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BACKGROUND: Aquaporin-1 (AQP1) channels are constitutively active water channels that allow rapid transmembrane osmotic water flux, and also serve as cyclic-GMP-gated ion channels. Tetraethylammonium chloride (TEA; 0.05 to 10 mM) was shown previously to inhibit the osmotic water permeability of human AQP1 channels expressed in Xenopus oocytes. The purpose of the present study was to determine if TEA blocks osmotic water flux of native AQP1 channels in kidney, and recombinant AQP1 channels expressed in a kidney derived MDCK cell line. We also demonstrate that TEA does not inhibit the cGMP-dependent ionic conductance of AQP1 expressed in oocytes, supporting the idea that water and ion fluxes involve pharmacologically distinct pathways in the AQP1 tetrameric complex.
RESULTS: TEA blocked water permeability of AQP1 channels in kidney and kidney-derived cells, demonstrating this effect is not limited to the oocyte expression system. Equivalent inhibition is seen in MDCK cells with viral-mediated AQP1 expression, and in rat renal descending thin limbs of Henle's loops which abundantly express native AQP1, but not in ascending thin limbs which do not express AQP1. External TEA (10 mM) does not block the cGMP-dependent AQP1 ionic conductance, measured by two-electrode voltage clamp after pre-incubation of oocytes in 8Br-cGMP (10-50 mM) or during application of the nitric oxide donor, sodium nitroprusside (2-4 mM).
CONCLUSIONS: TEA selectively inhibits osmotic water permeability through native and heterologously expressed AQP1 channels. The pathways for water and ions in AQP1 differ in pharmacological sensitivity to TEA, and are consistent with the idea of independent solute pathways within the channel structure. The results confirm the usefulness of TEA as a pharmacological tool for the analysis of AQP1 function.
Figure 1. Block by TEA of AQP1-mediated water permeability in monolayers of adenovirus-infected MDCK cells. (A) To account for differences in levels of AQP1 expression between different experimental preparations, data for "Percent water flux" were standardized to the mean water flux measured for AQP1-expressing monolayers without TEA on the same experimental day. Cells infected with empty adenovirus were untreated (Control) or treated with TEA (Con TEA). Cells infected with AQP1 adenovirus and expressing AQP1 were untreated (AQP) or treated with TEA (AQP TEA). Box plots show the distribution of data values; the gray box encloses 50% of the data points, the error bars show the full range, and the horizontal bar shows the modal value. Data for mean, standard error and n value are shown below each column. Significant differences were analyzed by T-test and are reported as p < 0.001 (*), and not significant (N.S.; p < 0.50) (B). Whole cells lysates were prepared from MDCK cells on filters and 10 μg of protein was loaded onto each lane. Shown is the expression of AQP1 protein in MDCK cells infected with empty (CON) or AQP1 adenovirus. No AQP1 protein was seen in CON lanes even after extended exposures to film (i.e., 30 minutes).
Figure 2. Immunocytochemical analysis confirms the expression of AQP1 channels in pure descending but not ascending thin limb preparations. Examples are shown for isolated preparations of rat inner medullary descending thin limb(upper panel) and inner medullary ascending thin limb (lower panel) labeled with a rabbit antibody to the AQP1 carboxy tail domain, and visualized with Cy5-labeled antirabbit antibody immunoglobulin-G. The scale bar is 100 μm.
Figure 3. Analysis of water flux in isolated perfused renal ascending and descending thin limbs of Henle's loop under osmotic gradient conditions.A. Compilation of Jv values obtained for descending thin limbs that were untreated (Desc cont) or exposed to 10 mM TEA (Desc TEA), and for ascending thin limbs that were untreated (Asc cont) or exposed to TEA (Asc TEA). B. Compilation of calculated osmotic water permeabilities (Pf) for the same tubules as analyzed in (A). Box plots show the distribution of data values; the gray box encloses 50% of the data points, the error bars show the full range, and the horizontal bar shows the modal value. Data for group means, standard errors and n values are shown below each column. However, since each tubule served as its own control, differences were analyzed by paired T-test and are reported as p < 0.02 (**), p < 0.05 (*), and not significant (N.S.).
Figure 4. Tetraethylammonium does not block the ionic conductance in cGMP-activated AQP1 channels expressed in Xenopus oocytes.A. Recordings of current responses to voltage steps analyzed by two-electrode voltage clamp in an AQP1-expressing oocyte before stimulation (initial), after cGMP-dependent activation in 100 mM Na+ saline (activated) and after perfusion with saline containing 10 mM TEA and 90 mM Na+ (+TEA). No block of the ionic current was observed. B. Plot of the current-voltage relationship for data illustrated in (A). Conductance values calculated from linear fits of the data from -50 to +30 mV were 3.0 μS initial, 57.8 μS after activation in Na+ saline, and 57.3 μS with 10 mM TEA.
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