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Maintenance of proper biomechanics of the eye lens is important for its structural integrity and for the process of accommodation to focus near and far objects. Several studies have shown that specialized cytoskeletal systems such as the beaded filament (BF) and spectrin-actin networks contribute to mammalian lens biomechanics; mutations or deletion in these proteins alters lens biomechanics. Aquaporin 0 (AQP0), which constitutes ∼45% of the total membrane proteins of lens fiber cells, has been shown to function as a water channel and a structural cell-to-cell adhesion (CTCA) protein. Our recent ex vivo study on AQP0 knockout (AQP0 KO) mouse lenses showed the CTCA function of AQP0 could be crucial for establishing the refractive index gradient. However, biomechanical studies on the role of AQP0 are lacking. The present investigation used wild type (WT), AQP5 KO (AQP5(-/-)), AQP0 KO (heterozygous KO: AQP0(+/-); homozygous KO: AQP0(-/-); all in C57BL/6J) and WT-FVB/N mouse lenses to learn more about the role of fiber cell AQPs in lens biomechanics. Electron microscopic images exhibited decreases in lens fiber cell compaction and increases in extracellular space due to deletion of even one allele of AQP0. Biomechanical assay revealed that loss of one or both alleles of AQP0 caused a significant reduction in the compressive load-bearing capacity of the lenses compared to WT lenses. Conversely, loss of AQP5 did not alter the lens load-bearing ability. Compressive load-bearing at the suture area of AQP0(+/-) lenses showed easy separation while WT lens suture remained intact. These data from KO mouse lenses in conjunction with previous studies on lens-specific BF proteins (CP49 and filensin) suggest that AQP0 and BF proteins could act co-operatively in establishing normal lens biomechanics. We hypothesize that AQP0, with its prolific expression at the fiber cell membrane, could provide anchorage for cytoskeletal structures like BFs and together they help to confer fiber cell shape, architecture and integrity. To our knowledge, this is the first report identifying the involvement of an aquaporin in lens biomechanics. Since accommodation is required in human lenses for proper focusing, alteration in the adhesion and/or water channel functions of AQP0 could contribute to presbyopia.

Activity of the epithelial Na+ channel (ENaC) is limiting for [Na.sup.+] reabsorption at the distal nephron. Gain-of-function mutations in ENaC cause Liddle's syndrome: a severe form of inheritable hypertension. Several polymorphisms in [alpha]-hENaC possibly associated with abnormal Na+ handling by the kidney and the salt-sensitive hypertension prevalent in black populations have been reported. The functional effects of [alpha]-hENaC polymorphisms on channel activity, however, remain controversial and have not been directly tested in a mammalian background. We ask here whether polymorphisms at positions 334, 618, and 663 in [alpha]-hENaC influence channel activity. Activity of wild-type (A334, C618, A663) and polymorphic ENaC expressed in Chinese hamster ovary cells was assessed with patch-clamp electrophysiology. While the A334T polymorphism had little effect on macroscopic ENaC currents, the C618F and A663T polymorphisms significantly increased ENaC activity >3.3- and 1.6-fold, respectively. Similarly, polymorphic ENaC had greater activity compared with wild-type channels in excised patches with activity of C618F and A663T channels increased 3.8- and 2.6-fold, respectively. Unitary channel conductances and reversal potentials were not different for polymorphic and wild-type ENaC. Increases in activity resulted primarily from increases in the apparent number of active (polymorphic) channels in the plasma membrane. Moreover, addition of a reducing agent to the cytosol significantly increased activity of wild-type ENaC equal to that of C618F polymorphic channels but had no effect on these latter channels. These results are consistent with the C618F and A663T polymorphisms leading to elevated ENaC activity with the possibility that they facilitate altered [Na.sup.+] handling by the kidney. hypertension; Liddle's syndrome; sequence variations

Evidence that lymphangiomyomatosis (LAM) is caused by TSC2 (ital) mutations has been found but genetic transmission of LAM has not been reported. On chromosome 16p13 loss of heterozygosity has been found in angiomyolipomas and lymph nodes of women with LAM, a rare disease which involves diffuse proliferation of smooth muscle cells and can only be treated by transplanting lungs. LAM may or may not be associated with tuberous sclerosis complex (TSC), an autosomal dominant tumor suppressor gene syndrome. Loss of heterozygosity is found in the chromosomal region for the TSC2 (ital) gene in 60% of tuberous sclerosis complex (TSC)-associated angiomyolipomas. It seems pathogenesis of sporadic LAM may be related to TSC2 (ital). Women with LAM may have low-penetrance germ-line TSC2 (ital) mutations or may be mosaic, having TSC2 (ital) mutations in the kidney and lungs and not in other organs.

We previously reported the presence of a novel variant ([Beta]-T594M) of the amiloride-sensitive [Na.sup.+] channel (ASSC) in which the threonine residue at position 594 in the [Beta]-subunit has been replaced by a methionine residue. Electrophysiological studies of the ASSC on Epstein-Barr virus (EBV)-transformed lymphocytes carrying this variant showed that the 8-(4-chlorophenyithio) adenosine 3[prime]:5[prime]-cyclic monophosphate (8cpt-cAMP)-induced responses were enhanced when compared to wild-type EBV-transformed lymphocytes. Furthermore, in wild-type EBV-transformed cells, the 8cpt-cAMP-induced response was totally blocked by the phorbol ester, phorbol 12-myristate 13-acetate (PMA). This inhibitory effect of PMA was blocked by a protein kinase C inhibitor, chelerythrine. We now have identified individuals who are homozygous for this variant, and showed that PMA had no effect on the 8cpt-cAMP-induced responses in the EBV-transformed lymphocytes from such individuals. Cells heterozygous for this variant showed mixed responses to PMA, with the majority of cells partially inhibited by PMA. Our results demonstrate that an alteration in a single amino acid residue in the [Beta]-subunit of the ASSC can lead to a total loss of inhibition to PMA, and establish the [Beta]-subunit as having an important role in conferring a regulatory effect on the ASSC of lymphocytes.

The identification of a candidate gene for neurofibromatosis 2 (NF2), a dominantly inherited disorder characterized by the occurrence of bilateral schwannomas and other central nervous system tumors such as multiple meningiomas, is reported. Previous studies have suggested the inactivation of a tumor suppressor gene in choromosome 22q12 as a potential cause of the disorder. The candidate NF2 gene encodes a 587-amino acid protein which is related to the membrane-organizing extension spike protein-ezrin-radixin family of proteins that link cytoskeletal proteins with proteins in the cell membrane.