Functional role of Na+–HCO3− cotransport in migration of transformed renal epithelial cells

Cell migration is crucial for immune defence, wound healing or formation of tumour metastases. It has been shown that the activity of the Na⁺–H⁺ exchanger (NHE1) plays an important role in cell migration. However, so far it is unknown whether Na⁺– HCO₃⁻ cotransport (NBC), which has similar functions in the regulation of intracellular pH (pH_i) as NHE1, is also involved in cell migration. We therefore isolated NHE-deficient Madin-Darby canine kidney (MDCK-F) cells and tested whether NBC compensates for NHE in pH_i and cell volume regulation as well as in migration. Intracellular pH was measured with the fluorescent pH indicator 2'7'-bis(carboxyethyl)-5-carboxyfluorescein (BCECF). The expression of NBC isoforms was determined with semiquantitative PCR. Migration was monitored with time-lapse video microscopy and quantified as the displacement of the cell centre. We found that MDCK-F cells express the isoform NBC1 ( SLCA4A gene product) at a much higher level than the isoform kNBC3 ( SLCA4A8 gene product). This difference is even more pronounced in NHE-deficient cells so that NBC1 is likely to be the major acid extruder in these cells and the major mediator of propionate-induced cell volume increase. NHE-deficient MDCK-F cells migrate more slowly than normal MDCK-F cells. NBC activity promotes migration during an acute intracellular acid load and increases migratory speed and displacement on a short timescale (< 30 min) whereas it has no effect on the long-term behaviour of migrating MDCK-F cells. Taken together, our results show that NBC actvity, despite many functional similarities, does not have the same importance for cell migration as NHE1 activity.     

pH dependence of melanoma cell migration: protons extruded by NHE1 dominate protons of the bulk solution

Migration and morphology of human melanoma cells (MV3) depend on extracellular pH (pH_e) and the activity of the Na⁺/H⁺ exchanger NHE1. To distinguish effects of NHE1 activity per se from effects of pH_e we compared an NHE1-deficient mutant with rescued and wild-type cells. Time lapse video microscopy was used to investigate migratory and morphological effects caused by pH_e and NHE1 activity, and a membrane-bound fluorescein conjugate was employed for ratiometric pH measurements at the outer leaflet of the cell membrane. As long as NHE1 remained inactive due to deficiency or inhibition by cariporide (HOE642) neither migration nor morphology was affected by changes in pH_e. Under these conditions pH at the outer leaflet of the plasma membrane was uniform all over the cell surface. The typical pH dependence of MV3 cell migration and morphology could be reconstituted by restoring NHE1 activity. At the same time the proton gradient at the outer leaflet of the plasma membrane with the higher proton concentration at the leading edge and the lower one at the cell rear was re-established as well. Hence, NHE1 activity generates a proton gradient at the cell surface accompanied by the cells' ability to respond to changes in pH_e (bulk pH). We conclude that NHE1 activity contributes to the generation of a well-defined cell surface pH by creating a proton gradient at the outer leaflet of the plasma membrane that is needed for (i) the development of a variety of morphologies including a distinct polarity and (ii) migration. A missing proton gradient at the cell surface cannot be compensated for by varying pH_e.     

Molecular aspects of acute inhibition of Na+-H+ exchanger NHE3 by A2-adenosine receptor agonists

Adenosine regulates Na⁺ homeostasis by its acute effects on renal Na⁺ transport. We have shown in heterologously transfected A6/C1 cells (renal cell line from Xenopus laevis ) that adenosine-induced natriuresis may be effected partly via A₂ adenosine receptor-mediated inactivation of the renal brush border membrane Na⁺-H⁺ exchanger NHE3. In this study we utilized A6/C1 cells stably expressing wild-type as well as mutated forms of NHE3 to assess the molecular mechanism underlying A₂-dependent control of NHE3 function. Cell surface biotinylation combined with immunoprecipitation revealed that NHE3 is targeted exclusively to the apical domain and that the endogenous Xenopus NHE is located entirely on the basolateral side of A6/C1 transfectants. Stimulation of A₂-adenosine receptors located on the basolateral side for 15 min with CPA ( N 6-cyclopentyladenosine) acutely decreased NHE3 activity (microspectrofluorimety). This effect was mimicked by 8-bromo-cAMP and entirely blocked by pharmacological inhibition of PKA (with H89) or singular substitution of two PKA target sites (serine 552 and serine 605) on NHE3. Downregulation of NHE3 activity by CPA was attributable to a reduction of NHE3 intrinsic transport activity without change in surface NHE3 protein at 15 min. At 30 min, the decrease in transport activity was associated with a decrease in apical membrane NHE3 antigen. In conclusion, two highly conserved target serine sites on NHE3 determine NHE3 modulation upon A₂-receptor activation and NHE3 inactivation by adenosine proceeds via two phases with distinct mechanisms.     

pH-dependent and -independent effects inhibit Ca2+-induced Ca2+ release during metabolic blockade in rat ventricular myocytes

We have investigated the role of changes of intracellular pH (pH_i) in the effects of metabolic blockade (cyanide plus 2-deoxyglucose) on Ca²⁺ release from the sarcoplasmic reticulum (SR) in rat ventricular myocytes. pH_i and cell length were measured simultaneously. Metabolic blockade decreased the frequency of Ca²⁺ waves, an effect previously shown to be due to inhibition of Ca²⁺ release from the SR. This was accompanied by an intracellular acidification. Intracellular acidification was produced in the absence of metabolic inhibition by application of sodium butyrate. A maintained intracellular acidosis produced a decrease of wave frequency. A hysteresis between pH_i and wave frequency was observed such that during the onset of the acidification the wave frequency decreased more than in the steady state. Comparison of the steady state relationship between pH_i and wave frequency showed that the decrease of wave frequency produced by metabolic blockade was greater than could be accounted for simply by the accompanying decrease of pH_i. In other experiments the buffering power of the solution was increased. Under these conditions, metabolic blockade produced no change of pH_i but the decrease of wave frequency persisted. We conclude that, although intracellular acidification occurs during metabolic blockade, it is not responsible for most of the inhibition of Ca²⁺ release from the SR.     

Expression of α5 (CD49e) and α6 (CD49f) Integrin Subunits on T Cells in the Circulation and the Lamina Propria of Normal and Inflammatory Bowel Disease Colonic Mucosa

Intestinal lamina propria T cells are believed to be derived, via the systemic circulation, from gut-associated lymphoid tissue. After migration into the lamina propria, T cells are capable of luminally directed migration following the loss of surface epithelial cells. For adhesion and migration within the extracellular matrix, T cells are likely to utilize the integrin family of adhesion molecules. The aim of this study was to quantitatively and qualitatively investigate the expression of α₅ and α₆ integrin subunits on the surface of human T cells that: (a) migrated out of the lamina propria, (b) remained resident within the matrix and (c) were present in the circulation. In both subpopulations of CD4 and CD8-positive T cells, from both normal and inflamed (inflammatory bowel disease) colonic mucosa, there were significantly fewer α₅ and α₆-positive cells than in the peripheral blood. In addition, there were significantly fewer α₆ integrin molecules on the surface of CD4 and CD8-positive lamina propria T-cell subpopulations, compared with those in the circulation. Our studies suggest that, following migration into the lamina propria, there is down-regulation of α₅ and α₆ integrin-subunit expression on the surface of T cells. Molecules other than members of very late activation antigen-5 (VLA-5) (α₅β₁) and VLA-6 (α₆β₁) families of adhesion molecules are likely to be important in interactions with extracellular components in the lamina propria of normal and inflamed human colonic mucosa.     

Activation of Cloned Human CD4+ Th1 and Th2 Cells by Blood Dendritic Cells

Dendritic cells (DC) have been reported to be the most potent antigen-presenting cells (APC) for the activation of naive T cells and to be 10–100-fold more potent APC than monocytes (Mφ) in the mixed lymphocyte reaction. In this study the authors compared human blood DC with Mφ and B cells for their ability to activate cloned rye grass allergen Lol p I specific CD4⁺ Th₁ and Th₂ cells. In the presence of Lol  p I, all three types of APC activated Th₁ and Th₂ cells to a similar extent, as shown by T-cell proliferation and interferon-γ, interleukin-2 (IL-2) or IL-4 secretion. However, at low APC : T cell ratios, Mφ were the most potent APC for both Th₁ and Th₂ cells followed in decreasing order by DC and B cells. This hierarchy was observed with APC preparations isolated by negative selection or highly purified by positive selection using fluorescent cell sorting for HLA-DR^high-DC, CD14^pos-Mφ and CD19^pos-B cells. The data demonstrate that, in contrast to what has been reported for naive T cells, human blood DC activate cloned memory Th₁ and Th₂ cells to a similar extent as Mφ and B cells presumably because the requirements for activation of memory type T cells are less stringent than those for naive T cells.     

Control of T-cell activation by CD4+ CD25+ suppressor T cells

Summary: Depletion of the minor (∼10%) subpopulation of CD4⁺ T cells that co-expresses CD25 (interleukin (IL)-2 receptor α-chain) by thymectomy of neonates on the third day of life or by treatment of adult CD4⁺ T cells with anti-CD25 and complement results in the development of organ-specific autoimmunity. Autoimmune disease can be prevented by reconstitution of the animals with CD4⁺ CD25⁺ cells. CD4⁺ CD25⁺-mediated protection of autoimmune gastritis does not require the suppressor cytokines IL-4, IL-10, or transforming growth factor (TGF)-β. Mice that express a transgenic T-cell receptor (TCR) derived from a thymectomized newborn that recognizes the gastric parietal cell antigen H/K ATPase all develop severe autoimmune gastritis very early in life. CD4⁺ CD25⁺ T cells are also powerful suppressors of the activation of both CD4⁺ and CD8⁺ T cells in vitro . Suppression is mediated by a cell contact-dependent, cytokine-independent T–T interaction. Activation of CD4⁺ CD25⁺ via their TCR generates suppressor effector cells that are capable of non-specifically suppressing the activation of any CD4⁺ or CD8⁺ T cell. Activation of suppressor effector function is independent of co-stimulation mediated by CD28/CTLA-4 interactions with CD80/CD86. We propose that CD4⁺ CD25⁺ T cells recognize organ-specific antigens, are recruited to sites of autoimmune damage where they are activated by their target antigen, and then physically interact with autoreactive CD4⁺ or CD8⁺ effector cells to suppress the development of autoimmune disease.     

Subunit composition and role of Na+,K+-ATPases in adrenal chromaffin cells

Adrenal medullary (AM) cells are exposed to high concentrations of cortical hormones, one of which is a ouabain-like substance. Thus, the effects of ouabain on catecholamine secretion and distribution of Na⁺,K⁺-ATPase α and β subunits in rat and guinea-pig AM cells were examined using amperometry and immunological techniques. While exposure to 1 μ m ouabain did not have a marked effect on resting secretion, it induced an increase in secretion due to mobilization of Ca²⁺ ions that were stored during a 4 min interval between muscarine applications. Immunocytochemistry revealed that Na⁺,K⁺-ATPase α1 subunit-like and β3 subunit-like immunoreactive (IR) materials were distributed ubiquitously at the cell periphery, whereas α2- and β2-like IR materials were present in restricted parts of the cell periphery. The α1 and α2 subunits were mainly immunoprecipitated from AM preparations by anti-β3 and anti-β2 antisera, respectively. Peripheral BODIPY-FL-InsP₃ binding sites were localized below membrane domains with α2- and β2-like IR materials. The results indicate that in AM cells, α1β3 isozymes of Na⁺,K⁺-ATPase were present ubiquitously in the plasma membrane, while α2β2 isozymes were in the membrane domain closely associated with peripheral Ca²⁺ store sites. This close association of the α2β2 isozyme with peripheral Ca²⁺ store sites may account for the facilitation of mobilization-dependent secretion in the presence of 1 μ m ouabain.     

Aquaporin-1 and HCO3−-Cl− transporter-mediated transport of CO2 across the human erythrocyte membrane

Recent studies have suggested that aquaporin-1 (AQP1) as well as the HCO₃⁻-Cl⁻ transporter may be involved in CO₂ transport across biological membranes, but the physiological importance of this route of gas transport remained unknown. We studied CO₂ transport in human red blood cell ghosts at physiological temperatures (37 °C). Replacement of inert with CO₂-containing gas above a stirred cell suspension caused an outside-to-inside directed CO₂ gradient and generated a rapid biphasic intracellular acidification. The gradient of the acidifying gas was kept small to favour high affinity entry of CO₂ passing the membrane. All rates of acidification except that of the approach to physicochemical equilibrium of the uncatalysed reaction were restricted to the intracellular environment. Inhibition of carbonic anhydrase (CA) demonstrated that CO₂-induced acidification required the catalytic activity of CA. Blockade of the function of either AQP1 (by HgCl₂ at 65 μM) or the HCO₃⁻-Cl⁻ transporter (by DIDS at 15 μM) completely prevented fast acidification. These data indicate that, at low chemical gradients for CO₂, nearly the entire CO₂ transport across the red cell membrane is mediated by AQP1 and the HCO₃⁻-Cl⁻ transporter. Therefore, these proteins may function as high affinity sites for CO₂ transport across the erythrocyte membrane.     

Expression of Integrin αvβ3 in Gliomas Correlates with Tumor Grade and Is not Restricted to Tumor Vasculature

In malignant gliomas, the integrin adhesion receptors seem to play a key role for invasive growth and angiogenesis. However, there is still a controversy about the expression and the distribution of α_vβ₃ integrin caused by malignancy. The aim of our study was to assess the extent and pattern of α_vβ₃ integrin expression within primary glioblastomas (GBMs) compared with low-grade gliomas (LGGs). Tumor samples were immunostained for the detection of α_vβ₃ integrin and quantified by an imaging software. The expression of α_vβ₃ was found to be significantly higher in GBMs than in LGGs, whereby focal strong reactivity was restricted to GBMs only. Subsequent analysis revealed that not only endothelial cells but also, to a large extent, glial tumor cells contribute to the overall amount of α_vβ₃ integrin in the tumors. To further analyze the integrin subunits, Western blots from histologic sections were performed, which demonstrated a significant difference in the expression of the β₃ integrin subunit between GBMs and LGGs. The presented data lead to new insights in the pattern of α_vβ₃ integrin in gliomas and are of relevance for the inhibition of α_vβ₃ integrin with specific RGD peptides and interfering drugs to reduce angiogenesis and tumor growth.     

Inhibition of HERG K+ Current and Prolongation of the Guinea-Pig Ventricular Action Potential by 4-Aminopyridine

4-Aminopyridine (4-AP) has been used extensively to study transient outward K⁺ current ( I _TO,1) in cardiac cells and tissues. We report here inhibition by 4-AP of HERG (the human ether-à-go-go -related gene) K⁺ channels expressed in a mammalian cell line, at concentrations relevant to those used to study I _TO,1. Under voltage clamp, whole cell HERG current ( I _HERG) tails following commands to +30 mV were blocked with an IC₅₀ of 4.4 ± 0.5 m m . Development of block was contingent upon HERG channel gating, with a preference for activated over inactivated channels. Treatment with 5 m m 4-AP inhibited peak I _HERG during an applied action potential clamp waveform by ∼59 %. It also significantly prolonged action potentials and inhibited resurgent I _K tails from guinea-pig isolated ventricular myocytes, which lack an I _TO,1. We conclude that by blocking the α-subunit of the I _Kr channel, millimolar concentrations of 4-AP can modulate ventricular repolarisation independently of any action on I _TO,1.     

Migration and differentiation of CD8+ T cells

Summary: Antigen-specific responses by CD8⁺ T cells require direct cell–cell interactions between T cells and antigen-presenting cells (APC). Initially, naïve T cells must communicate with APC in lymphoid organs. Once stimulated, the resulting effector cells interact with APC in peripheral tissues. To this end, T cells must migrate to discrete sites throughout the body where antigen may be found. Recent progress in the field has revealed that the migratory abilities of T cells are critically dependent on their differentiation state, which is shaped by a multitude of factors. Thus, naïve T cells are normally restricted to recirculate between the blood and secondary lymphoid tissues, although in some autoimmune diseases they may also accumulate in chronically inflamed tissues. When CD8⁺ T cells encounter antigen and differentiate into short-lived effector CTL, they lose the ability to home to lymph nodes but gain access to peripheral tissues and sites of inflammation. Long-lived memory cells exist in (at least) two flavors: central memory cells that migrate to both lymphoid organs and peripheral sites of inflammation, and effector memory cells that are preferentially localized in non-lymphoid tissues. Our current understanding of the interplay of T cell differentiation and migration has been boosted by the development of T-GFP mice, in which transgenic green fluorescent protein is expressed selectively in naïve and central memory T cells, but not in effector cytotoxic T cells (CTL). This review will focus on recent studies in which T-GFP mice were used to dissect the traffic signals for naïve T cell homing to secondary lymphoid organs, the factors that influence the differentiation of naïve CD8⁺ T cells into cytotoxic and memory cells, as well as the in vivo trafficking routes of antigen-experienced subsets.     

Establishment of cell polarity by afadin during the formation of embryoid bodies

Afadin directly links nectin, an immunoglobulin-like cell–cell adhesion molecule, to actin filaments (F-actin) at adherens junctions (AJs). The nectin–afadin complex is important for the formation of not only AJs but also tight junctions (TJs) in epithelial cells. Studies using afadin-knockout mice have revealed that afadin is indispensable for embryonic development by organizing the formation of cell–cell junctions. However, the molecular mechanism of cell–cell junction disorganization during embryonic development in afadin-knockout mice is poorly understood. To address this, we took advantage of embryoid bodies (EBs) as a model system. The formation of cell–cell junctions including AJs and TJs was impaired in afadin-null EBs. The proper accumulation of the Par complex and the activation of Cdc42 and atypical PKC (aPKC), which are crucial for the formation of cell polarity, were also inhibited by knockout of afadin. In addition, the disruption of afadin caused the abnormal deposition of laminin and the dislocalization of its receptors integrin α₆ and integrin β₁. These results indicate that afadin organizes the formation of cell–cell junctions by regulating cell polarization in early embryonic development.     

Characterization of Monoclonal Antibodies to the αIIbβ3 Integrin on Bovine Platelets

A set of monoclonal antibodies (MoAbs) to leucocyte antigens is an essential tool to identify different cell types and functional membrane molecules involved in immune responses. Since no MoAbs existed to bovine integrins, except against the β₂ subfamily, we generated MoAbs to β₃ integrin after the immunization of mice with bovine platelets. Two MoAbs, IL-A164 (IgG_2a) and IL-A166 (IgG₁), were selected that reacted specifically with bovine platelets and detected the same membrane molecule. The antigen was a heterodimer of two polypeptide chains of 122 kDa and 95 kDa as resolved by SDS-PAGE under reducing conditions. Although the Mr of the smaller subunit is identical to that of β₂ integrin, pre-absorption with an antibody to β₂ (or CD18) did not remove the bovine antigen. Comparing the molecular masses of the two subunits in reduced and non-reduced forms showed a pattern that was similar to that of human GPIIb/IIIa (also called α_IIbβ₃ or CD41a). Reduction of the bovine molecule increased the apparent Mr of the light chain from 76 kDa to 95 kDa, while the heavy subunit changed from 136 kDa to 122 kDa. As with human GPIIb, the decrease in Mr of the α-subunit is probably a result of a small disulphide-linked polypeptide, although no additional evidence for this was detected for the bovine integrin. Sequencing of the N-terminal amino acids of both bovine polypeptides showed identity of the bovine integrin with human GPIIb/IIIa.     

Selection during development of VH11+ B cells: a model for natural autoantibody-producing CD5+ B cells

Summary:  Natural autoantibodies constitute a large portion of serum immunoglobulin M (IgM) and bridge the adaptive and innate immune systems, serving as a rapid response to common pathogens. Many arise from a distinctive subset of B cells, termed B-1, that express CD5. Here, we describe our studies with a representative CD5⁺ B-cell-derived natural autoantibody, the V_H11V_κ9 B-cell receptor (BCR) that binds a determinant on senescent erythrocytes. This specificity represents 5–10% of the CD5⁺ B-cell subset, with a large portion accounted for by two novel BCRs, V_H11V_κ9 and V_H12V_κ4. We have found that the development of B-lineage cells with a V_H11 rearrangement is surprisingly restricted at several crucial bottlenecks: (i) one of the most common V_H11 rearrangements generates a heavy-chain protein that only inefficiently assembles a pre-BCR, key for recombinase-activating gene downregulation/allelic exclusion and pre-B-clonal expansion; (ii) cells containing V_H11-µ chains lacking N-addition are favored for progression to the B-cell stage, eliminating most bone marrow V_H11 rearrangements; and (iii) only a subset of V_κ-light chains combine with V_H11 heavy chain to foster progression to the mature B-cell stage. Together, these constrain V_H11 generation to fetal development and may favor production of B cells with the prototype V_H11V_κ9 BCR.     

Molecular basis of functional voltage-gated K+ channel diversity in the mammalian myocardium

In the mammalian heart, Ca²⁺-independent, depolarization-activated potassium (K⁺) currents contribute importantly to shaping the waveforms of action potentials, and several distinct types of voltage-gated K⁺ currents that subserve this role have been characterized. In most cardiac cells, transient outward currents, I _to,f and/or I _to,s, and several components of delayed reactivation, including I _Kr, I _Ks, I _Kur and I _K,slow, are expressed. Nevertheless, there are species, as well as cell-type and regional, differences in the expression patterns of these currents, and these differences are manifested as variations in action potential waveforms. A large number of voltage-gated K⁺ channel pore-forming (α) and accessory (β, minK, MiRP) subunits have been cloned from or shown to be expressed in heart, and a variety of experimental approaches are being exploited in vitro and in vivo to define the relationship(s) between these subunits and functional voltage-gated cardiac K⁺ channels. Considerable progress has been made in defining these relationships recently, and it is now clear that distinct molecular entities underlie the various electrophysiologically distinct repolarizing K⁺ currents (i.e. I _to,f, I _to,s, I _Kr, I _Ks, I _Kur, I _K,slow, etc.) in myocyardial cells.     

Ether-à-go-go-related gene K+ channels contribute to threshold excitability of mouse auditory brainstem neurons

The ionic basis of excitability requires identification and characterisation of expressed channels and their specific roles in native neurons. We have exploited principal neurons of the medial nucleus of the trapezoid body (MNTB) as a model system for examining voltage-gated K⁺ channels, because of their known function and simple morphology. Here we show that channels of the ether-à-go-go -related gene family (ERG, Kv11; encoded by kcnh ) complement Kv1 channels in regulating neuronal excitability around threshold voltages. Using whole-cell patch clamp from brainstem slices, the selective ERG antagonist E-4031 reduced action potential (AP) threshold and increased firing on depolarisation. In P12 mice, under voltage-clamp with elevated [K⁺]_o (20 m m ), a slowly deactivating current was blocked by E-4031 or terfenadine ( V _0.5,act=−58.4 ± 0.9 mV, V _0.5,inact=−76.1 ± 3.6 mV). Deactivation followed a double exponential time course (τ_slow= 113.8 ± 6.9 ms, τ_fast= 33.2 ± 3.8 ms at −110 mV, τ_fast 46% peak amplitude). In P25 mice, deactivation was best fitted by a single exponential (τ_fast= 46.8 ± 5.8 ms at −110 mV). Quantitative RT-PCR showed that ERG1 and ERG3 were the predominant mRNAs and immunohistochemistry showed expression as somatic plasma membrane puncta on principal neurons. We conclude that ERG currents complement Kv1 currents in limiting AP firing at around threshold; ERG may have a particular role during periods of high activity when [K⁺]_o is elevated. These ERG currents suggest a potential link between auditory hyperexcitability and acoustic startle triggering of cardiac events in familial LQT2.     

NHE3 inhibition by cAMP and Ca2+ is abolished in PDZ-domain protein PDZK1-deficient murine enterocytes

The PDZ-binding protein PDZK1 (NHERF3/CAP70/PDZ-dc-1) in vitro binds to NHE3, but its role in the regulation of NHE3 activity in native enterocytes is unknown. This study was undertaken to understand the physiological role of PDZK1 in regulating NHE3 activity in native murine colonic enterocytes. NHE3 transport rates were assessed fluorometrically in BCECF-loaded colonic crypts in the NHE3-expressing cryptal openings by measuring acid-activated, Na⁺-dependent, Hoe 642-insensitive proton efflux rates. NHE3 mRNA expression levels and NHE3 total enterocyte and brush border membrane (BBM) protein abundance were determined by quantitative PCR and Western analysis and immunohistochemistry. In pdzk1 −/− colonic surface cells, acid-activated NHE3 transport rates were strongly reduced, and the inhibitory effect of forskolin and ionomcyin was virtually abolished. Hyperosmolarity, on the other hand, still had an inhibitory effect. In addition, the NHE3-selective inhibitor S1611 inhibited acid-activated NHE3 activity in pdzk1 −/− and +/+ mice, suggesting that functional NHE3 is present in pdzk1 -deficient colonocytes. NHE1 and NHE2 activity was not altered in pdzk1 −/− colonic crypts. Immunohistochemistry revealed apical NHE3 staining in pdzk1 −/− and +/+ proximal colon, and Western blot analysis revealed no difference in NHE3 abundance in colonic enterocyte homogenate as well as brush border membrane. Lack of the PDZ-adaptor protein PDZK1 in murine proximal colonic enterocytes does not influence NHE3 abundance or targeting to the apical membrane, but abolishes NHE3 regulation by cAMPergic and Ca²⁺ -dependent pathways. It leaves NHE3 inhibition by hyperosmolarity intact, suggesting an important and selective role for PDZK1 in the agonist-mediated regulation of intestinal NHE3 activity.     

Modulation of Ca2+-activated K+ currents and Ca2+-dependent action potentials by exocytosis in goldfish bipolar cell terminals

Retinal bipolar cells convey light-evoked potentials from photoreceptors to ganglion cells and mediate the initial stages of visual signal processing. They do not fire Na⁺-dependent action potentials (APs) but the Mb1 class of goldfish bipolar cell exhibits Ca²⁺-dependent APs and regenerative potentials that originate in the axon terminal. I have examined the properties of Ca²⁺-dependent APs in isolated bipolar-cell terminals in goldfish retinal slices. All recorded terminals fired spontaneous or evoked APs at frequencies of up to 15 Hz. When an AP waveform was used as a voltage stimulus, exocytosis was evoked by single APs, maintained throughout AP trains and modulated by AP frequency. Furthermore, feedback inhibition of the Ca²⁺ current ( I _Ca) by released vesicular protons reduced depression of exocytosis during AP trains. In the absence of K⁺ current inhibition, step depolarizations and AP waveforms evoked a rapidly activated outward current that was dependent on Ca²⁺ influx ( I _K(Ca)). I therefore investigated whether proton-mediated feedback inhibition of I _Ca affected the activation of I _K(Ca). A transient inhibition of I _K(Ca) was observed that was dependent on exocytosis, blocked by high-pH extracellular buffer, of similar magnitude to inhibition of I _Ca but occurred with a delay of 2.7 ms. In addition, the amplitude of APs evoked under current clamp was inhibited by the action of vesicular protons released by the APs. Protons released via exocytosis may therefore be a significant modulator of Ca²⁺-dependent currents and regenerative potentials in bipolar-cell terminals.     

A Ca2+-activated Cl− conductance in interstitial cells of Cajal linked to slow wave currents and pacemaker activity

Interstitial cells of Cajal (ICC) are unique cells that generate electrical pacemaker activity in gastrointestinal (GI) muscles. Many previous studies have attempted to characterize the conductances responsible for pacemaker current and slow waves in the GI tract, but the precise mechanism of electrical rhythmicity is still debated. We used a new transgenic mouse with a bright green fluorescent protein (copGFP) constitutively expressed in ICC to facilitate study of these cells in mixed cell dispersions. We found that ICC express a specialized 'slow wave' current. Reversal of tail current analysis showed this current was due to a Cl⁻ selective conductance. ICC express ANO1, a Ca²⁺-activated Cl⁻ channel. Slow wave currents are not voltage dependent, but a secondary voltage-dependent process underlies activation of these currents. Removal of extracellular Ca²⁺, replacement of Ca²⁺ with Ba²⁺, or extracellular Ni²⁺ (30 μ m ) blocked the slow wave current. Single Ca²⁺-activated Cl⁻ channels with a unitary conductance of 7.8 pS were resolved in excised patches of ICC. These are similar in conductance to ANO1 channels (8 pS) expressed in HEK293 cells. Slow wave current was blocked in a concentration-dependent manner by niflumic acid (IC₅₀= 4.8 μ m ). Slow wave currents are associated with transient depolarizations of ICC in current clamp, and these events were blocked by niflumic acid. These findings demonstrate a role for a Ca²⁺-activated Cl⁻ conductance in slow wave current in ICC and are consistent with the idea that ANO1 participates in pacemaker activity.