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. 2006 Jul 26;26(30):7811-9.
doi: 10.1523/JNEUROSCI.0525-06.2006.

Ion channel function of aquaporin-1 natively expressed in choroid plexus

Daniela Boassa  1 , W Daniel Stamer, Andrea J Yool
Affiliations

Ion channel function of aquaporin-1 natively expressed in choroid plexus

Daniela Boassa et al. J Neurosci. .

Abstract

Aquaporins are known as water channels; however, an additional ion channel function has been observed for several including aquaporin-1 (AQP1). Using primary cultures of rat choroid plexus, a brain tissue that secretes CSF and abundantly expresses AQP1, we confirmed the ion channel function of AQP1 and assessed its functional relevance. The cGMP-gated cationic conductance associated with AQP1 is activated by an endogenous receptor guanylate cyclase for atrial natriuretic peptide (ANP). Fluid transport assays with confluent polarized choroid plexus cultures showed that AQP1 current activation by 4.5 mum ANP decreases the normal basal-to-apical fluid transport in the choroid plexus; conversely, AQP1 block with 500 mum Cd2+ restores fluid transport. The cGMP-gated conductance in the choroid plexus is lost with targeted knockdown of AQP1 by small interfering RNA (siRNA), as confirmed by immunocytochemistry and whole-cell patch electrophysiology of transiently transfected cells identified by enhanced green fluorescent protein. The properties of the current (permeability to Na+, K+, TEA+, and Cs+; voltage insensitivity; and dependence on cGMP) matched properties characterized previously in AQP1-expressing oocytes. Background K+ and Cl- currents in the choroid plexus were dissected from AQP1 currents using Cs-methanesulfonate recording salines; the background currents recorded in physiological salines were not affected by AQP1-siRNA treatment. These results confirm that AQP1 can function as both a water channel and a gated ion channel. The conclusion that the AQP1-associated cation current contributes to modulating CSF production resolves a lingering concern as to whether an aquaporin ionic conductance can have a physiologically relevant function.

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Figures

Figure 1.
Figure 1.
AQP1 expression in the choroid plexus in vivo and in vitro and knockdown by AQP1–siRNA treatment. a, Reverse transcription-PCR confirmation of AQP1 expression in vitro. Lane 1, Low mass DNA marker; lanes 2–6, 8, PCR products for AQP1 using cDNA from reverse-transcribed (RT) RNA (lanes 2, 3) at 114 and 400 ng, respectively, non-RT-RNA (lane 4; showing lack of genomic contamination), cloned AQP1 cDNA (lanes 5, 6), and no template (lane 8); lane 7, PCR product for cyclophilin (cyclop) using cDNA from reverse-transcribed RNA as a constitutively expressed control marker. b, Cryostat-sectioned fourth ventricle rat choroid plexus immunostained for AQP1 (green) and prealbumin (red). c, Confocal image of primary cultures showing AQP1 (green) and prealbumin (red). d–f, AQP1–siRNA knockdown. d, e, Phase contrast superimposed with epifluorescent images 48 h after cotransfection of AQP1-mixed siRNA and eGFP marker (green), immunolabeled for AQP1 (red). f, Fluorescence image showing AQP1 (red) is absent in the eGFP–siRNA-transfected cell (blue arrowhead). The top panel shows AQP1 and eGFP; the bottom panel is the same image for AQP1 alone. Scale bars, 10 μm. g–i, eGFP-transfected control. g, h, Control cells transfected with eGFP alone (48 h) show that both AQP1 (red) and eGFP (green) are present in the transfected cell. h, Top, AQP1 and eGFP. Bottom, AQP1 alone. i, Confocal image of a control eGFP-transfected cell (green) expressing AQP1 (red). A z-scan by confocal microscopy (bottom) along the indicated axis (white line; top) shows eGFP filling the cytoplasm of a cell in a full cross section (arrows), with AQP1 expression appropriately restricted to the apical surface.
Figure 2.
Figure 2.
Properties of the cGMP-dependent cation current in cultured choroid plexus cells analyzed by whole-cell patch clamp. a–c, Ionic currents in Cs-methanesulfonate salines (pipette and bath) were activated by SNP and blocked by extracellular Cd2+. a, Traces of currents recorded in a cell before SNP (initial), after a bolus application of SNP, and after subsequent additions of Cd2+ to the bath saline. Concentrations are estimated final values. Voltage steps are +40 to −110, from a −30 mV holding potential. b, Current–voltage relationship for the cell shown in a. c, Summary of the mean amplitude of the initial conductance (init) and the SNP-induced conductances before and after block by Cd2+ (n = 5 each). d, Monovalent cation selectivity determined by the measured reversal potentials, Erev, of SNP-induced currents (n = 6 each) in various ion-substituted salines (circle) compared with calculated equilibrium potentials for Cl− (asterisk) and nonselective cation (triangle) conductances. e, Block of the response to SNP but not to ANP by the soluble guanylate cyclase inhibitor ODQ (14 μm; 15 min). f, h, Ionic currents in Cs+ salines activated by ANP. f, ANP-dependent activation of a voltage-insensitive conductance comparable to that seen with SNP. Voltage steps were +40 to −110 from a holding potential of −30 mV. g, Summary histogram of the mean whole-cell conductances measured after preincubation with ODQ for initial recordings (init), and after treatment with SNP or ANP (n = 5 per treatment group). h, Current amplitude at +40 mV measured by brief pulses from a holding potential of −30 mV every 10 s. SNP, ANP, and Cd were applied as boluses over times indicated by bars, with estimated final concentrations shown. ODQ preincubation prevented the response to SNP but not ANP. The highest dose of ANP appeared to decrease current amplitude, perhaps reflecting desensitization. The current was blocked by Cd2+.
Figure 3.
Figure 3.
Excised inside-out patch recording from choroid plexus in vitro, illustrating activity of a Cd2+-sensitive AQP1-like channel. a, Consecutive sweeps at +60 mV after application of 8Br-cGMP (1 mm). The numbered boxes mark three classes of channels distinguished by unitary conductances. b, Selective block of the large-conductance, AQP1-like channel by Cd2+ (500 μm). c, Recovery after washout of Cd2+. d, Current amplitudes determined from all-points histograms. Unitary channel current levels are identified at +60 mV by numbered boxes; other amplitudes reflect combinations of channel events.
Figure 4.
Figure 4.
Loss of the cGMP-dependent cationic conductance by siRNA knockdown of AQP1 in the choroid plexus in vitro. a, Whole-cell currents in Cs-methanesulfonate saline before and after SNP and after subsequent application of Cd2+ in cells transfected with AQP1-mixed siRNA (15 nm), commercial control siRNA (60 nm), or eGFP alone. b, Currents recorded without (left) and with (right) 8Br-cGMP in the pipette from cells transfected with 15 nm AQP1–siRNA mixture, 60 nm control siRNA mixture, or eGFP alone, and from nontransfected cells. Voltage steps are +60 to −110 mV from a holding potential of −60 mV. c, Summary of the results of treatments with AQP1-mixed siRNAs and commercial control siRNA. Conductances (G/Gmax) were standardized to the mean cGMP-evoked conductance for all nontransfected cells within the same set of cultures. ∗∗p < 0.0001, statistically significant difference from all other cGMP groups (unpaired two-tailed Student’s t test). d, Summary of the results of treatments with synthetic sequence-specific AQP1–siRNAs [labeled as “a” and “b” to match the published data of Splinter et al. (2003)] at different concentrations and control siRNAs synthesized as scrambled versions of AQP1–siRNA-a and -b. Conductances (G/Gmax) of Cs+ currents with 10 mm 8Br-cGMP in the pipette saline were standardized to the mean cGMP-evoked conductance for all nontransfected cells within the same set of cultures. ∗p < 0.05 and ∗∗p < 0.006, statistically significant differences from the nontransfected group (unpaired two-tailed Student’s t test). NS, Not significant; no transf, no transfection.
Figure 5.
Figure 5.
Lack of appreciable effects of AQP1–siRNA treatment on endogenous background ion currents in the choroid plexus, measured with physiological salines in the absence of cGMP stimulation. a, Comparison of traces of ionic currents from choroid plexus cells that were untreated (control; top row) or transfected with 15 nm AQP1-mixed siRNA (bottom row). Currents were measured without leak subtraction (total currents) and with an on-line P/4 leak subtraction protocol (voltage-sensitive currents), with NaCl bath saline and K-gluconate pipette saline. b, Averaged current–voltage relationships compiled for all cells tested in the two treatment groups, with current amplitudes standardized to the maximal outward current amplitude measured at +60 mV, show no differences in rectification and reversal potentials. c, Data from b plotted as absolute rather than standardized amplitudes show no significant differences in mean current amplitudes between the control and siRNA treatment groups. Control group, n = 13–14; AQP1–siRNA-treated group, n = 6.
Figure 6.
Figure 6.
Net fluid transport assay in confluent monolayers before and after treatment with 4.5 μm ANP, with and without 500 μm Cd2+ in the apical solution. The baseline rate defined as 100% indicates the net fluid flow rate in the monolayer culture before treatment with ANP; baseline flow rates averaged 50 μl/h and ranged from 35 to 117 μl/h for different preparations. Data for flow rates after treatments were standardized as a percentage of the corresponding baseline rate in the same monolayer culture. ∗p < 0.05 and ∗∗p < 0.001, statistically significant differences (unpaired two-tailed Student’s t test). NS, Nonsignificant. ANP, n = 4; ANP + Cd2+, n = 4. Data are mean ± SD.
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