     

Proteolytic modification of swelling-activated Cl- current in LNCaP prostate cancer epithelial cells

The effects of intracellular application of trypsin on the Cl^– current induced by hypotonic cell swelling (I _Cl,swell) in human prostate cancer epithelial cells (LNCaP) was studied using the patch-clamp technique. In cells predialyzed with 1 mg/mL trypsin, I _Cl,swell developed and diminished in response to the application and withdrawal of hypotonic solution about three times faster than that in control cells. In trypsin-infused cells, I _Cl,swell also had about twofold higher current density and displayed considerably slowed voltage-dependent inactivation, which was quite pronounced in control cells at potentials above +60 mV. Trypsin-induced modification of I _Cl,swell could be prevented by coinfusion of 10 mg/mL soybean trypsin inhibitor, suggesting that proteolytic cleavage of essential intracellular structural domains of the I _Cl,swell-carrying volume-regulated anion channel (VRAC) was responsible for this functional modification. The effect of trypsin was not dependent on the presence of intracellular ATP. We conclude that VRACs, similarly to voltage-gated Na⁺, K⁺, and Cl^– channels, possess intracellular inactivation domain(s) subjected to proteolytic cleavage that may function in conformity with the classical ball-and-chain inactivation model.     

Phosphatidylinositol-4,5 bisphosphate (PIP2) inhibits apo-calmodulin binding to protein 4.1

Membrane skeletal protein 4.1R⁸⁰ plays a key role in regulation of erythrocyte plasticity. Protein 4.1R⁸⁰ interactions with transmembrane proteins, such as glycophorin C (GPC), are regulated by Ca²⁺-saturated calmodulin (Ca²⁺/CaM) through simultaneous binding to a short peptide (pep11; A²⁶⁴KKLWKVCVEHHTFFRL) and a serine residue (Ser¹⁸⁵), both located in the N-terminal 30 kDa FERM domain of 4.1R⁸⁰ (H·R30). We have previously demonstrated that CaM binding to H·R30 is Ca²⁺-independent and that CaM binding to H·R30 is responsible for the maintenance of H·R30 β-sheet structure. However, the mechanisms responsible for the regulation of CaM binding to H·R30 are still unknown. To investigate this, we took advantage of similarities and differences in the structure of Coracle, the Drosophila sp. homologue of human 4.1R⁸⁰, i.e. conservation of the pep11 sequence but substitution of the Ser¹⁸⁵ residue with an alanine residue. We show that the H·R30 homologue domain of Coracle, Cor30, also binds to CaM in a Ca²⁺-independent manner and that the Ca²⁺/CaM complex does not affect Cor30 binding to the transmembrane protein GPC. We also document that both H·R30 and Cor30 bind to phosphatidylinositol-4,5 bisphosphate (PIP₂) and other phospholipid species and that that PIP₂ inhibits Ca²⁺-free CaM but not Ca²⁺-saturated CaM binding to Cor30. We conclude that PIP₂ may play an important role as a modulator of apo-CaM binding to 4.1R⁸⁰ throughout evolution.     

The Swelling-Activated Anion Conductance in the Mouse Renal Inner Medullary Collecting Duct Cell Line mIMCD-K2

Swelling-activated Cl⁻ currents (I _Cl,swell ) have been characterized in a mouse renal inner medullary collecting duct cell line (mIMCD-K2). Currents activated by exposing the cells to hypotonicity exhibited characteristic outward rectification and time- and voltage-dependent inactivation at positive potentials and showed an anion selectivity of I⁻ > Br⁻ > Cl⁻ > Asp⁻. NPPB (100 μm) inhibited the current in a voltage independent manner, as did exposure to 10 μm tamoxifen and 500 μm niflumic acid (NFA). In contrast, DIDS (100 μm) blocked the current with a characteristic voltage dependency. These characteristics of I _Cl,swell in mIMCD-K2 cells are essentially identical to those of heterologously expressed cardiac CLC-3. A defining feature of CLC-3 is that activation of PKC by PDBu inhibits the conductance. In mIMCD-K2 cells preincubation with PDBu (100 nm) prevented the activation of I _Cl,swell by hypotonicity. However, PDBu inhibition of I _Cl,swell was reversed after PDBu withdrawal, but this was refractory to subsequent PDBu inhibition. Activation of either the cystic fibrosis transmembrane conductance regulator (CFTR) or Ca²⁺ activated Cl⁻ conductance (CaCC), which are coexpressed in mIMCD-K2 cells prior to PDBu treatment, abolished the PDBu inhibition of I _Cl,swell . Control of I _Cl,swell by PKC therefore depends on the physiological status of the cell. In intact mIMCD-K2 layers in Ussing chambers, forskolin stimulation of an inward short-circuit current (due to transepithelial Cl⁻ secretion via apical CFTR) was inhibited by cell swelling upon hypotonic exposure at the basolateral surface. Activation of I _Cl,swell is therefore capable of regulating transepithelial Cl⁻ secretion and suggests that I _Cl,swell is located at the basolateral membrane. PDBu exposure prior to or during hypotonic challenge was ineffective in reversing the swelling-activated inhibition of Cl⁻ secretion, but tamoxifen (100 μm) abolished the hypotonic inhibition of forskolin-stimulated short-circuit current (I _sc ). RT-PCR analysis confirmed expression of mRNA for members of the CLC family, including both CLC-2 and 3, in the mIMCD-K2 cell line. Received: 24 February 2000/Revised: 26 May 2000     

Swelling‐induced chloride current in glioblastoma proliferation,migration, and invasion

Glioblastoma (GBM) remains as the most common and aggressive brain tumor. The survival of GBM has been linked to the aberrant activation of swelling‐induced chloride current I_Cl,swell. In this study, we investigated the effects of I_Cl,swell on cell viability, proliferation, and migration in the human GBM cell lines, U251 and U87, using a combination of patch clamp electrophysiology, MTT, colony formation, wound healing assays and Western immunoblotting. First, we showed that the specific inhibitor of I_Cl,swell, DCPIB, potently reduced the I_Cl,swell in U87 cells. Next, in both U87 and U251 cells, we found that DCPIB reduced GBM viability, proliferation, colony formation, migration, and invasion. In addition, our Western immunoblot assay showed that DCPIB‐treated U251 cells had a reduction in JAK2, STAT3, and Akt phosphorylation, thus, suggesting that DCPIB potentially suppresses GBM functions through inhibition of the JAK2/STAT3 and PI3K/Akt signaling pathways. Therefore, the I_Cl,swell may be a potential drug target for GBM.     

Caveolae Act as Membrane Reserves Which Limit Mechanosensitive I Cl,swell Channel Activation during Swelling in the Rat Ventricular Myocyte

Background

Many ion channels are preferentially located in caveolae where compartmentalisation/scaffolding with signal transduction components regulates their activity. Channels that are mechanosensitive may be additionally dependent on caveolar control of the mechanical state of the membrane. Here we test which mechanism underlies caveolar-regulation of the mechanosensitive I _Cl,swell channel in the adult cardiac myocyte.

Methodology/Principal Findings

Rat ventricular myocytes were exposed to solution of 0.02 tonicity (T; until lysis), 0.64T for 10–15 min (swelling), and/or methyl-β-cyclodextrin (MBCD; to disrupt caveolae). MBCD and 0.64T swelling reduced the number of caveolae visualised by electron microscopy by 75 and 50% respectively. MBCD stimulated translocation of caveolin 3 from caveolae-enriched buoyant membrane fractions, but both caveolin 1 and 3 remained in buoyant fractions after swelling. I _Cl,swell inhibition in control cells decreased time to half-maximal volume (t _0.5,vol; 0.64T), consistent with a role for I _Cl,swell in volume regulation. MBCD-treated cells showed reduced time to lysis (0.02T) and t _0.5,vol (0.64T) compared with controls. The negative inotropic response to swelling (an index of I _Cl,swell activation) was enhanced by MBCD.

Conclusions/Significance

These data show that disrupting caveolae removes essential membrane reserves, which speeds swelling in hyposmotic conditions, and thereby promotes activation of I _Cl,swell. They illustrate a general principle whereby caveolae as a membrane reserve limit increases in membrane tension during stretch/swelling thereby restricting mechanosensitive channel activation.     

Protein 4.1R self-association: identification of the binding domain

Erythroid protein 4.1 (4.1R) stabilizes the spectrin-actin network and anchors it to the plasma membrane. To contribute to the characterization of non-erythroid protein 4.1R, we used sedimentation, pull-down and co-immunoprecipitation assays to investigate the ability of protein 4.1R to establish inter-/intra-molecular associations. We demonstrated that the small 4.1R isoforms of 60 kDa (4.1R60), but not the larger isoforms of 80 and 135 kDa (4.1R80 and 4.1R135), were self-associated, and that a domain contained in all 4.1R isoforms, the core region, was responsible for 4.1R self-association. Results from denaturing-renaturing experiments, in which an initially non-self-associated 4.1R80 isoform became self-associated, suggested that an initially hidden core region was subsequently exposed. This hypothesis was supported by results from pull-down assays, which showed that the core region interacted with the N-terminal end of the FERM (4.1, ezrin, radixin, moesin) domain that is present in 4.1R80 and 4.1R135 isoforms but absent from 4.1R60 isoforms. Consistently, 4.1R80 isoforms bound neither to each other nor to 4.1R60 isoforms. We propose that 4.1R60 isoforms are constitutively self-associated, whereas 4.1R80 and 4.1R135 self-association is prevented by intramolecular interactions.     

Anion-Sensitive Fluorophore Identifies the Drosophila Swell-Activated Chloride Channel in a Genome-Wide RNA Interference Screen

When cells swell in hypo-osmotic solutions, chloride-selective ion channels (Cl_swell) activate to reduce intracellular osmolality and prevent catastrophic cell rupture. Despite intensive efforts to assign a molecular identity to the mammalian Cl_swell channel, it remains unknown. In an unbiased genome-wide RNA interference (RNAi) screen of Drosophila cells stably expressing an anion-sensitive fluorescent indicator, we identify Bestrophin 1 (dBest1) as the Drosophila Cl_swell channel. Of the 23 screen hits with mammalian homologs and predicted transmembrane domains, only RNAi specifically targeting dBest1 eliminated the Cl_swell current (I_Clswell). We further demonstrate the essential contribution of dBest1 to Drosophila I_Clswell with the introduction of a human Bestrophin disease-associated mutation (W94C). Overexpression of the W94C construct in Drosophila cells significantly reduced the endogenous I_Clswell. We confirm that exogenous expression of dBest1 alone in human embryonic kidney (HEK293) cells creates a clearly identifiable Drosophila–like I_Clswell. In contrast, activation of mouse Bestrophin 2 (mBest2), the closest mammalian ortholog of dBest1, is swell-insensitive. The first 64 residues of dBest1 conferred swell activation to mBest2. The chimera, however, maintains mBest2-like pore properties, strongly indicating that the Bestrophin protein forms the Cl_swell channel itself rather than functioning as an essential auxiliary subunit. dBest1 is an anion channel clearly responsive to swell; this activation depends upon its N-terminus.     

Activation of Swelling-activated Chloride Current by Tumor Necrosis Factor-α Requires ClC-3-dependent Endosomal Reactive Oxygen Production

ClC-3 is a Cl⁻/H⁺ antiporter required for cytokine-induced intraendosomal reactive oxygen species (ROS) generation by Nox1. ClC-3 current is distinct from the swelling-activated chloride current (ICl_swell), but overexpression of ClC-3 can activate currents that resemble ICl_swell. Because H₂O₂ activates ICl_swell directly, we hypothesized that ClC-3-dependent, endosomal ROS production activates ICl_swell. Whole-cell perforated patch clamp methods were used to record Cl⁻ currents in cultured aortic vascular smooth muscle cells from wild type (WT) and ClC-3 null mice. Under isotonic conditions, tumor necrosis factor-α (TNF-α) (10 ng/ml) activated outwardly rectifying Cl⁻ currents with time-dependent inactivation in WT but not ClC-3 null cells. Inhibition by tamoxifen (10 μm) and by hypertonicity (340 mosm) identified them as ICl_swell. ICl_swell was also activated by H₂O₂ (500 μm), and the effect of TNF-α was completely inhibited by polyethylene glycol-catalase. ClC-3 expression induced ICl_swell in ClC-3 null cells in the absence of swelling or TNF-α, and this effect was also blocked by catalase. ICl_swell activation by hypotonicity (240 mosm) was only partially inhibited by catalase, and the size of these currents did not differ between WT and ClC-3 null cells. Disruption of endosome trafficking with either mutant Rab5 (S34N) or Rab11 (S25N) inhibited TNF-α-mediated activation of ICl_swell. Thrombin also activates ROS production by Nox1 but not in endosomes. Thrombin caused H₂O₂-dependent activation of ICl_swell, but this effect was not ClC-3- or Rab5-dependent. Thus, activation of ICl_swell by TNF-α requires ClC-3-dependent endosomal H₂O₂ production. This demonstrates a functional link between two distinct anion currents, ClC-3 and ICl_swell.     

On the endothelial cell ISOC

Ca²⁺ store depletion activates both Ca²⁺ selective and non-selective currents in endothelial cells. Recently, considerable progress has been made in understanding the molecular make-up and regulation of an endothelial cell thapsigargin-activated Ca²⁺ selective current, I_SOC. Indeed, I_SOC is a relatively small inward Ca²⁺ current that exhibits an approximate +40 mV reversal potential and is strongly inwardly rectifying. This current is sensitive to organization of the actin-based cytoskeleton. Transient receptor potential (TRP) proteins 1 and 4 (TRPC1 and TRPC4, respectively) each contribute to the molecular basis of I_SOC, although it is TRPC4 that appears to be tethered to the cytoskeleton through a dynamic interaction with protein 4.1. Activation of I_SOC requires association between protein 4.1 and the actin-based cytoskeleton (mediated through spectrin), suggesting protein 4.1 mediates the physical communication between Ca²⁺ store depletion and channel activation. Thus, at present findings indicate a TRPC4–protein 4.1 physical linkage regulates I_SOC activation following Ca²⁺ store depletion.     

Marked difference in membrane-protein-binding properties of the two isoforms of protein 4.1R expressed at early and late stages of erythroid differentiation

Two major isoforms of protein 4.1R, a 135 kDa isoform (4.1R(135)) and an 80 kDa isoform (4.1R(80)), are expressed at distinct stages of terminal erythroid differentiation. The 4.1R(135) isoform is exclusively expressed in early erythroblasts and is not present in mature erythrocytes, whereas the 4.1R(80) isoform is expressed at late stages of erythroid differentiation and is the principal component of mature erythrocytes. These two isoforms differ in that the 4.1R(135) isoform includes an additional 209 amino acids designated as the HP (head-piece) at the N-terminus of 4.1R(80). In the present study, we performed detailed characterization of the interactions of the two 4.1R isoforms with various membrane-binding partners and identified several isoform-specific differences. Although both 4.1R(135) and 4.1R(80) bound to cytoplasmic domains of GPC (glycophorin C) and band 3, there is an order of magnitude difference in the binding affinities. Furthermore, although both isoforms bound CaM (calmodulin), the binding of 4.1R(80) was Ca2+-independent, whereas the binding of 4.1R(135) was strongly Ca2+-dependent. The HP of 4.1R(135) mediates this Ca2+-dependent binding. Ca2+-saturated CaM completely inhibited the binding of 4.1R(135) to GPC, whereas it strongly reduced the affinity of its binding to band 3. Interestingly, in spite of the absence of spectrin-binding activity, the 4.1R(135) isoform was able to assemble on to the membrane of early erythroblasts suggesting that its ability to bind to membrane proteins is sufficient for its membrane localization. These findings enable us to offer potential new insights into the differential contribution of 4.1R isoforms to membrane assembly during terminal erythroid differentiation.     

TMEM16 Proteins Produce Volume-regulated Chloride Currents That Are Reduced in Mice Lacking TMEM16A

All vertebrate cells regulate their cell volume by activating chloride channels of unknown molecular identity, thereby activating regulatory volume decrease. We show that the Ca²⁺-activated Cl⁻ channel TMEM16A together with other TMEM16 proteins are activated by cell swelling through an autocrine mechanism that involves ATP release and binding to purinergic P2Y₂ receptors. TMEM16A channels are activated by ATP through an increase in intracellular Ca²⁺ and a Ca²⁺-independent mechanism engaging extracellular-regulated protein kinases (ERK1/2). The ability of epithelial cells to activate a Cl⁻ conductance upon cell swelling, and to decrease their cell volume (regulatory volume decrease) was dependent on TMEM16 proteins. Activation of I_Cl,swell was reduced in the colonic epithelium and in salivary acinar cells from mice lacking expression of TMEM16A. Thus TMEM16 proteins appear to be a crucial component of epithelial volume-regulated Cl⁻ channels and may also have a function during proliferation and apoptotic cell death.Regulation of cell volume is fundamental to all cells, particularly during cell growth and division. External hypotonicity leads to cell swelling and subsequent activation of volume-regulated chloride and potassium channels, to release intracellular ions and to re-shrink the cells, a process termed regulatory volume decrease (RVD)³ (1). Volume-regulated chloride currents (I_Cl,swell) have dual functions during cell proliferation as well as apoptotic volume decrease (AVD), preceding apoptotic cell death (2). Although I_Cl,swell is activated in swollen cells to induce RVD, AVD takes place under normotonic conditions to shrink cells (3, 4). Early work suggested intracellular Ca²⁺ as an important mediator for activation of I_Cl,swell and volume-regulated K⁺ channels (5), whereas subsequent studies only found a permissive role of Ca²⁺ for activation of I_Cl,swell (6), reviewed in Ref. 1. In addition, a plethora of factors and signaling pathways have been implicated in activation of I_Cl,swell, making cell volume regulation an extremely complex process (reviewed in Refs. 1, 3, and 7). These factors include intracellular ATP, the cytoskeleton, phospholipase A2-dependent pathways, and protein kinases such as extracellular-regulated kinase ERK1/2 (reviewed in Refs. 1 and 7). Previous approaches in identifying swelling-activated Cl⁻ channels have been unsuccessful or have produced controversial data. Thus none of the previous candidates such as pI_Cln, the multidrug resistance protein, or ClC-3 are generally accepted to operate as volume-regulated Cl⁻ channels (reviewed in Refs. 8 and 9). Notably, the cystic fibrosis transmembrane conductance regulator (CFTR) had been shown in earlier studies to influence I_Cl,swell and volume regulation (10–12). The variable properties of I_Cl,swell suggest that several gene products may affect I_Cl,swell in different cell types.The TMEM16 transmembrane protein family consists of 10 different proteins with numerous splice variants that contain 8–9 transmembrane domains and have predicted intracellular N- and C-terminal tails (13, 16–18). TMEM16A (also called ANO1) is required for normal development of the murine trachea (14) and is associated with different types of tumors, dysplasia, and nonsyndromic hearing impairment (13, 15). TMEM16A has been identified as a subunit of Ca²⁺-activated Cl⁻ channels that are expressed in epithelial and non-epithelial tissues (16–18). Interestingly, members of the TMEM16 family have been suggested to play a role in osmotolerance in Saccharomyces cerevisiae (19). Here we show that TMEM16 proteins also contribute to I_Cl,swell and regulatory volume decrease.     

Modulation of Voltage-dependent Properties of a Swelling-activated Cl? Current

We used the patch-clamp technique to study the voltage-dependent properties of the swelling-activated Cl⁻ current (I _Cl,swell) in BC₃H1 myoblasts. This Cl⁻ current is outwardly rectifying and exhibits time-dependent inactivation at positive potentials (potential for half-maximal inactivation of +75 mV). Single-channel Cl⁻ currents with similar voltage-dependent characteristics could be measured in outside-out patches pulled from swollen cells. The estimated single-channel slope conductance in the region between +60 and +140 mV was 47 pS. The time course of inactivation was well described by a double exponential function, with a voltage-independent fast time constant (∼60 ms) and a voltage-dependent slow time constant (>200 ms). Recovery from inactivation, which occurred over the physiological voltage range, was also well described by a double exponential function, with a voltage-dependent fast time constant (10–80 ms) and a voltage-dependent slow time constant (>100 ms). The inactivation process was significantly accelerated by reducing the pH, increasing the Mg²⁺ concentration or reducing the Cl⁻ concentration of the extracellular solution. Replacing extracellular Cl⁻ by other permeant anions shifted the inactivation curve in parallel with their relative permeabilities (SCN⁻ > I⁻ > NO₃ ⁻ > Cl⁻ >> gluconate). A leftward shift of the inactivation curve could also be induced by channel blockers. Additionally, the permeant anion and the channel blockers, but not external pH or Mg²⁺, modulated the recovery from inactivation. In conclusion, our results show that the voltage-dependent properties of I _Cl,swell are strongly influenced by external pH , external divalent cations, and by the nature of the permeant anion.     

Differential distribution of cAMP-dependent protein kinase isoforms in the mantle of the bivalve mollusc <Emphasis Type="Italic">Mytilus galloprovincialis</Emphasis>

Two different isoforms of the regulatory (R) subunit of cAMP-dependent protein kinase (PKA), named R_myt1 and R_myt2, had previously been identified in the sea mussel Mytilus galloprovincialis. R_myt1 and R_myt2 were differentially distributed in the various cell types comprising the mussel mantle. R_myt1 was found the only isoform to be present in the auxiliary cells of female follicles and the cubic epithelium of the middle fold of mantle edge. In contrast, only the R_myt2 isoform was detected in the subepithelial connective tissue, the endothelium of haemolymph vessels, the adipogranular and vesicular cells of reserve connective tissue, and the spermatozoa. Finally, both R_myt1 and R_myt2 coexist in some cell types but they show a different cellular localization: R_myt1 was localized in the cytoplasm whereas R_myt2 was mainly detected in the cell apical edge, cell periphery, cilia and sperm flagella. Interestingly, both R_myt1 and R_myt2 were absent in oocytes within the female gonadal follicles but, in contrast, they were highly expressed in follicle-released oocytes collected after spawning. Taken together the results show that mussel PKA isoforms are differentially distributed in the mantle cell types, which suggests that they are involved in the regulation of distinct cellular functions. On the other hand, the expression of R_myt1 and R_myt2 proteins is associated with the meiosis resumption of oocytes at the prophase I stage, which occurs in parallel to spawning.     

Halide Permeation in Wild-Type and Mutant Cystic Fibrosis Transmembrane Conductance Regulator Chloride Channels

Permeation of cystic fibrosis transmembrane conductance regulator (CFTR) Cl⁻ channels by halide ions was studied in stably transfected Chinese hamster ovary cells by using the patch clamp technique. In cell-attached patches with a high Cl⁻ pipette solution, the CFTR channel displayed outwardly rectifying currents and had a conductance near the membrane potential of 6.0 pS at 22°C or 8.7 pS at 37°C. The current–voltage relationship became linear when patches were excised into symmetrical, N-tris(hydroxymethyl)methyl-2-aminomethane sulfonate (TES)-buffered solutions. Under these conditions, conductance increased from 7.0 pS at 22°C to 10.9 pS at 37°C. The conductance at 22°C was ∼1.0 pS higher when TES and HEPES were omitted from the solution, suggesting weak, voltage-independent block by pH buffers. The relationship between conductance and Cl⁻ activity was hyperbolic and well fitted by a Michaelis-Menten–type function having a K _m of ∼38 mM and maximum conductance of 10 pS at 22°C. Dilution potentials measured with NaCl gradients indicated high anion selectivity (P_Na/P_Cl = 0.003–0.028). Biionic reversal potentials measured immediately after exposure of the cytoplasmic side to various test anions indicated P_I (1.8) > P_Br (1.3) > P_Cl (1.0) > P_F (0.17), consistent with a “weak field strength” selectivity site. The same sequence was obtained for external halides, although inward F⁻ flow was not observed. Iodide currents were protocol dependent and became blocked after 1–2 min. This coincided with a large shift in the (extrapolated) reversal potential to values indicating a greatly reduced I⁻/Cl⁻ permeability ratio (P_I/P_Cl < 0.4). The switch to low I⁻ permeability was enhanced at potentials that favored Cl⁻ entry into the pore and was not observed in the R347D mutant, which is thought to lack an anion binding site involved in multi-ion pore behavior. Interactions between Cl⁻ and I⁻ ions may influence I⁻ permeation and be responsible for the wide range of P_I/P_Cl ratios that have been reported for the CFTR channel. The low P_I/P_Cl ratio usually reported for CFTR only occurred after entry into an altered permeability state and thus may not be comparable with permeability ratios for other anions, which are obtained in the absence of iodide. We propose that CFTR displays a “weak field strength” anion selectivity sequence.     

Recombinant pICln Forms Highly Cation-selective Channels when Reconstituted into Artificial and Biological Membranes

pI_Cln has been proposed to be the swelling-activated anion channel responsible for I_{Cl, swell}, or a channel regulator. We tested the anion channel hypothesis by reconstituting recombinant pI_Cln into artificial and biological membranes. Single channels were observed when pI_Cln was reconstituted into planar lipid bilayers. In the presence of symmetrical 300 mM KCl, the channels had a high open probability and a slope conductance of 48 pS, and were outwardly rectifying. Reduction of trans KCl to 50 mM shifted the reversal potential by −31.2 ± 0.06 mV, demonstrating that the channel is at least seven times more selective for cations than for anions. Consistent with this finding, channel conductance was unaffected by substitution of Cl⁻ with glutamate, but was undetectable when K⁺ was replaced by N-methyl-d-glucamine. Reconstitution of pI_Cln into liposomes increased ⁸⁶Rb⁺ uptake by three- to fourfold, but had no effect on ³⁶Cl⁻ uptake. Phosphorylation of pI_Cln with casein kinase II or mutation of G54, G56, and G58 to alanine decreased channel open probability and ⁸⁶Rb⁺ uptake. When added to the external medium bathing Sf9 cells, pI_Cln inserted into the plasma membrane and increased cell cation permeability. Taken together, these observations demonstrate that channel activity is due to pI_Cln and not minor contaminant proteins. However, these findings do not support the hypothesis that pI_Cln is the anion-selective I_{Cl, swell} channel. The observed cation channel activity may reflect an as yet to be defined physiological function of pI_Cln, or may be a consequence of in vitro reconstitution of purified, recombinant protein.     

Phosphorylation of P-glycoprotein by PKA and PKC modulates swelling-activated Cl- currents

Several proteins belonging to the ATP-binding cassettesuperfamily can affect ion channel function. These include the cystic fibrosis transmembrane conductance regulator, the sulfonylurea receptor, and the multidrug resistance protein P-glycoprotein (MDR1).We measured whole cell swelling-activatedCl currents(I_Cl,swell) inparental cells and cells expressing wild-type MDR1 or aphosphorylation-defective mutant (Ser-661, Ser-667, and Ser-671replaced by Ala). Stimulation of protein kinase C (PKC) with a phorbolester reduced the rate of increase inI_Cl,swell only incells that express MDR1. PKC stimulation had no effect on steady-stateI_Cl,swell.Stimulation of protein kinase A (PKA) with 8-bromoadenosine3',5'-cyclic monophosphate reduced steady-state I_Cl,swell only inMDR1-expressing cells. PKA stimulation had no effect on the rate ofI_Cl,swellactivation. The effects of stimulation of PKA and PKC onI_Cl,swell wereadditive (i.e., decrease in the rate of activation and reduction insteady-stateI_Cl,swell). The effects of PKA and PKC stimulation were absent in cells expressing thephosphorylation-defective mutant. In summary, it is likely thatphosphorylation of MDR1 by PKA and by PKC alters swelling-activated Cl channels by independentmechanisms and that Ser-661, Ser-667, and Ser-671 are involved in theresponses ofI_Cl,swell tostimulation of PKA and PKC. These results support the notion that MDR1phosphorylation affectsI_Cl,swell.     

An internal ribosome entry site element directs the synthesis of the 80 kDa isoforms of protein 4.1R

Background  

In red blood cells, protein 4.1 (4.1R) is an 80 kDa protein that stabilizes the spectrin-actin network and anchors it to the plasma membrane through its FERM domain. While the expression pattern of 4.1R in mature red cells is relatively simple, a rather complex array of 4.1R protein isoforms varying in N-terminal extensions, internal sequences and subcellular locations has been identified in nucleated cells. Among these, 135 kDa and 80 kDa isoforms have different N-terminal extensions and are expressed either from AUG1- or AUG2-containing mRNAs, respectively. These two types of mRNAs, varying solely by presence/absence of 17 nucleotides (nt) which contain the AUG1 codon, are produced by alternative splicing of the 4.1R pre-mRNA. It is unknown whether the 699 nt region comprised between AUG1 and AUG2, kept as a 5' untranslated region in AUG2-containing mRNAs, plays a role on 4.1R mRNA translation.     

Spontaneous Excitation Patterns Computed for Axons with Injury-like Impairments of Sodium Channels and Na/K Pumps

In injured neurons, “leaky” voltage-gated sodium channels (Nav) underlie dysfunctional excitability that ranges from spontaneous subthreshold oscillations (STO), to ectopic (sometimes paroxysmal) excitation, to depolarizing block. In recombinant systems, mechanical injury to Nav1.6-rich membranes causes cytoplasmic Na⁺-loading and “Nav-CLS”, i.e., coupled left-(hyperpolarizing)-shift of Nav activation and availability. Metabolic injury of hippocampal neurons (epileptic discharge) results in comparable impairment: left-shifted activation and availability and hence left-shifted I_Na-window. A recent computation study revealed that CLS-based I_Na-window left-shift dissipates ion gradients and impairs excitability. Here, via dynamical analyses, we focus on sustained excitability patterns in mildly damaged nodes, in particular with more realistic Gaussian-distributed Nav-CLS to mimic “smeared” injury intensity. Since our interest is axons that might survive injury, pumps (sine qua non for live axons) are included. In some simulations, pump efficacy and system volumes are varied. Impacts of current noise inputs are also characterized. The diverse modes of spontaneous rhythmic activity evident in these scenarios are studied using bifurcation analysis. For “mild CLS injury”, a prominent feature is slow pump/leak-mediated E_Ion oscillations. These slow oscillations yield dynamic firing thresholds that underlie complex voltage STO and bursting behaviors. Thus, Nav-CLS, a biophysically justified mode of injury, in parallel with functioning pumps, robustly engenders an emergent slow process that triggers a plethora of pathological excitability patterns. This minimalist “device” could have physiological analogs. At first nodes of Ranvier and at nociceptors, e.g., localized lipid-tuning that modulated Nav midpoints could produce Nav-CLS, as could co-expression of appropriately differing Nav isoforms.