scholarly journals Developmental Regulation of the Direct Interaction between the Intracellular Loop of Connexin 45.6 and the C Terminus of Major Intrinsic Protein (Aquaporin-0)

2005 ◽  
Vol 280 (23) ◽  
pp. 22081-22090 ◽  
Author(s):  
Xun Sean Yu ◽  
Xinye Yin ◽  
Eileen M. Lafer ◽  
Jean X. Jiang
Keyword(s):  
Gap Junction ◽  
Intercellular Communication ◽  
Direct Interaction ◽  
Lens Development ◽  
Major Intrinsic Protein ◽  
Intracellular Loop ◽  
Intrinsic Protein ◽  
Lens Fibers ◽  
Loop Region ◽  
C Terminus

The eye lens is dependent upon a network of gap junction-mediated intercellular communication to facilitate its homeostasis and development. Three gap junction-forming proteins are expressed in the lens of which two are in lens fibers, namely connexin (Cx) 45.6 and 56. Major intrinsic protein (MIP), also known as aquaporin-0 (AQP0), is the most abundant membrane protein in lens fibers. However, its role in the lens is not clear. Our previous studies show that MIP(AQP0) associates with gap junction plaques formed by Cx45.6 and Cx56 during the early stages of embryonic chick lens development but not in late embryonic and adult lenses. We report here that MIP(AQP0) directly interacts with Cx45.6 but not with Cx56. We further identified the intracellular loop of Cx45.6 as the interacting domain for the MIP(AQP0) C terminus. Surface plasmon resonance experiments indicated that the C-terminal domain of MIP(AQP0) interacts with two binding sites within the intracellular loop region of Cx45.6 with a KD(app) of 7.5 and 10.3 μm, respectively. The KD(app) for the full-length loop region is 7.7 μm. The cleavage at the intracellular loop of Cx45.6 was observed during lens development, and the C terminus of MIP(AQP0) did not interact with the loop-cleaved form of Cx45.6. Thus, the dissociation between these two proteins that occurs in the mature fibers of late lens development is likely caused by this cleavage. Finally this interaction had no impact on Cx45.6-mediated intercellular communication, suggesting that the Cx45.6-MIP(AQP0) interaction plays a novel unidentified role in lens fibers.

scholarly journals Heterologous expression in Escherichia coli of native and mutant forms of the major intrinsic protein of rat eye lens (MIP26)

1995 ◽  
Vol 305 (3) ◽  
pp. 753-759 ◽  
Author(s):  
N Dilsiz ◽  
M J C Crabbe
Keyword(s):  
Escherichia Coli ◽  
Monoclonal Antibody ◽  
Cytoplasmic Membrane ◽  
Amino Acid Level ◽  
Site Directed Mutagenesis ◽  
Eye Lens ◽  
Major Intrinsic Protein ◽  
Intrinsic Protein ◽  
C Terminus ◽  
Cell Plasma

The complete cDNA of rat eye lens major intrinsic protein (MIP26) was sequenced using the dideoxy chain termination method. The sequence displayed 89% nucleotide identity and 95% identity at the amino acid level with bovine MIP26 [Gorin, Yancey, Cline, Revel and Horwitz (1984) Cell, 39, 49-54]. Both native and mutant cDNAs coding for rat MIP26 were amplified by PCR and subcloned into the pPOW expression vector for expression of Escherichia coli. A membrane signal peptide (PelB) was used for secretion of MIP26 into the cytoplasmic membrane. A hydrophilic octapeptide tail (FLAG) was fused to either the N- or C-terminus of MIP26 to aid monoclonal antibody-mediated identification and purification. Heterologously expressed MIP26 was identified by using a monoclonal antibody corresponding to the FLAG peptide located at the termini of MIP26. Immunofluorescently labelled monoclonal antibody was used to determine the localization of MIP26 in the cytoplasmic membrane. The majority of the protein was integrated into cell plasma membrane. MIP26 was extracted with n-octyl beta-D-glucopyranoside and then purified on an affinity gel column. Rat MIP26 cDNA contains an -Asn-Gly- sequence at the C-terminus, which has been shown in other proteins to be particularly susceptible to spontaneous deamidation [Takemoto and Emmons (1991) Curr. Eye Res. 10, 863-869]. We therefore modified the MIP26 molecule using a site-directed mutagenesis method to generate a mutant MIP26 at the appropriate asparagine residue (Asn244-->Asp) near the C-terminus. The mutation was confirmed by DNA sequencing. The mutant MIP26 protein was also expressed in E. coli and incorporated predominantly into the cytoplasmic membrane.

scholarly journals Properties of channels reconstituted from the major intrinsic protein of lens fiber membranes.

1990 ◽  
Vol 96 (3) ◽  
pp. 631-664 ◽  
Author(s):  
G R Ehring ◽  
G Zampighi ◽  
J Horwitz ◽  
D Bok ◽  
J E Hall
Keyword(s):  
Lipid Bilayers ◽  
High Performance ◽  
Single Channel ◽  
Freeze Fracture ◽  
Boltzmann Distribution ◽  
Major Intrinsic Protein ◽  
Time Constants ◽  
Intrinsic Protein ◽  
Lens Fibers ◽  
Conductance States

Detergent-solubilized plasma membrane protein of either adult bovine or calf lens and high-performance liquid chromatography-purified major intrinsic protein (MIP) of the lens were reconstituted into unilamellar vesicles and planar lipid bilayers. Freeze-fracture studies showed that the density of intramembrane particles in the vesicles was proportional to the protein/lipid ratio. At high ratios, these particles crystallized into tetragonal arrays as does MIP in lens fibers. Channels induced by either purified MIP or detergent-solubilized protein had essentially identical properties. The conductance of multichannel membranes was maximal near 0 mV and decreased to 0.49 +/- 0.08 of the maximum value at voltages greater than 80 mV. The dependence of the conductance on voltage was well fit by a two-state Boltzmann distribution. Voltage steps greater than 30 mV elicited an ohmic current step followed by a slow (seconds) biexponential decrease. The amplitudes and time constants depended on the magnitude but not the sign of the voltage. Steps from 100 mV to voltages less than 30 mV caused the channels to open exponentially with a millisecond time constant. Analysis of latency to first closure after a voltage step gave nearly the same time constants as multichannel kinetics. Single-channel conductance is proportional to salt concentration from 0.1 to 1.0 M in KCl. In 0.1M KCl, the channel had two preferred conductance states with amplitudes of 380 and 160 pS, as well as three additional substates. Multi- and single-channel data suggest that the channel has two kinetically important open states. The channel is slightly anion selective. The properties of the channel do not vary appreciably from pH 7.4 to 5.8 or from pCa 7 to 2. We propose that a channel with these properties could contribute to maintenance of lens transparency and fluid balance.

Genome-wide Characterization of Major Intrinsic Protein (MIP) Gene Family in Brachypodium distachyon

2018 ◽  
Vol 13 (5) ◽  
pp. 536-552 ◽  
Author(s):  
Ankush Ashok Saddhe ◽  
Shweta ◽  
Kareem A. Mosa ◽  
Kundan Kumar ◽  
Manoj Prasad ◽  
...  
Keyword(s):  
Gene Family ◽  
Brachypodium Distachyon ◽  
Major Intrinsic Protein ◽  
Intrinsic Protein ◽  
Genome Wide

Connexin expression by alveolar epithelial cells is regulated by extracellular matrix

2001 ◽  
Vol 280 (2) ◽  
pp. L191-L202 ◽  
Author(s):  
Yihe Guo ◽  
Cara Martinez-Williams ◽  
Clare E. Yellowley ◽  
Henry J. Donahue ◽  
D. Eugene Rannels
Keyword(s):  
Extracellular Matrix ◽  
Epithelial Cells ◽  
Gap Junction ◽  
Intercellular Communication ◽  
Translational Regulation ◽  
Alveolar Epithelial Cells ◽  
Alveolar Type ◽  
Type Ii ◽  
Gap Junction Intercellular Communication ◽  
Type Ii Cell

Extracellular matrix (ECM) proteins promote attachment, spreading, and differentiation of cultured alveolar type II epithelial cells. The present studies address the hypothesis that the ECM also regulates expression and function of gap junction proteins, connexins, in this cell population. Expression of cellular fibronectin and connexin (Cx) 43 increase in parallel during early type II cell culture as Cx26 expression declines. Gap junction intercellular communication is established over the same interval. Cells plated on a preformed, type II cell-derived, fibronectin-rich ECM demonstrate accelerated formation of gap junction plaques and elevated gap junction intercellular communication. These effects are blocked by antibodies against fibronectin, which cause redistribution of Cx43 protein from the plasma membrane to the cytoplasm. Conversely, cells cultured on a laminin-rich ECM, Matrigel, express low levels of Cx43 but high levels of Cx26, reflecting both transcriptional and translational regulation. Cx26 and Cx43 thus demonstrate reciprocal regulation by ECM constituents.

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