Blockade of an Inward Sodium Current Facilitates Pharmacological Study of Hemi-Gap-Junctional Currents in Xenopus Oocytes

2002 ◽  
Vol 203 (2) ◽  
pp. 192-194 ◽  
Author(s):  
Harris Ripps ◽  
Haohua Qian ◽  
Jane Zakevicius
Keyword(s):  
Xenopus Oocytes ◽  
Sodium Current ◽  
Pharmacological Study ◽  
Gap Junctional

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

2011 ◽  
Vol 300 (5) ◽  
pp. C1055-C1064 ◽  
Author(s):  
Jun-Jie Tong ◽  
Peter J. Minogue ◽  
Wenji Guo ◽  
Tung-Ling Chen ◽  
Eric C. Beyer ◽  
...  
Keyword(s):  
Amino Acids ◽  
Plasma Membrane ◽  
Hela Cells ◽  
Xenopus Oocytes ◽  
Dominant Negative ◽  
Wild Type ◽  
Gap Junction Channels ◽  
Dominant Negative Inhibitor ◽  
Intercellular Channels ◽  
Gap Junctional

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.

scholarly journals Properties of a nonjunctional current expressed from a rat connexin46 cDNA in Xenopus oocytes.

1993 ◽  
Vol 102 (1) ◽  
pp. 59-74 ◽  
Author(s):  
L Ebihara ◽  
E Steiner
Keyword(s):  
Xenopus Oocytes ◽  
Inactivation Kinetics ◽  
Induced Current ◽  
Strongly Coupled ◽  
Deactivation Kinetics ◽  
Voltage Dependent ◽  
Junctional Protein ◽  
Gap Junctional ◽  
Kinetics Of ◽  
External Calcium

Connexin46 (cxn46) is a gap junctional protein that was cloned from a rat lens cDNA library. Expression of cxn46 in solitary Xenopus oocytes resulted in the development of a large time- and voltage-dependent current that was not observed in noninjected control oocytes or in oocytes injected with mRNA for cxn43 or cxn32. The cxn46-induced current activated at potentials positive to -20 mV. On repolarization to -40 mV, the current deactivated over a period of several seconds. Removal of external calcium caused a marked increase in the amplitude of the cxn46-induced current, shifted the steady-state activation curve to more negative potentials, and altered the kinetics of activation and deactivation. Increasing external calcium had the opposite effect. The ability of cxn46 to induce the formation of cell-to-cell channels was tested in the oocyte pair system. Oocyte pairs injected with cxn46 mRNA + antisense oligonucleotides for Xenopus cxn38 were strongly coupled. In contrast, oocyte pairs injected with antisense alone showed no coupling. The inactivation kinetics of the gap junctional channels resembled the deactivation kinetics of the cxn46-induced current in solitary oocytes.

Pharmacological enhancement of hemi-gap-junctional currents in Xenopus oocytes

2002 ◽  
Vol 121 (1) ◽  
pp. 81-92 ◽  
Author(s):  
Harris Ripps ◽  
Haohua Qian ◽  
Jane Zakevicius
Keyword(s):  
Xenopus Oocytes ◽  
Gap Junctional

Potentiation of rat brain sodium channel currents by PKA inXenopus oocytes involves the I-II linker

2000 ◽  
Vol 278 (4) ◽  
pp. C638-C645 ◽  
Author(s):  
Raymond D. Smith ◽  
Alan L. Goldin
Keyword(s):  
Sodium Channel ◽  
Xenopus Oocytes ◽  
Sodium Current ◽  
Sodium Currents ◽  
Voltage Gated Sodium Channels ◽  
Single Region ◽  
Functional Modulation ◽  
Excitability Of Neurons ◽  
Channel Currents ◽  
The Brain

Functional modulation of voltage-gated sodium channels affects the electrical excitability of neurons. Protein kinase A (PKA) can decrease sodium currents by phosphorylation at consensus sites in the cytoplasmic I-II linker. Once the sites are phosphorylated, however, additional PKA activity can increase sodium currents by an unknown mechanism. When the PKA sites were eliminated by substitutions of alanine for serine, peak sodium current amplitudes were increased by 20–80% when PKA was activated in Xenopus oocytes either by stimulation of a coexpressed β2-adrenergic receptor or by perfusion with reagents that increase cAMP. Potentiation required the I-II linker of the brain channel, in that a chimeric channel in which the brain linker was replaced with the comparable linker from the skeletal muscle channel did not demonstrate potentiation. Using a series of chimeric and deleted channels, we demonstrate that potentiation is not dependent on any single region of the linker and that the extent of potentiation varies depending on the total length and the residues throughout the linker. These data are consistent with the hypothesis that potentiation by PKA is an indirect process involving phosphorylation of an accessory protein that interacts with the I-II linker of the sodium channel.

Molecular mechanism underlying a Cx50-linked congenital cataract

1999 ◽  
Vol 276 (6) ◽  
pp. C1443-C1446 ◽  
Author(s):  
J. D. Pal ◽  
V. M. Berthoud ◽  
E. C. Beyer ◽  
D. Mackay ◽  
A. Shiels ◽  
...  
Keyword(s):  
Congenital Cataract ◽  
Xenopus Oocytes ◽  
Dominant Negative ◽  
Wild Type ◽  
Autosomal Dominant Fashion ◽  
Junctional Coupling ◽  
Type Coupling ◽  
Connexin 50 ◽  
Gap Junctional ◽  
Gap Junctional Coupling

Mutations in gap junctional channels have been linked to certain forms of inherited congenital cataract (D. Mackay, A. Ionides, V. Berry, A. Moore, S. Bhattacharya, and A. Shiels. Am. J. Hum. Genet. 60: 1474–1478, 1997; A. Shiels, D. Mackay, A. Ionides, V. Berry, A. Moore, and S. Bhattacharya. Am. J. Hum. Genet. 62: 526–532, 1998). We used the Xenopus oocyte pair system to investigate the functional properties of a missense mutation in the human connexin 50 gene (P88S) associated with zonular pulverulent cataract. The associated phenotype for the mutation is transmitted in an autosomal dominant fashion. Xenopus oocytes injected with wild-type connexin 50 cRNA developed gap junctional conductances of ∼5 μS 4–7 h after pairing. In contrast, the P88S mutant connexin failed to form functional gap junctional channels when paired homotypically. Moreover, the P88S mutant functioned in a dominant negative manner as an inhibitor of human connexin 50 gap junctional channels when coinjected with wild-type connexin 50 cRNA. Cells injected with 1:5 and 1:11 ratios of P88S mutant to wild-type cRNA exhibited gap junctional coupling of ∼8% and 39% of wild-type coupling, respectively. Based on these findings, we conclude that only one P88S mutant subunit is necessary per gap junctional channel to abolish channel function.

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

2000 ◽  
Vol 279 (3) ◽  
pp. C596-C602 ◽  
Author(s):  
Jay D. Pal ◽  
Xiaoqin Liu ◽  
Donna Mackay ◽  
Alan Shiels ◽  
Viviana M. Berthoud ◽  
...  
Keyword(s):  
Xenopus Oocytes ◽  
Dominant Negative ◽  
Metabolic Labeling ◽  
Loss Of Function ◽  
Wild Type ◽  
Reading Frame ◽  
Gap Junction Channel ◽  
Intercellular Channels ◽  
Normal Lens ◽  
Gap Junctional

Human connexin46 (hCx46) forms gap junctional channels interconnecting lens fiber cells and appears to be critical for normal lens function, because hCx46 mutations have been linked to congenital cataracts. We studied two hCx46 mutants, N63S, a missense mutation in the first extracellular domain, and fs380, a frame-shift mutation that shifts the translational reading frame at amino acid residue 380. We expressed wild-type Cx46 and the two mutants in Xenopus oocytes. Production of the expressed proteins was verified by SDS-PAGE after metabolic labeling with [35S]methionine or by immunoblotting. Dual two-microelectrode voltage-clamp studies showed that hCx46 formed both gap junctional channels in paired Xenopus oocytes and hemi-gap junctional channels in single oocytes. In contrast, neither of the two cataract-associated hCx46 mutants could form intercellular channels in paired Xenopus oocytes. The hCx46 mutants were also impaired in their ability to form hemi-gap-junctional channels. When N63S or fs380 was coexpressed with wild-type connexins, both mutations acted like “loss of function” rather than “dominant negative” mutations, because they did not affect the gap junctional conductance induced by either wild-type hCx46 or wild-type hCx50.

Sign in / Sign up

Export Citation Format

Share Document