Boassa D , Yool AJ .

R01 GM59986 NIGMS NIH HHS , R01 GM059986 NIGMS NIH HHS

	Figure 1. Osmotically induced swelling in oocytes expressing AQP1 wild type and C-terminal mutant channels. A. Relative volume increases were determined by videomicroscopy as a function of time after the transfer of the oocytes from 200 mOsM into 100 mOsM saline at time 0. The change in cross-sectional area of the oocyte was converted to volume to calculate relative volume increase and standardized to the initial volume at time zero. Data are shown for representative individual oocytes. Panels show data for control and AQP1 wild type- or mutant-expressing oocytes from within the same batches of oocytes. B. Summary of the osmotic water permeability (Pf) values for wild type and mutant channels. Data for Pf (cm/s × 10-4) are mean ± SEM; n values are shown in parentheses (right). Unpaired two-tailed Student's t tests were used to analyze significance. An asterisk (*) indicates a value that is significantly different (p < 0.0005) from control; the pound sign (#) indicates a value that is significantly different (p < 0.009) from wild type AQP1.
	Figure 2. Wild type AQP1 channels show a dose-dependent ionic conductance response to the nitric oxide donor, sodium nitroprusside. A. Comparison of representative currents before and after 14 mM SNP in AQP1-expressing and control oocytes. Voltage steps were +60 mV to -110 mV in -10 mV intervals, from -40 mV holding potential. The net current is (final)-(initial). B. Dose-dependence of net current responses of wild type AQP1-expressing oocytes, with the same voltage protocol as in (A). C. Summary of data for conductance responses (mean ± SEM, n = 9–34 oocytes per group) at different doses of SNP (indicated in mM on horizontal axis). 'Initial' data are the conductance levels of the oocytes recorded prior to SNP application, and represent total conductance rather than net values. The net conductance responses were calculated by subtracting the matched initial values individually from the SNP response for each oocyte.D. Mean values ± SEM (n = 7 oocytes per group within the same batch of oocytes) are shown for net conductance responses to SNP for control (square) and AQP1-expressing oocytes (circle). The difference between mean net AQP1 and control conductances (diamond) was fit to estimate an EC50 value of 7.8 mM SNP; see text for details. E. Dose-dependent rate of activation of ionic current by SNP in representative AQP1-expressing oocytes, measured at 5 sec intervals with voltage steps to +40 mV from a holding potential of -40 mV. The arrow indicates the time of application of SNP in the recording chamber bath. Data prior to the application are superimposed.
	Figure 3. Intracellular cGMP produced in oocytes in response to SNP. Oocytes were tested in conditions comparable to those used for electrophysiological analyses, being assayed after 4 minutes of treatment with various concentrations of SNP in K+ recording saline. The amount of cGMP per oocyte was measured by enzyme immunoassay. Each point represents the mean of duplicate or triplicate measurements from four different experiments (separate batches of oocytes). Values shown are mean ± SEM.
	Figure 4. Inhibition of soluble guanylate cyclase prevents the AQP1 conductance response to SNP. Wild type AQP1-expressing and control oocytes were incubated three hours in ND96 saline with or without an inhibitor of soluble guanylate cyclase (1H-[1,2,4]oxadiazolo [4,3-a]quinoxalin-1-one, ODQ, 10 μM) prior to electrophysiological analysis of the response to SNP (doses indicated in mM on horizontal axis). Data are mean ± SEM; n = 3 or 4 oocytes per group. An asterisk (*) indicates values that are significantly different (p < 0.04) from control; the pound sign (#) indicates values that are significantly different (p < 0.04) from wild type AQP1 (unpaired two-tailed Student's t test).
	Figure 5. Summary of the effects of AQP1 C-terminal mutations on the net conductance responses evoked by SNP. Data are grouped to compare control, AQP1 wild type and mutant responses from within the same batches of oocytes. The mutants D237S, D237N (A) and D237K (B) showed dose-dependent responses to SNP (mM) comparable to that of wild type AQP1. However, the mutations D237A (A) and K243C (C) showed decreased conductance responses at all doses of SNP as compared with wild type. 'Initial' data are the conductance levels of the oocytes recorded prior to SNP application, and represent total conductance rather than net values. The net conductance responses were calculated by subtracting the matched initial values individually from the SNP response for each oocyte. Data are mean ± SEM; n = 3–28 oocytes per group. An asterisk (*) indicates values that are significantly different (p < 0.02) from control; the pound sign (#) indicates values that are significantly different (p < 0.04) from wild type AQP1 (unpaired two-tailed Student's t test).
	Figure 6. Double mutation D237A + K243C in the AQP1 carboxyl terminal domain shows a decreased conductance response to SNP as compared with wild type. A. Net current responses of control oocytes, and oocytes expressing AQP1 wild type and mutant channels were measured for voltage steps from +60 mV to -110 mV in -10 mV intervals, from -40 mV holding potential, after 4 minutes in 8 mM SNP. B. Summary of conductances (mean ± SEM, n = 9–18 oocytes per group) measured at different doses of SNP (mM). 'Initial' data are the conductance levels of the oocytes recorded prior to SNP application. The net conductance responses were calculated by subtracting the matched initial values individually from the SNP response for each oocyte. An asterisk (*) indicates values that are significantly different (p < 0.006) from control; the pound sign (#) indicates values that are significantly different (p < 0.0001) from wild type AQP1 (unpaired two-tailed Student's t test).
	Figure 7. Effects of preincubation with the reducing agent dithiothreitol (DTT) on SNP-induced ionic conductances. Oocytes expressing AQP1 wild type and double mutant D237A + K243C channels, and control oocytes were preincubated in ND96 saline with and without 10 mM DTT for 1 hour prior to electrophysiological recording of the response to SNP. Initial data are prior to SNP application; net conductances are (final)-(initial) for the SNP doses (mM) as indicated. An asterisk (*) indicates values that are significantly different (p < 0.006) from control; the pound sign (#) indicates values that are significantly different (p < 0.0001) from wild type AQP1 (unpaired two-tailed Student's t test) for comparisons within the same treatment group (with or without DTT). For other statistical comparisons, see details in text.

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