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Biophys Rep (N Y)
2023 Mar 08;31:100100. doi: 10.1016/j.bpr.2023.100100.
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Proteoliposomes reconstituted with human aquaporin-1 reveal novel single-ion-channel properties.
Henderson SW
,
Nakayama Y
,
Whitelaw ML
,
Bruning JB
,
Anderson PA
,
Tyerman SD
,
Ramesh SA
,
Martinac B
,
Yool AJ
.
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Human aquaporin 1 (hAQP1) forms homotetrameric channels that facilitate fluxes of water and small solutes across cell membranes. In addition to water channel activity, hAQP1 displays non-selective monovalent cation-channel activity gated by intracellular cyclic GMP. Dual water and ion-channel activity of hAQP1, thought to regulate cell shape and volume, could offer a target for novel therapeutics relevant to controlling cancer cell invasiveness. This study probed properties of hAQP1 ion channels using proteoliposomes, which, unlike conventional cell-based systems such as Xenopus laevis oocytes, are relatively free of background ion channels. Histidine-tagged recombinant hAQP1 protein was synthesized and purified from the methylotrophic yeast, Pichia pastoris, and reconstituted into proteoliposomes for biophysical analyses. Osmotic water channel activity confirmed correct folding and channel assembly. Ion-channel activity of hAQP1-Myc-His6 was recorded by patch-clamp electrophysiology with excised patches. In symmetrical potassium, the hAQP1-Myc-His6 channels displayed coordinated gating, a single-channel conductance of approximately 75 pS, and multiple subconductance states. Applicability of this method for structure-function analyses was tested using hAQP1-Myc-His6 D48A/D185A channels modified by site-directed mutations of charged Asp residues estimated to be adjacent to the central ion-conducting pore of the tetramer. No differences in conductance were detected between mutant and wild-type constructs, suggesting the open-state conformation could differ substantially from expectations based on crystal structures. Nonetheless, the method pioneered here for AQP1 demonstrates feasibility for future work defining structure-function relationships, screening pharmacological inhibitors, and testing other classes in the broad family of aquaporins for previously undiscovered ion-conducting capabilities.
Figure 1. Typical unilamellar blisters used for patch-clamp electrophysiology in this study. Phase contrast micrograph of unilamellar membrane blisters (black arrows) within solution that have developed from a cluster of collapsed proteoliposomes in the presence of Mg2+. Blisters are amenable to GΩ seal formation with a borosilicate glass micropipette (patch pipette, white asterisk). Dashed lines flanking the patch pipette have been added for clarity.
Figure 2. Water channel activity of hAQP1-Myc-His6 in X. laevis oocytes. (A) Oocytes expressing tagged human hAQP1 variants, or control oocytes, were exposed to 50% hypotonic saline at t = 0. Relative volume change was measured from the time-lapse images. Data are mean ± SEM of three untagged wild-type (WT), 13 (variant A), 12 (variant B), and seven (control) oocytes. (B) Boxplots showing the swelling rates of oocytes measured in (A). Significant differences were determined by one-way ANOVA. ∗∗∗∗p < 0.0001; ns, not significant. (C) Immunoblot of plasma-membrane-enriched fractions from 15 pooled oocytes injected with hAQP1-Myc-His6-B or from 15 water-injected controls. Bands were visualized using an anti-His antibody. (D) Water permeability increased linearly when oocytes were injected with increasing amounts of cRNA (1–10 ng) encoding hAQP1-Myc-His6-B. Data are mean ± SD of 10 oocytes (except 2.5 ng, which are 11 oocytes) after the mean water permeability of 12 control oocytes (injected with 50 nL of water) was subtracted. Data were fitted with a linear regression (R2 = 0.59).
Figure 3. Ion-channel activity of hAQP1-Myc-His6 in X. laevis oocytes. (A and B) Current-voltage relationships of oocytes injected with hAQP1-Myc-His6 (A) or water-injected controls (B) in isotonic Ca2+-free saline at the start of the recording (initial), after activation with 10 μM CPT-cGMP (labeled “cGMP”), and after application of 600 μM CdCl2 to the activated cells (Cd2+). Data are mean ± SEM for six (hAQP1-Myc-His6) or three (water-injected control) oocytes. (C and D) Slope conductance values for individual oocytes expressing hAQP1-Myc-His6 (C) or water-injected controls (D) before activation (initial), after activation (cGMP), and after block (Cd2+). Different letters indicate statistically significant differences between the means (ANOVA, p < 0.001). (E) Whole-cell current trace recordings illustrate the CPT-cGMP activation of hAQP1-Myc-His6-mediated ion currents. Dashed lines indicate zero current levels.
Figure 4. Expression and purification of functional recombinant hAQP1-Myc-His6 water channels from P. pastoris. (A) Dot-blot screening of crude extracts from independent P. pastoris transformants harboring hAQP1-Myc-His6 from pPICZ-A (upper) or pPICZ-B (lower). X33 WT control shows low background signal. Asterisks indicates strains used for immunoblot. (B) hAQP1-Myc-His6 detection by immunoblot. P. pastoris X33 containing hAQP1-Myc-His6 from pPICZ-A or -B, untransformed negative control (X33), or BcAP8 (positive control) were induced in BMMY for 6 h (left) or 24 h (right). Total protein (10 μg) from crude extracts was loaded onto TGX gels, transferred to nitrocellulose, and probed with an anti-His antibody. Total protein loading is demonstrated by Ponceau S staining. (C) Coomassie-stained SDS-PAGE gel electrophoresis demonstrates highly purified hAQP1-Myc-His6 from pPICZ-B. Fractions were eluted from Ni-NTA resin using 100, 250, or 475 mM imidazole. Approximately 1 μg of total protein was loaded per lane. MW, molecular weight marker. (D) Stopped-flow spectrometry measurements of water fluxes in proteoliposomes reconstituted with hAQP1-Myc-His6 (blue trace), control liposomes without protein (gray trace), and proteoliposomes containing hAQP1-Myc-His6 that were pre-treated with 500 μM HgCl2 for 20 min (red trace). Averages of five traces are shown as colored lines and represent the light scatter of vesicles. Fitted exponential curves are shown as black lines.
Figure 5. cGMP-stimulated electrical properties of proteoliposomes reconstituted with hAQP1-Myc-His6. (A) Gap-free recording of an inside-out patch from a proteoliposome containing hAQP1-Myc-His6 in symmetrical (112 mM) K+ solutions at −100 mV. Arrow indicates time of CPT-cGMP addition (labeled “cGMP”). (B) Increased-resolution view of (A). (C) Gap-free recording of a control (empty) proteoliposome at −60 mV in the presence of 10 μM CPT-cGMP. Dotted lines indicate zero current levels.
Figure 6. Single-channel properties of hAQP1-Myc-His6 in patch-clamped proteoliposomes. (A) Gap-free recording of an inside-out patch from a proteoliposome containing hAQP1-Myc-His6 in symmetrical (112 mM) K+ solutions at 0 and −100 mV (shown below the trace). Dotted line indicates zero current level; dashed lines denote channel configurations where C is closed and O is open. (B) Single-channel bursts of hAQP1-Myc-His6 activity recorded during steps to different holding potentials (indicated at the right of each trace) for 2.7 s (total). Dashed lines indicate different open-state levels. Open probability (Po) ± SD is shown for each voltage. Po analyses were done specifically on recorded segments that had active channels. (C) Recordings of control patches from liposomes without hAQP1-Myc-His6 at ±100 mV showed no single-channel activity. Dotted lines indicate zero current levels. (D) Amplitude histogram of idealized events at −100 mV fitted with a Gaussian function that indicates two open-state levels and a single-channel amplitude of 6.5 pA (± 0.48 SD). (E) Current-voltage relationships of hAQP1-Myc-His6 in symmetrical (112 mM) K+ solutions. Unitary amplitudes were plotted against pipette voltage (× −1) for three independent inside-out patches, as shown with different symbols. The mean unitary conductance (ɣ) ± SD (see inset), was determined from the slope of the linear regression line fits.
Figure 7. hAQP1-Myc-His6 displays subconductance states. (A) Gap-free recording of an inside-out patch from a proteoliposome containing hAQP1-Myc-His6 in symmetrical (112 mM) K+ solutions at +200 mV with 10 μM CPT-cGMP. Dashed lines show the different channel conductance levels; O is open and S is subconductance. (B) Amplitude histogram of data in (A) fitted with Gaussians at open (O) levels 1, 2, and 3. SC, subconductance.
Figure 8. Application of the patch-clamp method to investigate a double-mutant variant hAQP1-Myc-His6D48A/D185A in proteoliposomes. (A) Structural view of hAQP1 homotetramer (PDB: 1IH5) displaying a ring of charged Asp (D) residues (red) near the ion-conducting central pore, generated using the National Center for Biotechnology Information interactive iCN3D viewer (https://www.ncbi.nlm.nih.gov/Structure). Left, full view; right, higher-magnification inset. (B) Current-voltage relationships of oocytes injected with hAQP1-Myc-His6D48A/D185A in isotonic Ca2+-free saline (initial), and after activation with 10 μM CPT-cGMP. Data are mean ± SEM of six oocytes. (C) Current-voltage relationship of purified hAQP1-Myc-His6D48A/D185A in symmetrical (112 mM) K+ solutions in a single inside-out proteoliposome patch (blue circles). Data for WT hAQP1-Myc-His6 with error bars (SD) are displayed for comparison (dashed line). (D) Example of a gap-free recording of an inside-out patch from a proteoliposome containing hAQP1-Myc-His6D48A/D185A in symmetrical (112 mM) K+ solutions at 120 mV with CPT-cGMP displaying channel activity.
Figure S1: Expression constructs used to drive hAQP1-Myc-His6 expression in
P. pastoris.
Upper panel: pPICZ-A expression vector containing human Aquaporin 1 (hAQP1)
with C-terminal Myc and 6 x His tags in frame (hAQP1-Myc-His6-A). Gene
expression is driven by the alcohol oxidase 1 (AOX1) promoter. Lower panel:
pPICZ-B vector containing hAQP1-Myc-His6-B. Note the recombinant proteins differ
by two residues before the Myc tag.
Figure S2: Ion channel activity of untagged hAQP1 in X. laevis oocytes.
Current-voltage relationships of oocytes injected with hAQP1 in isotonic Ca2+
-free
saline at the start of the recording (initial); after activation with 10 µM CPT-cGMP
(labeled 'cGMP'); and after application of 600 µM CdCl2 to the activated cells (Cd2+).
Data are mean ± SEM for 4 oocytes.
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