Micropipette Aspiration‐Based Assessment of Single Channel Water Permeability

Aquaporin-4 (AQP4) water channels exist as heterotetramers of M1 and M23 splice variants and appear to be present in orthogonal arrays of intramembraneous particles (OAPs) visualized by freeze-fracture microscopy. We report that AQP4 forms OAPs in rat gastric parietal cells but not in parietal cells from the mouse or kangaroo rat. Furthermore, the organization of principal cell OAPs in Brattleboro rat kidney is perturbed by vasopressin (arginine vasopressin). Membranes of LLC-PK1 cells expressing M23-AQP4 showed large, abundant OAPs, but none were detectable in cells expressing M1-AQP4. Measurements of osmotic swelling of transfected LLC-PK1 cells using videomicroscopy, gave osmotic water permeability coefficient ( Pf) values (in cm/s) of 0.018 (M1-AQP4), 0.019 (M23-AQP4), and 0.003 (control). Quantitative immunoblot and immunofluorescence showed an eightfold greater expression of M1- over M23-AQP4 in the cell lines, suggesting that single-channel pf (cm3/s) is much greater for the M23 variant. Somatic fusion of M1- and M23-AQP4 cells ( Pf = 0.028 cm/s) yielded OAPs that were fewer and smaller than in M23 cells alone, and M1-to-M23 expression ratios (∼1:4) normalized to AQP4 in M1 or M23 cells indicated a reduced single-channel pf for the M23 variant. Expression of an M23-AQP4-Ser111E mutant produced ∼1.5-fold greater single-channel pf and OAPs that were up to 2.5-fold larger than wild-type M23-AQP4 OAPs, suggesting that a putative PKA phosphorylation site Ser111 is involved in OAP formation. We conclude that the higher-order organization of AQP4 in OAPs increases single-channel osmotic water permeability by one order of magnitude and that differential cellular expression levels of the two isoforms could regulate this organization.

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Tetrameric assembly of CHIP28 water channels in liposomes and cell membranes: a freeze-fracture study.

The Journal of Cell Biology ◽

10.1083/jcb.123.3.605 ◽

1993 ◽

Vol 123 (3) ◽

pp. 605-618 ◽

Cited By ~ 166

Author(s):

J M Verbavatz ◽

D Brown ◽

I Sabolić ◽

G Valenti ◽

D A Ausiello ◽

...

Keyword(s):

Water Permeability ◽

Cho Cells ◽

Cell Membranes ◽

Plasma Membranes ◽

Single Channel ◽

Water Channel ◽

Freeze Fracture ◽

Water Channels ◽

Straight Tubules ◽

Tetrameric Assembly

Channel forming integral protein of 28 kD (CHIP28) functions as a water channel in erythrocytes, kidney proximal tubule and thin descending limb of Henle. CHIP28 morphology was examined by freeze-fracture EM in proteoliposomes reconstituted with purified CHIP28, CHO cells stably transfected with CHIP28k cDNA, and rat kidney tubules. Liposomes reconstituted with HPLC-purified CHIP28 from human erythrocytes had a high osmotic water permeability (Pf0.04 cm/s) that was inhibited by HgCl2. Freeze-fracture replicas showed a fairly uniform set of intramembrane particles (IMPs); no IMPs were observed in liposomes without incorporated protein. By rotary shadowing, the IMPs had a diameter of 8.5 +/- 1.3 nm (mean +/- SD); many IMPs consisted of a distinct arrangement of four smaller subunits surrounding a central depression. IMPs of similar size and appearance were seen on the P-face of plasma membranes from CHIP28k-transfected (but not mock-transfected) CHO cells, rat thin descending limb (TDL) of Henle, and S3 segment of proximal straight tubules. A distinctive network of complementary IMP imprints was observed on the E-face of CHIP28-containing plasma membranes. The densities of IMPs in the size range of CHIP28 IMPs, determined by non-linear regression, were (in IMPs/microns 2): 2,494 in CHO cells, 5,785 in TDL, and 1,928 in proximal straight tubules; predicted Pf, based on the CHIP28 single channel water permeability of 3.6 x 10(-14) cm3/S (10 degrees C), was in good agreement with measured Pf of 0.027 cm/S, 0.075 cm/S, and 0.031 cm/S, respectively, in these cell types. Assuming that each CHIP28 monomer is a right cylindrical pore of length 5 nm and density 1.3 g/cm3, the monomer diameter would be 3.2 nm; a symmetrical arrangement of four cylinders would have a greatest diameter of 7.2 nm, which after correction for the thickness of platinum deposit, is similar to the measured IMP diameter of approximately 8.5 nm. These results provide a morphological signature for CHIP28 water channels and evidence for a tetrameric assembly of CHIP28 monomers in reconstituted proteoliposomes and cell membranes.

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Single-Channel Water Permeabilities of Escherichia coli Aquaporins AqpZ and GlpF

Biophysical Journal ◽

10.1529/biophysj.105.073965 ◽

2006 ◽

Vol 90 (7) ◽

pp. 2270-2284 ◽

Cited By ~ 91

Author(s):

Morten Ø. Jensen ◽

Ole G. Mouritsen

Keyword(s):

Escherichia Coli ◽

Single Channel ◽

Channel Water

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Functional analysis of aquaporin-2 mutants associated with nephrogenic diabetes insipidus by yeast expression

AJP Renal Physiology ◽

10.1152/ajprenal.1999.277.5.f734 ◽

1999 ◽

Vol 277 (5) ◽

pp. F734-F741 ◽

Cited By ~ 8

Author(s):

Itsuki Shinbo ◽

Kiyohide Fushimi ◽

Michihiro Kasahara ◽

Kazushi Yamauchi ◽

Sei Sasaki ◽

...

Keyword(s):

Diabetes Insipidus ◽

Water Permeability ◽

Nephrogenic Diabetes Insipidus ◽

Water Channel ◽

Osmotic Water Permeability ◽

Wild Type ◽

The Third ◽

Aquaporin 2 ◽

Channel Water ◽

Osmotic Water

Mutations of aquaporin-2 (AQP2) vasopressin water channel cause nephrogenic diabetes insipidus (NDI). It has been suggested that impaired routing of AQP2 mutants to the plasma membrane causes the disease; however, no determinations have been made of mutation-induced alterations of AQP2 channel water permeability. To address this issue, a series of AQP2 mutants were expressed in yeast, and the osmotic water permeability ( Pf) of the isolated vesicles was measured. Wild-type and mutant AQP2 were expressed equally well in vesicles. Pfof the vesicles containing wild-type AQP2 was 22 times greater than that of the control, which was sensitive to mercury and weakly dependent on the temperature. Pfmeasurements and mercury inhibition examinations suggested that mutants L22V and P262L are fully functional, whereas mutants N68S, R187C, and S216P are partially functional. In contrast, mutants N123D, T125M, T126M, A147T, and C181W had very low water permeability. Our results suggest that the structure between the third and fifth hydrophilic loops is critical for the functional integrity of the AQP2 water channel and that disruption of AQP2 water permeability by mutations may cause NDI.

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