Connexin46 mutations linked to congenital cataract show loss of gap junction channel function

Human connexin46 (hCx46) forms gapjunctional channels interconnecting lens fiber cells and appears to becritical for normal lens function, because hCx46 mutations have beenlinked to congenital cataracts. We studied two hCx46 mutants, N63S, amissense mutation in the first extracellular domain, and fs380, aframe-shift mutation that shifts the translational reading frame atamino acid residue 380. We expressed wild-type Cx46 and the two mutantsin Xenopus oocytes. Production of the expressed proteins wasverified by SDS-PAGE after metabolic labeling with[³⁵S]methionine or by immunoblotting. Dualtwo-microelectrode voltage-clamp studies showed that hCx46 formed bothgap junctional channels in paired Xenopus oocytes andhemi-gap junctional channels in single oocytes. In contrast, neither ofthe two cataract-associated hCx46 mutants could form intercellularchannels in paired Xenopus oocytes. The hCx46 mutants werealso impaired in their ability to form hemi-gap-junctional channels.When N63S or fs380 was coexpressed with wild-type connexins, bothmutations acted like "loss of function" rather than "dominantnegative" mutations, because they did not affect the gap junctionalconductance induced by either wild-type hCx46 or wild-type hCx50.

     

Loss of function and impaired degradation of a cataract-associated mutant connexin50

A mutant human connexin50 (hCx50), hCx50P88S, has been linked to cataracts inherited as an autosomal dominant trait. The functional, biochemical and cellular behavior of wild-type and mutant hCx50 were examined in transfected cells. hCx50P88S was unable to induce gap junctional currents by itself, and it abolished gap junctional currents when co-expressed with wild-type (wt) hCx50. Cells transfected with hCx50P88S showed cytoplasmic accumulations of Cx50 immunoreactivity in addition to staining at appositional membranes; these accumulations did not significantly co-localize with markers for the endoplasmic reticulum, Golgi apparatus, lysosomes, endosomes or vimentin filaments. Immunoelectron microscopy studies localized hCx50P88S to cytoplasmic membrane stacks in close vicinity to the endoplasmic reticulum. In contrast, aggresome-like accumulations were induced by treatment of wt hCx50-transfected cells with proteasomal inhibitors. The formation of hCx50P88S accumulations in transiently transfected cells was not blocked by treatment with Brefeldin A suggesting that they form before Cx50 transits through the Golgi apparatus to the plasma membrane. Treatment of HeLa-hCx50P88S cells with cycloheximide demonstrated the presence of a very stable pool of hCx50P88S. Taken together, these results suggest that the P to S mutation at amino acid residue 88 causes a defect that leads to decreased degradation and subsequent accumulation of hCx50P88S in a cellular structure different from aggresomes.     

Comparison of intestinal folate carrier clone expressed in IEC-6 cells and in Xenopus oocytes

We recently identified a cDNA clone frommouse small intestine, which appears to be involved in folate transportwhen expressed in Xenopus oocytes. Theopen reading frame of this clone is identical to that of the reducedfolate carrier (RFC) (K. H. Dixon, B. C. Lanpher, J. Chiu, K. Kelley,and K. H. Cowan. J. Biol. Chem. 269: 17-20,1994). The characteristics of this cDNA clone [previously referred toas intestinal folate carrier 1 (IFC-1)] expressed inXenopus oocytes, however, were foundto be different from the characteristics of folate transport in nativesmall intestinal epithelial cells. To further study these differences,we determined the characteristics of RFC when expressed in anintestinal epithelial cell line, IEC-6, and compared the findings toits characteristics when expressed inXenopus oocytes. RFC was stablytransfected into IEC-6 cells by electroporation; its cRNA wasmicroinjected into Xenopus oocytes.Northern blot analysis of poly(A)⁺RNA from IEC-6 cells stably transfected with RFC cDNA (IEC-6/RFC) showed a twofold increase in RFC mRNA levels over controls. Similarly, uptake of folic acid and 5-methyltetrahydrofolate (5-MTHF) by IEC-6/RFCwas found to be fourfold higher than uptake in control sublines. Thisincrease in folic acid and 5-MTHF uptake was inhibited by treatingIEC-6/RFC cells with cholesterol-modified antisense DNAoligonucleotides. The increase in uptake was found to be mainly mediated through an increase in the maximal velocity(V_max) of theuptake process [the apparent Michaelis-Menten constant(K_m) alsochanged (range was 0.31 to 1.56 µM), but no specific trend wasseen]. In both IEC-6/RFC and control sublines, the uptake of bothfolic acid and 5-MTHF displayed 1)pH dependency, with a higher uptake at acidic pH 5.5 compared with pH7.5, and 2) inhibition to the sameextent by both reduced and oxidized folate derivatives. Thesecharacteristics are very similar to those seen in native intestinalepithelial cells. In contrast, RFC expressed inXenopus oocytes showed1) higher uptake at neutral andalkaline pH 7.5 compared with acidic pH 5.5 and2) higher sensitivity to reducedcompared with oxidized folate derivatives. Results of these studiesdemonstrate that the characteristics of RFC vary depending on the cellsystem in which it is expressed. Furthermore, the results may suggestthe involvement of cell- or tissue-specific posttranslationalmodification(s) and/or the existence of an auxiliary proteinthat may account for the differences in the characteristics of theintestinal RFC when expressed inXenopus oocytes compared with whenexpressed in intestinal epithelial cells.

     

Differential phosphorylation of the gap junction protein connexin43 in junctional communication-competent and -deficient cell lines

Connexin43 is a member of the highly homologous connexin family of gap junction proteins. We have studied how connexin monomers are assembled into functional gap junction plaques by examining the biosynthesis of connexin43 in cell types that differ greatly in their ability to form functional gap junctions. Using a combination of metabolic radiolabeling and immunoprecipitation, we have shown that connexin43 is synthesized in gap junctional communication-competent cells as a 42-kD protein that is efficiently converted to a approximately 46-kD species (connexin43-P2) by the posttranslational addition of phosphate. Surprisingly, certain cell lines severely deficient in gap junctional communication and known cell-cell adhesion molecules (S180 and L929 cells) also expressed 42-kD connexin43. Connexin43 in these communication-deficient cell lines was not, however, phosphorylated to the P2 form. Conversion of S180 cells to a communication-competent phenotype by transfection with a cDNA encoding the cell-cell adhesion molecule L-CAM induced phosphorylation of connexin43 to the P2 form; conversely, blocking junctional communication in ordinarily communication-competent cells inhibited connexin43-P2 formation. Immunohistochemical localization studies indicated that only communication-competent cells accumulated connexin43 in visible gap junction plaques. Together, these results establish a strong correlation between the ability of cells to process connexin43 to the P2 form and to produce functional gap junctions. Connexin43 phosphorylation may therefore play a functional role in gap junction assembly and/or activity.     

Different consequences of cataract-associated mutations at adjacent positions in the first extracellular boundary of connexin50

Gap junction channels, which are made of connexins, are critical for intercellular communication, a function that may be disrupted in a variety of diseases. We studied the consequences of two cataract-associated mutations at adjacent positions at the first extracellular boundary in human connexin50 (Cx50), W45S and G46V. Both of these mutants formed gap junctional plaques when they were expressed in HeLa cells, suggesting that they trafficked to the plasma membrane properly. However, their functional properties differed. Dual two-microelectrode voltage-clamp studies showed that W45S did not form functional intercellular channels in paired Xenopus oocytes or hemichannel currents in single oocytes. When W45S was coexpressed with wild-type Cx50, the mutant acted as a dominant negative inhibitor of wild-type function. In contrast, G46V formed both functional gap junctional channels and hemichannels. G46V exhibited greatly enhanced currents compared with wild-type Cx50 in the presence of physiological calcium concentrations. This increase in hemichannel activity persisted when G46V was coexpressed with wild-type lens connexins, consistent with a dominant gain of hemichannel function for G46V. These data suggest that although these two mutations are in adjacent amino acids, they have very different effects on connexin function and cause disease by different mechanisms: W45S inhibits gap junctional channel function; G46V reduces cell viability by forming open hemichannels.     

The Clouston syndrome mutation connexin30 A88V leads to hyperproliferation of sebaceous glands and hearing impairments in mice

Distinct mutations in the gap junction protein connexin30 (Cx30) can cause the ectodermal dysplasia Clouston syndrome in humans. We have generated a new mouse line expressing the Clouston syndrome mutation Cx30A88V under the control of the endogenous Cx30 promoter. Our results show that the mutated Cx30A88V protein is incorporated in gap junctional plaques of the epidermis. Homozygous Cx30A88V mice reveal hyperproliferative and enlarged sebaceous glands as well as a mild palmoplantar hyperkeratosis. Additionally, homozygous mutant mice show an altered hearing profile compared to control mice. We conclude that the Cx30A88V mutation triggers hyperproliferation in the skin and changes the cochlear homeostasis in mice.     

Asymmetry in the Osmotic Response of a Rat Cortical Collecting Duct Cell Line: Role of Aquaporin-2

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.     

Diverse gap junctions modulate distinct mechanisms for fiber cell formation during lens development and cataractogenesis

Different mutations of alpha3 connexin (Cx46 or Gja8) and alpha8 connexin (Cx50 or Gja8), subunits of lens gap junction channels, cause a variety of cataracts via unknown mechanisms. We identified a dominant cataractous mouse line (L1), caused by a missense alpha8 connexin mutation that resulted in the expression of alpha8-S50P mutant proteins. Histology studies showed that primary lens fiber cells failed to fully elongate in heterozygous alpha8(S50P/+) embryonic lenses, but not in homozygous alpha8(S50P/S50P), alpha8-/- and alpha3-/- alpha8-/- mutant embryonic lenses. We hypothesized that alpha8-S50P mutant subunits interacted with wild-type alpha3 or alpha8, or with both subunits to affect fiber cell formation. We found that the combination of mutant alpha8-S50P and wild-type alpha8 subunits specifically inhibited the elongation of primary fiber cells, while the combination of alpha8-S50P and wild-type alpha3 subunits disrupted the formation of secondary fiber cells. Thus, this work provides the first in vivo evidence that distinct mechanisms, modulated by diverse gap junctions, control the formation of primary and secondary fiber cells during lens development. This explains why and how different connexin mutations lead to a variety of cataracts. The principle of this explanation can also be applied to mutations of other connexin isoforms that cause different diseases in other organs.     

Inversion of both gating polarity and CO2 sensitivity of voltage gating with D3N mutation of Cx50

The effect of CO₂-induced acidification on transjunctional voltage (V_j) gating was studied by dual voltage-clamp in oocytes expressing mouse connexin 50 (Cx50) or a Cx50 mutant (Cx50-D3N), in which the third residue, aspartate (D), was mutated to asparagine (N). This mutation inverted the gating polarity of Cx50 from positive to negative. CO₂ application greatly decreased the V_j sensitivity of Cx50 channels, and increased that of Cx50-D3N channels. CO₂ also affected the kinetics of V_j dependent inactivation of junctional current (I_j), decreasing the gating speed of Cx50 channels and increasing that of Cx50-D3N channels. In addition, the D3N mutation increased the CO₂ sensitivity of chemical gating such that even CO₂ concentrations as low as 2.5% significantly lowered junctional conductance (G_j). With Cx50 channels G_j dropped by 78% with a drop in intracellular pH (pH_i) to 6.83, whereas with Cx50-D3N channels G_j dropped by 95% with a drop in pH_i to just 7.19. We have previously hypothesized that the way in which V_j gating reacts to CO₂ might be related to connexin’s gating polarity. This hypothesis is confirmed here by evidence that the D3N mutation inverts the gating polarity as well as the effect of CO₂ on V_j gating sensitivity and speed. cell communication; lens; gap junctions; chemical gating; channel gating; Xenopus oocytes     

Patch-clamp study reveals that the importance of connexin43-mediated gap junctional communication for ovarian folliculogenesis is strain specific in the mouse

Genetic ablation of connexin37 (Cx37) or connexin43 (Cx43), the two gap junction proteins expressed by mouse ovarian granulosa cells, has been shown to result in impaired follicle development. We used patch-clamp techniques to evaluate quantitatively the contribution of these connexins to gap junctional intercellular communication (GJIC) among granulosa cells. The coupling conductance derived from a voltage step-induced capacitive current transient was used as a measure of GJIC in cultured granulosa cells. Using this method, we determined that the conductance of wild-type (84.1 ± 28.6 nS; n = 6) and Cx37-deficient granulosa cells (83.7 ± 6.4 nS; n = 11) does not differ significantly (P = 0.35), suggesting a limited contribution, if any, of Cx37 to granulosa cell coupling. In contrast, the conductance between granulosa cells of Cx43-deficient mice (2.6 ± 0.8 nS; n = 5) was not significantly different from that of single, isolated wild-type granulosa cells (2.5 ± 0.7 nS, n = 5; P = 0.83), indicating that Cx43-deficient granulosa cells were not electrically coupled. A direct measurement of transjunctional conductance between isolated granulosa cell pairs using a dual patch-clamp technique confirmed this conclusion. Interestingly, a partial rescue of folliculogenesis was observed when the Cx43-null mutation in C57BL/6 mice was crossed into the CD1 strain, and capacitive current measurement demonstrated that this rescue was not due to reestablishment of GJIC. These results demonstrate that folliculogenesis is impaired in the absence of GJIC between granulosa cells, but they also indicate that the severity is dependent on genetic background, a phenomenon that cannot be attributed to the expression of additional connexins. ovarian follicle; oogenesis; connexin37; intercellular communication     

Cloning and characterization of a type III Na-dependent phosphate cotransporter from mouse intestine

Intestinal and renalabsorption of inorganic phosphate (P_i) is critical forphosphate homeostasis in mammals. We have isolated a cDNA that encodesa type III Na-dependent phosphate cotransporter from mouse smallintestine (mPit-2). The nucleotide sequence of mPit-2 predicts aprotein of 653 amino acids with at least 10 putative transmembranedomains. Kinetic studies, carried out in Xenopus oocytes,showed that mPit-2 cRNA induces significant Na-dependent P_iuptake with an apparent Michaelis constant (K_m)for phosphate of 38 µM. The transport of phosphate by mPit-2 isinhibited at high pH. Northern blot analysis demonstrated the presenceof mPit-2 mRNA in various tissues, including intestine, kidney, heart,liver, brain, testis, and skin. The highest expression of mPit-2 in the intestine was found in the jejunum. In situ hybridization revealed thatmPit-2 mRNA is expressed throughout the vertical crypt-villus axis ofthe intestinal epithelium. The presence of mPit-2 in the mouseintestine and its unique transport characteristics suggest thatmultiple Na-dependent cotransporters may contribute to phosphate absorption in the mammalian small intestine.

     

Aberrant hemichannel properties of Cx26 mutations causing skin disease and deafness

Mutations in the human GJB2 gene, which encodes connexin26 (Cx26), underlie various forms of hereditary deafness and skin disease. While it has proven difficult to discern the exact pathological mechanisms that cause these disorders, studies have shown that the loss or abnormal function of Cx26 protein has a profound effect on tissue homeostasis. Here, we used the Xenopus oocyte expression system to examine the functional characteristics of a Cx26 mutation (G45E) that results in keratitis-ichthyosis-deafness syndrome (KIDS) with a fatal outcome. Our data showed that oocytes were able to express both wild-type Cx26 and its G45E variant, each of which formed hemichannels and gap junction channels. However, Cx26-G45E hemichannels displayed significantly greater whole cell currents than wild-type Cx26, leading to cell lysis and death. This severe phenotype could be rescued in the presence of elevated Ca²⁺ levels in the extracellular milieu. Cx26-G45E could also form intercellular channels with a similar efficiency as wild-type Cx26, however, with increased voltage sensitive gating. We also compared Cx26-G45E with a previously described Cx26 mutant, A40V, which has an overlapping human phenotype. We found that both dominant Cx26 mutants elicited similar functional consequences and that cells coexpressing mutant and wild-type connexins predominantly displayed mutant-like behavior. These data suggest that mutant hemichannels may act on cellular homeostasis in a manner that can be detrimental to the tissues in which they are expressed. connexin     

Human connexin26 (GJB2) deafness mutations affect the function of gap junction channels at different levels of protein expression.

Mutations in the connexin26 (GJB2) gene account for about half of inherited non-syndromic deafness cases in Western countries. The connexin26 protein is a subunit of gap junctions that form a network of intercellular communication among supporting cells and fibrocytes in the mammalian inner ear. Here we describe functional implications of mutations in the coding region of connexin26 genes (M1V, M34T, L90P, R127H, F161S, P173R, and R184P), identified in patients and stably transfected in human HeLa cells. While all mutated connexin26 cDNAs were transcribed, only M34T, L90P, R127H, F161S, and R184P were translated in HeLa cells. Analysis of indirect immunofluorescence showed membranous localization, strong for M34T, L90P, R127H, and very weak for F161S, but no signal corresponding to M1V, P173R and R184P. Tracer coupling experiments revealed diffusion of microinjected neurobiotin into neighbouring cells in the case of M34T and R127H, whereas M1V, L90P, F161S, P173R and R184P mutants did not show intercellular coupling. The results of oligomerisation studies suggested a partly disturbed assembly of hemichannels in M34T and L90P mutants but complete absence of hemichannel formation in the R184P mutant. The R127H mutation did not affect channel formation and is likely to represent a polymorphism. Our results show that mutations in the connexin26 gene can affect gap junctional intercellular communication at the level of protein translation, trafficking or assembly of hemichannels.     

Functional Role of the Carboxyl Terminal Domain of Human Connexin 50 in Gap Junctional Channels

Gap junction channels formed by connexin 50 (Cx50) are critical for maintenance of lens transparency. Because the C-terminus of Cx50 can be cleaved post-translationally, we hypothesized that channels formed by the truncated Cx50 exhibit altered properties or regulation. We used the dual whole-cell patch-clamp technique to investigate the macroscopic and single-channel properties of gap junctional channels formed by wild-type human Cx50 and a truncation mutant (Cx50A294stop) after transfection of N2A cells. Our results show that wild-type Cx50 formed functional gap junctional channels. The macroscopic Gjss-Vj relationship was well described by a Boltzmann equation with A of 0.10, V0 of 43.8 mV and Gjmin of 0.23. The single-channel conductance was 212 +/- 5 pS. Multiple long-lasting substates were observed with conductances ranging between 31 and 80 pS. Wild-type Cx50 gap junctional channels were reversibly blocked when pHi was reduced to 6.3. Truncating the C-terminus at amino acid 294 caused a loss of pHi sensitivity, but there were no significant changes in single-channel current amplitude or Gjss-Vj relationship. These results suggest that the C-terminus of human Cx50 is involved in pHi sensitivity, but has little influence over single-channel conductance, voltage dependence, or gating kinetics.     

Effect of PCMBS on CO2 permeability of Xenopus oocytes expressing aquaporin 1or its C189S mutant

A recent study onXenopus oocytes [N. L. Nakhoul,M. F. Romero, B. A. Davis, and W. F. Boron. Am. J. Physiol. 274 (CellPhysiol. 43): C543-548, 1998] injected withcarbonic anhydrase showed that expressing aquaporin 1 (AQP1) increasesby ~40% the rate at which exposing the cell toCO₂ causes intracellular pH tofall. This observation is consistent with several interpretations.Overexpressing AQP1 might increase apparentCO₂ permeability by1) allowingCO₂ to pass through AQP1,2) stimulating injected carbonicanhydrase, 3) enhancing theCO₂ solubility of the membrane'slipid, or 4) increasing theexpression of a native "gas channel." The purpose of the presentstudy was to distinguish among these possibilities. We found thatexpressing the H₂O channel AQP1 inXenopus oocytes increases theCO₂ permeability of oocytes in anexpression-dependent fashion, whereas expressing theK⁺ channel ROMK1 has no effect.The mercury derivativep-chloromercuriphenylsulfonic acid(PCMBS), which inhibits the H₂Omovement through AQP1, also blocks the AQP1-dependent increase inCO₂ permeability. Themercury-insensitive C189S mutant of AQP1 increases theCO₂ permeability of the oocyte tothe same extent as does the wild-type channel. However, the C189S-dependent increase in CO₂permeability is unaffected by treatment with PCMBS. These data rule outoptions 2-4 listed above. Thusour results suggest that CO₂passes through the pore of AQP1 and are the first data to demonstratethat a gas can enter a cell by a means other than diffusing through themembrane lipid.

     

Biochemical analysis of connexin43 intracellular transport, phosphorylation, and assembly into gap junctional plaques

We previously demonstrated that the gap junction protein connexin43 is translated as a 42-kD protein (connexin43-NP) that is efficiently phosphorylated to a 46,000-Mr species (connexin43-P2) in gap junctional communication-competent, but not in communication-deficient, cells. In this study, we used a combination of metabolic radiolabeling and immunoprecipitation to investigate the assembly of connexin43 into gap junctions and the relationship of this event to phosphorylation of connexin43. Examination of the detergent solubility of connexin43 in communication-competent NRK cells revealed that processing of connexin43 to the P2 form was accompanied by acquisition of resistance to solubilization in 1% Triton X-100. Immunohistochemical localization of connexin43 in Triton-extracted NRK cells demonstrated that connexin43-P2 (Triton-insoluble) was concentrated in gap junctional plaques, whereas connexin43-NP (Triton-soluble) was predominantly intracellular. Using either a 20 degrees C intracellular transport block or cell-surface protein biotinylation, we determined that connexin43 was transported to the plasma membrane in the Triton-soluble connexin43-NP form. Cell-surface biotinylated connexin43-NP was processed to Triton-insoluble connexin43-P2 at 37 degrees C. Connexin43-NP was also transported to the plasma membrane in communication defective, gap junction-deficient S180 and L929 cells but was not processed to Triton-insoluble connexin43-P2. Taken together, these results demonstrate that gap junction assembly is regulated after arrival of connexin43 at the plasma membrane and is temporally associated with acquisition of insolubility in Triton X-100 and phosphorylation to the connexin43-P2 form.     

Cloning, expression, and characterization of the trout cardiac Na+/Ca2+ exchanger

Isoform 1 of the cardiacNa⁺/Ca²⁺exchanger (NCX1) is an important regulator of cytosolicCa²⁺ concentration in contractionand relaxation. Studies with trout heart sarcolemmal vesicles haveshown NCX to have a high level of activity at 7°C, and this uniqueproperty is likely due to differences in protein structure. In thisstudy, we describe the cloning of an NCX (NCX-TR1) from a Lambda ZAPII cDNA library constructed from rainbow trout(Oncorhynchus mykiss) heart RNA. TheNCX-TR1 cDNA has an open reading frame that codes for a protein of 968 amino acids with a deduced molecular mass of 108 kDa. A hydropathy plotindicates the protein contains 12 hydrophobic segments (of which thefirst is predicted to be a cleaved leader peptide) and a largecytoplasmic loop. By analogy to NCX1, NCX-TR1 is predicted to have ninetransmembrane segments. The sequences demonstrated to be the exchangerinhibitory peptide site and the regulatoryCa²⁺ binding site in thecytoplasmic loop of mammalian NCX1 are almost completely conserved inNCX-TR1. NCX-TR1 cRNA was injected into Xenopus oocytes, and after 3-4days currents were measured by the giant excised patch technique.NCX-TR1 currents measured at ~23°C demonstratedNa⁺-dependent inactivation andCa²⁺-dependent activation in amanner qualitatively similar to that for NCX1 currents.

     

The cataract causing Cx50-S50P mutant inhibits Cx43 and intercellular communication in the lens epithelium

Mutations in Connexin50 (Cx50) cause cataracts in both humans and mice. The mechanism(s) behind how mutated connexins lead to a variety of cataracts have yet to be fully elucidated. Here, we tested whether the cataract inducing Cx50-S50P mutant interacts with wild-type Connexin43 (Cx43) to form mixed channels with attenuated function. Using dual whole-cell voltage clamp, immunofluorescent microscopy and in situ dye transfer analysis we identified a unique interaction between the mutant subunit and wild-type Cx43. In paired Xenopus oocytes, co-expression of Cx50-S50P with Cx43 reduced electrical coupling ≥ 90%, without a reduction in protein expression. In transfected cells, Cx50-S50P did not target to cell-cell interfaces by itself, but co-expression of Cx50-S50P with Cx43 resulted in its localization at areas of cell-cell contact. We used Cx43 conditional knockout, Cx50 knockout and Cx50-S50P mutant mice to examine this interaction in vivo. Mice expressing both Cx43 and Cx50-S50P in the lens epithelium revealed a unique expression pattern for Cx43 and a reduction in Cx43 protein. In situ dye transfer experiments showed that the Cx50-S50P mutant, but not the Cx50, or Cx43 conditional knockout, greatly inhibited epithelial cell gap junctional communication in a manner similar to a double knockout of Cx43 and Cx50. The inhibitory affects of Cx50-S50P lead to diminished electrical coupling in vitro, as well as a discernable reduction in epithelial cell dye permeation. These data suggest that dominant inhibition of Cx43 mediated epithelial cell coupling may play a role in the lens pathophysiology caused by the Cx50-S50P mutation.     

Regulation of the epithelial Na(+) channel by extracellular acidification

The effect of extracellular acidification wastested on the native epithelial Na⁺ channel (ENaC) in A6epithelia and on the cloned ENaC expressed in Xenopusoocytes. Channel activity was determined utilizing blocker-inducedfluctuation analysis in A6 epithelia and dual electrode voltage clampin oocytes. In A6 cells, a decrease of extracellular pH(pH_o) from 7.4 to 6.4 caused a slow stimulation of theamiloride-sensitive short-circuit current (I_Na)by 68.4 ± 11% (n = 9) at 60 min. This increaseof I_Na was attributed to an increase of openchannel and total channel (N_T) densities. Similar changes were observed with pH_o 5.4. The effects ofpH_o were blocked by buffering intracellularCa²⁺ with 5 µM1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid. Inoocytes, pH_o 6.4 elicited a small transient increase of theslope conductance of the cloned ENaC (11.4 ± 2.2% at 2 min)followed by a decrease to 83.7 ± 11.7% of control at 60 min (n = 6). Thus small decreases of pH_ostimulate the native ENaC by increasing N_T butdo not appreciably affect ENaC expressed in Xenopus oocytes.These effects are distinct from those observed with decreasingintracellular pH with permeant buffers that are known to inhibit ENaC.

     

Connexin phosphorylation as a regulatory event linked to gap junction channel assembly

Gap junctions, composed of proteins from the connexin family, allow for intercellular communication between cells and are important in development and maintenance of cell homeostasis. Phosphorylation has been implicated in the regulation of gap junctional communication at several stages of the cell cycle and the connexin “lifecycle”, such as trafficking, assembly/disassembly, degradation, as well as in the gating of “hemi” channels or intact gap junction channels. This review focuses on how phosphorylation can regulate the early stages of the connexin life cycle through assembly of functional gap junctional channels. The availability of sequences from the human genome databases has indicated that the number of connexins in the gene family is approximately 20, but we know mostly about how connexin43 (Cx43) is regulated. Recent technologies and investigations of interacting proteins have shown that activation of several kinases including protein kinase A, protein kinase C (PKC), p34^cdc2/cyclin B kinase, casein kinase 1 (CK1), mitogen-activated protein kinase (MAPK) and pp60^src kinase can lead to phosphorylation of the majority of the 21 serine and two of the tyrosine residues in the C-terminal region of Cx43. While many studies have correlated changes in kinase activity with changes in gap junctional communication, further research is needed to directly link specific phosphorylation events with changes in connexin oligomerization and gap junction assembly.