|
Figure 1
Sequence analysis. A, alignment of amino acid sequences of AQPxlo, human AQP3, human AQP10, and the bacterial glycerol facilitator GlpF. Sequences were aligned using the Megalign module (Clustal method) of the Lasergene program suite (DNAstar). Membrane-spanning segments of GlpF, derived form the crystal structure (6), are underlined, and identical amino acids are highlighted. B, dendrogram of AQPs. We used Megalign (Clustal method) to align the amino acid sequences of AQPxlo, mouse AQP 0â5, 7, and 8, rat AQP 6 and 9, human AQP10,Xenopus AQP3, and GlpF. % divergence is indicated by the summed horizontal lengths of line segments between labels.C, hydropathy analysis of AQPxlo. Hydropathy analysis (Kyte-Doolittle algorithm, window size 9) is consistent with six membrane-spanning regions (numbered 1â6) and five connecting loops (AâE).
|
|
Figure 2
Northern blot analysis. X. laevis total RNA was probed with a 32P-labeled cDNA probe corresponding to the full coding region of AQPxlo. The RNA was run on two separate denaturing agarose gels, and the autoradiographs were apposed for display. The positions and sizes of molecular weight markers are indicated on the left. The molecular weight of the single band in fat body, oocyte,
|
|
Figure 3
P f values of oocytes expressing different AQPs. Oocytes were injected with water or cRNA encoding AQP1 (2.5 ng/oocyte), AQP3 (10 ng/oocyte), AQP7 (10 ng/oocyte), or AQPxlo (20 ng/oocyte). Error bars, S.E.,n = 5–7. Asterisk indicates that the difference is statistically significant (p < 0.05) compared with water-injected oocytes in a two-tailed ttest.
|
|
Figure 4
Effect of pHo changes onP f and pHi of AQPxlo-expressing oocytes. A, P f. Oocytes were preincubated for 2 min at the appropriate pHo before switching to a hypotonic solution with the same pH. Closed circles, AQPxlo-expressing oocytes; closed diamonds, water-injected oocytes. n = 5 (H2O) and 8–14 (AQPxlo).B, pHi. Microelectrodes were used to measure pHi and V m of oocytes exposed to solutions of different pHo values. Similar recordings were obtained in one other AQPxlo-expressing oocyte and two water-injected oocytes.A, asterisk indicates that the difference is statistically significant (p < 0.05) compared with the value obtained at pHo 7.5 in a two-tailed t test.Vertical bars indicate S.E. but are omitted when they are smaller than the symbol.
|
|
Figure 5
Effect of pHo changes onP f, glycerol, and urea uptake in oocytes expressing different AQPs. A, P f. B, glycerol uptake. C,urea uptake. Measurements were carried out on oocytes injected with water or expressing AQP3, AQP7, or AQPxlo. Dark gray bars, pHo 7.5; light gray bars, pHo 9.5.n = 7–12. Error bars, S.E.Asterisk indicates that the difference between control and AQP-expressing oocytes, at the relevant pHo, is statistically significant (p < 0.05). Dagger indicates that the difference between pHo 7.5 and 9.5, comparing oocytes expressing the same AQP, is statistically significant in a two-tailedt test.
|
|
Figure 6
Effect of HgCl2 onP f. Oocytes injected with water (closed diamonds) or expressing either AQP1 (open squares) or AQPxlo (closed circles) were treated with the indicated concentrations of HgCl2. Data are expressed as % of control (i.e. absence of HgCl2, indicated by a dotted line). n = 6–9 (H2O), 4–6 (AQP1), and 5–12 (AQPxlo). Error bars, S.E. Asterisk indicates that the difference between (un-normalized) values in HgCl2 and controls is statistically significant (p < 0.05) in a two-tailedt test.
|
|
Figure 7
HgCl2 inhibition ofP f, effect of pHo changes, and reversibility. A, effect of pHo. AQPxlo-expressing oocytes were either untreated or pretreated with 1 μM HgCl2 at pHo 7.5 for 5 min. Subsequently, P f was measured (i) in untreated oocytes at pHo 7.5 or 9.5 (black bars); (ii) in pretreated oocytes in the continued presence of HgCl2 at pHo 7.5 (light gray bar); or (iii) in pretreated oocytes in the absence of HgCl2 either at pHo 7.5 or 9.5 (dark gray bars). B, reversibility of HgCl2 inhibition. P f was measured in the same oocyte before HgCl2 treatment, after treatment with HgCl2 (1 μM), and after treatment with β-mercaptoethanol (5 mM). Error bars, S.E.Asterisk indicates that the difference between HgCl2-treated and -untreated oocytes is statistically significant (p < 0.05) in a two-tailed ttest.
|
|
Figure 8
Effect of HgCl2 and phloretin on glycerol and urea uptake. A, effect of HgCl2 on glycerol uptake in water-injected oocytes and oocytes expressing AQP3 or AQPxlo. B, effect of HgCl2 on urea uptake in water-injected oocytes and in oocytes expressing AQPxlo. Black bars indicate control condition; light gray bar, 1 μMHgCl2; and dark gray bars, 300 μMHgCl2. C, effect of phloretin on urea uptake in water-injected oocytes and oocytes expressing UT-A2 or AQPxlo.Black bars indicate control condition; gray barsindicate 500 μM phloretin. A and B,error bars, S.E. n = 6–16; C,n = 4–8. Asterisk indicates that the difference between that value and water-injected control is statistically significant (p < 0.05) in a two-tailedt test. Dagger indicates that the difference between that value and no-drug control (black bar) is statistically significant (p < 0.05) in a two-tailedt test.
|
|
Figure 9
Reflection coefficients ς for osmolytes. A, ς measured for straight chain polyols (glycerol, xylitol, ribitol, and mannitol), urea and thiourea in AQPxlo-expressing oocytes. B, ς measured for glycerol and urea in water-injected oocytes (black bars) and oocytes expressing AQP1 (light gray bars) or AQP3 (dark gray bars). A, error bars, S.E. n = 6;B, n = 4–6. Asterisk indicates that the value of ς is significantly (p < 0.05) smaller than 1.
|
