Kunyan Kuang

Autosomal Dominant Glut-1 Deficiency Syndrome and Familial Epilepsy

Glut‐1 deficiency syndrome was first described in 1991 as a sporadic clinical condition, later shown to be the result of haploinsufficiency. We now report a family with Glut‐1 deficiency syndrome affecting 5 members over 3 generations. The syndrome behaves as an autosomal dominant condition. Affected family members manifested mild to severe seizures, developmental delay, ataxia, hypoglycorrhachia, and decreased erythrocyte 3‐O‐methyl‐D‐glucose uptake. Seizure frequency and severity were aggravated by fasting, and responded to a carbohydrate load. Glut‐1 immunoreactivity in erythrocyte membranes was normal. A heterozygous R126H missense mutation was identified in the 3 patients available for testing, 2 brothers (Generation 3) and their mother (Generation 2). The sister and her father were clinically and genotypically normal. In vitro mutagenesis studies in Xenopus laevis oocytes demonstrated significant decreases in the transport of 3‐O‐methyl‐D‐glucose and dehydroascorbic acid. Xenopus oocyte membranes expressed high amounts of the R126H mutant Glut‐1. Kinetic analysis indicated that replacement of arginine‐126 by histidine in the mutant Glut‐1 resulted in a lower Vmax. These studies demonstrate the pathogenicity of the R126H missense mutation and transmission of Glut‐1 deficiency syndrome as an autosomal dominant trait.

Effects of ambient bicarbonate, phosphate and carbonic anhydrase inhibitors on fluid transport across rabbit corneal endothelium

Bicarbonate has been long held to be indispensable for fluid pumping by the endothelium; however, such need has been disputed recently. We investigated this issue and found that: (1) the corneal endothelium pumps fluid equally well (at 6-8 microliters hr-1 cm-2) whether the bathing solution contains 43 mM bicarbonate or 10 mM phosphate, (2) if bicarbonate and most of the phosphate are absent, fluid pumping is noticeably lowered (2-4 microliters hr-1 cm-2), (3) carbonic anhydrase inhibitors (5 mM acetazolamide; 0.1, 0.2 and 0.3 mM ethoxzolamide) block this lowered fluid pumping, and (4) in the absence of external bicarbonate, 20 mM HEPES is insufficient to preserve adequate fluid pumping. These results are consistent with existing models for endothelial transport in which exogenous and endogenous CO2 are converted to HCO3- by carbonic anhydrase, with HCO3- fueling the transport mechanism and therefore the fluid pump.

Transport of fluid by lens epithelium

We report for the first time that cultured lens epithelial cell layers and rabbit lenses in vitro transport fluid. Layers of the alphaTN4 mouse cell line and bovine cell cultures were grown to confluence on permeable membrane inserts. Fluid movement across cultured layers and excised rabbit lenses was determined by volume clamp (37 degrees C). Cultured layers transported fluid from their basal to their apical sides against a pressure head of 3 cmH2O. Rates were (in microliter. h-1. cm-2) 3.3 +/- 0.3 for alphaTN4 cells (n = 27) and 4.7 +/- 1.0 for bovine layers (n = 6). Quinidine, a blocker of K+ channels, and p-chloromercuribenzenesulfonate and HgCl2, inhibitors of aquaporins, inhibited fluid transport. Rabbit lenses transported fluid from their anterior to their posterior sides against a 2.5-cmH2O pressure head at 10.3 +/- 0.62 microliter. h-1. lens-1 (n = 5) and along the same pressure head at 12.5 +/- 1.1 microliter. h-1. lens-1 (n = 6). We calculate that this flow could wash the lens extracellular space by convection about once every 2 h and therefore might contribute to lens homeostasis and transparency.We report for the first time that cultured lens epithelial cell layers and rabbit lenses in vitro transport fluid. Layers of the αTN4 mouse cell line and bovine cell cultures were grown to confluence on permeable membrane inserts. Fluid movement across cultured layers and excised rabbit lenses was determined by volume clamp (37°C). Cultured layers transported fluid from their basal to their apical sides against a pressure head of 3 cmH2O. Rates were (in μl ⋅ h-1 ⋅ cm-2) 3.3 ± 0.3 for αTN4 cells ( n = 27) and 4.7 ± 1.0 for bovine layers ( n = 6). Quinidine, a blocker of K+ channels, and p-chloromercuribenzenesulfonate and HgCl2, inhibitors of aquaporins, inhibited fluid transport. Rabbit lenses transported fluid from their anterior to their posterior sides against a 2.5-cmH2O pressure head at 10.3 ± 0.62 μl ⋅ h-1 ⋅ lens-1( n = 5) and along the same pressure head at 12.5 ± 1.1 μl ⋅ h-1 ⋅ lens-1( n = 6). We calculate that this flow could wash the lens extracellular space by convection about once every 2 h and therefore might contribute to lens homeostasis and transparency.

Evidence for a central role for electro-osmosis in fluid transport by corneal endothelium.

The mechanism of transepithelial fluid transport remains unclear. The prevailing explanation is that transport of electrolytes across cell membranes results in local concentration gradients and transcellular osmosis. However, when transporting fluid, the corneal endothelium spontaneously generates a locally circulating current of approximately 25 microA cm(-2), and we report here that electrical currents (0 to +/-15 microA cm(-2)) imposed across this layer induce fluid movements linear with the currents. As the imposed currents must be approximately 98% paracellular, the direction of induced fluid movements and the rapidity with which they follow current imposition (rise time < or =3 sec) is consistent with electro-osmosis driven by sodium movement across the paracellular pathway. The value of the coupling coefficient between current and fluid movements found here (2.37 +/- 0.11 microm cm(2) hr(-1) microA (-1), suggests that: 1) the local endothelial current accounts for spontaneous transendothelial fluid transport; 2) the fluid transported becomes isotonically equilibrated. Ca(++)-free solutions or endothelial damage eliminate the coupling, pointing to the cells and particularly their intercellular junctions as a main site of electro-osmosis. The polycation polylysine, which is expected to affect surface charges, reverses the direction of current-induced fluid movements. Fluid transport is proportional to the electrical resistance of the ambient medium. Taken together, the results suggest that electro-osmosis through the intercellular junctions is the primary process in a sequence of events that results in fluid transport across this preparation.

Immunocytochemical Localization of Aquaporin-1 in Bovine Corneal Endothelial Cells and Keratocytes

For immunocytochemistry, cultured bovine corneal endothelial cells (CBCEC) and bovine corneal cryosections were utilized. Preparations were fixed, permeabilized, and incubated with primary rabbit anti-rat aquaporin 1 (AQP1) antibody followed by rhodamine-conjugated secondary antibody, and were counter-stained with Sytox nuclear acid stain. Confocal microscopy of CBCEC in the x, y, and z planes showed rhodamine fluorescence, indicating the presence of AQP1 antibody localized to the apical and basolateral domains of the plasma membrane, but not to the membranes of intracellular compartments or other subcellular locations. Preabsorption with control antigenic peptide yielded no positive staining. Similar results were obtained using freshly dissected bovine corneas; in addition, these images showed AQP1 distributed to the plasma membranes of keratocytes. No AQP1 staining was seen in corneal epithelium, and no staining was observed in CBCEC layers exposed to AQP3, AQP4, and AQP5 antibodies.

Are most transporters and channels beta barrels

Given the sequence of transporters or channels of unknown secondary structure, it is usual to predict their putative transmembrane regions as α-helical. However, recent evidence for a facilitative glucose transporter (GLUT1_ appears inconsistent with such predictions, which has led us to propose an alternative folding model for GLUTs based on the 16-stranded antiparallel β-barrel of porins. Here we apply the same predictive algorithms we used for GLUTs to several other membrane proteins. For some of them, a high-resolution structure has been derived (β-barrels: Rhodobacter capsulatus andEscherichia coli porins; multihelical: colicin A, bacteriorhodopsin, and reaction center L chain); we use them to test the prediction procedures. The other proteins we analyze (GLUT1, CHIP28, acetylcholine receptor alpha subunit, lac permease, Na+-glucose cotransporter, shaker K+ channel, sarcoplasmic reticulum Ca2+-ATPase) are representative of classes of similar membrane proteins. As with GLUTs, we find that the predicted transmembrane segments of these proteins are consistently shorter than expected for transmembrane spanning α-helices, but are of the correct length and number for the proteins to fold instead as porin-like β-barrels.

Macular holes: migratory gaps and vitreous as obstacles to glial closure.

Abstract• Purpose: Retinal glia may play an important role in the closure of macular holes. This in vitro study examines whether and how the specific pathoanatomy, including foveal eversion and foveal vitreous, may interfere with glial closure of macular holes. • Methods: Culture dishes used to grow glial cells were modified by the placement of slopes, vertical steps, and gaps to mimic the in vivo migratory surface in and surrounding macular holes. In separate experiments, defects were made in a rodent glial monolayer. These defects were exposed to hyaluronic acid (HA) and to rabbit (RV) and bovine (BV) vitreous gel. The migratory behavior and completeness of closure of defects were compared to controls. • Results: As expected, glial cells migrated further and in greater numbers on a smooth surface. Slopes and steps were mode-rate obstacles to migration; gaps in the surface were absolute obstacles. HA modified the pattern of adhesion of cells at the bottom of defects. Defects in the glial monolayer were repaired in 5–7 days. Compared to these controls, repair was inhibited by 11 % (n. s.), 28% (P=0.02), and 58% (P=0.004) after direct exposure of defects to HA, RV and BV respectively. • Conclusion: The elevated and everted margins of macular holes represent slope, step, and gap-like obstacles to the migration of glial cells and hence to the healing of defects. The defect allows extension of extracellular matrix into it and the subretinal space. Our results indicate that gaps in the migratory surface caused and aggravated by eversion and the presence of vitreous present obstacles to glial migration and closure of macular holes.

Solutions containing miotic agents: Effects on corneal transendothelial electrical potential difference

Abstract• Background: Anterior chamber miotic solutions are widely used during anterior chamber surgery. We examined the effects of solutions containing miotic agents such as carbachol and/or acetylcholine on corneal endothelial pumping activity. • Methods: We monitored, in vitro, the transendothelial electrical potential difference of isolated rabbit corneal endothelial preparations. As controls, we used solutions without miotics. • Results: We found that a solution containing 55 mM acetylcholine and minimal amounts of salts (Miochol E) maintains transendothelial electrical potential difference some 30% above control levels for up to 4 h. Two other solutions, one including balanced salts and 0.55 mM carbachol (Miostat), the other a mixture of 0.19 mM carbachol and 55 mM acetylcholine plus minimal salts, are adequate to maintain the potential difference at control levels. Lastly, a solution with acetylcholine but without any salts (Miochol) greatly decreases the potential difference, to 30% of the control level, in 100 min. • Conclusion: Our results indicate that: (1) 55 mM (1%) acetylcholine stimulates the endothelial electrical potential difference; (2) addition of 0.19 mM (0.003%) carbachol negates the stimulatory effect of acetylcholine; and (3) absence of electrolytes severely depresses the endothelial electrical activity.

Predictive Evidence for a Porin-Type .BETA.-Barrel Fold in CHIP28 and other Members of the MIP Family. A Restricted-Pore Model Common to Water Channels and Facilitators.

Water channels are the subject of much current attention, as they may be central for cell functions in a host of tissues. We have analyzed the possible fold of facilitators and water channels of the MIP family based on structural predictions, on findings about the topology of CHIP28, and on the biophysical characteristics of water channels. We developed predictions for the following proteins: MIP26, NOD26, GLP, BIB, γ-TIP, FA-CHIP, CHIP28k, WCH-CD1, and CHIP28. We utilized Kyte Doolittle hydrophobicity, Eisenbergs amphiphilicity, Chou-Fasman-Prevelige propensities, and our own Union algorithm. We found that hydrophobic amphiphilic segments likely to be transmembrane were consistently shorter than required for α-helical segments, but of the correct length for β-strands. Turn propensity was high at frequent intervals, consistent with transmembrane β-strands. We propose that these proteins fold as porin-like 16-stranded antiparallel β-barrels. In water channels, from the size of molecules excluded, an extramembrane loop(s) would enter the pore and restrict it to a bottleneck with a width 4 Å ⩽w ⩽5 Å. A similar but more mobile loop(s) would act as gate and binding site for the facilitators of the MIP family.

Mercurial sensitivity of aquaporin 1 endofacial loop B residues

The water channel protein aquaporin‐1 (AQP1) has two asparagine‐proline‐alanine (NPA) repeats on loops B and E. From recent structural information, these loops are on opposite sides of the membrane and meet to form a pore. We replaced the mercury‐sensitive residue cysteine 189 in AQP1 by serine to obtain a mercury‐insensitive template (C189S). Subsequently, we substituted three consecutive cysteines for residues 71–73 near the first NPA repeat (76–78) in intracellular loop B, and investigated whether they were accessible to extracellular mercurials. AQP1 and its mutants were expressed in Xenopus laevis oocytes, and the osmotic permeability (Pf) of the oocytes was determined. C189S had wild‐type Pf but was not sensitive to HgCl2. Expression of all three C189S cysteine mutants resulted in increased Pf, and all three mutants regained mercurial sensitivity. These results, especially the inhibitions by the large mercurial p‐chloromercunbenzene‐sulfonic acid (pCMBS) (∼6Å wide), suggest that residues 71–73 at the pore are accessible to extracellular mercurials. A 30‐ps molecular dynamics simulation (at 300 K) starting with crystallographic coordinates of AQP1 showed that the width of the pore bottleneck (between Connolly surfaces) can vary (wavg = 3.9 Å, σ = 0.75; hydrated AQP1). Thus, although the pore width would be ≥ 6 Å only for 0.0026 of the time, this might suffice for pCMBS to reach residues 71–73. Alternative explanations such as passage of pCMBS across the AQP1 tetramer center or other unspecified transmembrane pathways cannot be excluded.