Monitoring Single-channel Water Permeability in Polarized Cells

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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Calcium and related channels in fertilization and early development of Fucus

Philosophical Transactions of the Royal Society B Biological Sciences ◽

10.1098/rstb.1992.0133 ◽

1992 ◽

Vol 338 (1283) ◽

pp. 97-104 ◽

Cited By ~ 6

Keyword(s):

Plasma Membrane ◽

Single Channel ◽

Axis Formation ◽

Patch Clamping ◽

New Developments ◽

Inward Current ◽

Polarized Cells ◽

Single Channel Currents ◽

Axis Fixation ◽

Channel Currents

Unfertilized eggs of Fucus serratus are primed to respond rapidly to the fertilizing sperm. The unfertilized egg plasma membrane is excitable due to the presence of voltage-regulated Ca 2+ and K + channels. Sperm-egg interaction elicits a fertilization potential as the first observable fertilization event. It is speculated that sperm-gated Na + channels are responsible for the initial depolarization phase, leading to opening of Ca 2+ channels, allowing Ca 2+ influx and further depolarizing the membrane to the threshold for outward K + channels. K + efflux repolarizes the membrane and the zygote plasmalemma quickly becomes dominated by a large K + conductance. The involvement of Ca 2+ in axis formation and fixation is not clear. Ca 2+ carries a proportion of the inward current at the future rhizoid pole and asymmetric 45 Ca influx has been detected in polarizing zygotes. However, there is no requirement for external Ca 2+ in axis fixation. In contrast, Ca 2+ influx is required for expression of polarity and rhizoid growth. New developments in patch clamping can now enable localized areas of the plasma membrane in polarized cells to be studied. So far, both inward and outward single channel currents have been observed in the growing rhizoid tip, most probably carrying Cl - and K + respectively. These channels can be related to the currents identified by previous studies using the extracellular vibrating probe.

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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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