Membrane organization and function of M1 and M23 isoforms of aquaporin-4 in epithelial cells

Abstract

(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 (cm 3 /s) is much greater for the M23 variant. Somatic fusion of M1-and M23-AQP4 cells (Pf ϭ 0.028 cm/s) yielded OAPs that were fewer and smaller than in M23 cells alone, and M1-to-M23 expression ratios (ϳ1:4) normalized to AQP4 in M1 or M23 cells indicated a reduced single-channel pf for the M23 variant. Expression of an M23-AQP4-Ser 111E mutant produced ϳ1.5-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 Ser 111 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. water transport; freeze-fracture; LLC-PK1 cells; orthogonal arrays; intramembraneous particles THE MAMMALIAN FAMILY OF AQUAPORIN water channels consists of 12 known members, each with a specific tissue distribution and membrane localization pattern. However, the role of aquaporin-4 (AQP4) in water transport physiology is not well understood. AQP4 was the first aquaporin to be observed and identified in biological membranes, because when examined by freeze-fracture electron microscopy, it forms characteristic arrays of intramembraneous particles (IMPs) in the form of checkerboard aggregates or orthogonal arrays of intramembraneous particles (OAPs). These OAPs were described in various cell membranes long before a role in water permeability was suspected (3,18,27,31,32). The relationship of OAPs to water channels was first suggested by earlier studies showing that membranes that contained OAPs, including astrocytes, gastric parietal cells, and collecting duct principal cells were immunostained by an antibody raised against the whole AQP1 (then called CHIP28) protein (37). A different antibody raised against skeletal muscle OAP-containing membranes also recognized an ϳ30-kDa protein in these membranes (15,41). In this way, a protein initially called basolateral intrinsic membrane protein (BLIP) was ...

MicroRNA-130a Represses Transcriptional Activity of Aquaporin 4 M1 Promoter

“…Water permeability studies showed that the M1 isoform conducts water at a higher rate than the M23 isoform (4,6). Quantitative immunoblotting and immunofluorescence studies showed that expression of AQP4 M1 was 8-fold higher than expression of M23 (7). On the contrary, Hirt et al (8) observed higher expression of M23 mRNA levels in mouse brain.…”

“…Expression of M1 is possibly crucial in cerebral homeostasis as its single channel water permeability rates are much higher than M23. Furthermore, M23 is often implicated with structural organization of the membrane (7,22).…”

Live Cell Analysis of Aquaporin-4 M1/M23 Interactions and Regulated Orthogonal Array Assembly in Glial Cells

“…Freezefracture electron microscopy of cells transfected with AQP4-M23 shows large OAPs, sometimes containing Ͼ100 particles, whereas OAPs are largely absent in cells transfected with AQP4-M1 (33). OAP size is reduced when AQP1-M1 is coexpressed with AQP4-M23, suggesting an interaction between these two isoforms in vivo (21,33).…”

Hydrocephalus: the role of cerebral aquaporin-4 channels and computational modeling considerations of cerebrospinal fluid

“…The single-channel water permeability of M23 is approximately 8 times greater than that of M1, likely due to the ability of M23 to form stable square arrays. 118 It has been further suggested that the larger the array, the more it is able to accommodate a higher water flux through the membrane induced by an osmotic gradient, consistent with the speculation that AQP4 could play an adhesive role at gap junctions. 48 At the end-foot cellular membrane near the perivascular space, the anchoring of AQP4 requires a-syntrophin, a member of the dystrophin complex.…”

“…46 Silberstein et al found that a phosphomimetic M23-Ser 111E forms 2.5-times larger square arrays compared with wild-type M23 and possesses a 1.5-fold increase in single-channel water permeability. 118 However, Assentoft et al did not find an increase in water permeability in oocytes transfected with AQP4 with a phosphomimetic Ser 111 site or in oocytes transfected with AQP4 and treated with protein kinase A (PKA) or PKG activators. 9 They also did not detect evidence of Ser 111 phosphorylation in vivo after treating animals with an mGluR1/5 analog.…”