A comparative analysis reveals electrogenic properties of PfCRT and pendrin

Abstract

Ion translocation is an essential process in all living cells. Most traditional approaches studying ion-translocating systems have employed cellular systems replete with native proteins that potentially interfere with the functional assessment of the protein of interest. The reconstitution of purified functional target proteins into proteoliposomes (PLs), artificial membrane systems of defined lipid composition, allows for their characterization without these intricacies. Targeting three distinct proteins, NhaA, pendrin, and the Plasmodium falciparum chloroquine resistance transporter (PfCRT), upon their reconstitution into PLs with a combined array of experimental approaches centered around solid-supported membrane electrophysiology, we show the advantage of the PL study system over cell-based approaches to assess protein-specific functional features. Using NhaA, the well-characterized archetype of Na⁺/H⁺ antiporters (exchangers), as a molecular ruler, our studies reveal that pendrin, a clinically relevant anion transporter in the thyroid, ear, kidney, and lungs, catalyzes the electrogenic exchange of its transported anions, opposing a long-standing dogma of the electroneutral activity of pendrin. We also provide direct evidence that PfCRT-a key contributor in multidrug resistance that thwarts efforts to combat malaria-mediates H⁺-coupled drug transport.

Keywords: NhaA; PfCRT; anion transport; drug transport; electrophysiology; ion transport; membrane transport; pendrin; sodium-proton exchange; solid-supported membrane.

Figure 1

Measurements in proteoliposomes containing NhaA. A, representative recording of an SSM electrophysiological measurement of WT NhaA performed in the SURFE²R N1 system. Inset, recorded currents of WT NhaA and the no functional mutant, D163N, after 10 mM NaCl was added at time 1 s. The black bar shows the addition of different concentrations of NaCl as indicated in the legend. B, transport kinetics of NaCl using proteoliposomes containing NhaA at different pHs. C, transport kinetics of NaCl using the area under the currents obtained from SSM electrophysiological measurement of WT NhaA (control liposomes used for signal correction) at pH 8.0. Fitting the data to a nonlinear regression model yielded an apparent affinity for Na⁺ of 0.87 ± 0.37 mM. D, transport kinetics of NaCl using the maximal currents obtained from SSM electrophysiological measurement of WT NhaA (control liposomes used for signal correction) at pH 8.0. Fitting the data to a nonlinear regression model yielded an apparent affinity for Na⁺ of 0.99 ± 0.36 mM. All data are mean ± SD (n = 5–7 independent biological replicates), and the currents of three individual sensors were integrated over time to determine the transfer of charges (Coulombs) associated with protein-specific transport by determining the area under the curve using GraphPad Prism 10. SSM, solid-supported membrane.

Figure 2

Pendrin-mediated anion transport.A, representative SSM recordings of pendrin-specific on-signal and control proteoliposome (PL) (absence of protein) current traces in response to the addition of 50 mM NaI (black bar) to assay buffer at time 1 s. Inset, schematic representation of the concept of the SSM measurements (10). B, transport kinetics of Nal using the currents obtained from SSM electrophysiological measurement of PLs containing pendrin. Integration of the on-signal currents (control liposomes used for signal correction) over time reveals the pendrin-specific charge transfer. Fitting the data (mean ± SD, n = 6 independent biological replicates) to a nonlinear regression model yielded an apparent affinity for iodide anions of 12.3 ± 3.6 mM. C, integration of the on-signal currents over time reveals the pendrin-specific charge transfer in response to Na⁺-salts. Inset, a representative SSM recording of pendrin PL. Data are mean ± SD (n = 6 independent biological replicates) of the area under the peak. D, 1-min uptake of 10 μM Na¹²⁵I was measured in pendrin-PLs in the absence (−) or presence of 50 mM Cl^-, CHO₂^-, HCO₃^-, or l^-. Data (mean ± SD, n = 6 independent biological replicates) uptake was corrected for unspecific uptake in control liposomes and normalized with regard to the 1-min uptake in the presence of l^-. ∗p < 0.05, ∗∗p < 0.01. Statistical tests and exact p values are provided in Table S1. SSM, solid-supported membrane.

Figure 3

Pendrin-mediated ¹²⁵I^- transport is electrogenic and pH dependent.A, normalized charge transfer elicited by the addition of 25 mM I^- to external assay buffer (pH 7.5) in response to varying the internal pH in the proteoliposomes (PLs). Inset, representative pendrin-specific on-signal current traces in response to the addition of 25 mM NaI to assay buffer composed of 200 mM Tris–Gly-Gly, at pH 5.5, 7.5, or 8.5. Fitting the data (mean ± SD, n = 4 independent biological replicates) to a nonlinear regression model yielded an apparent affinity for hydroxide anions (OH^-) of 0.86 ± 0.08 μM (or a pOH of 6.07 or a pH of 7.93). B, transport of 10 μM ¹²⁵I^- was measured in pendrin-containing PLs for 1-min periods in assay buffer composed of 200 mM Tris–Gly-Gly, at pH 6.5, 7.5, or 8.5. Data (mean ± SD, n = 6 independent biological replicates) are normalized with regard to pH 8.5. C, transport of 10 μM ¹²⁵I^- was measured in pendrin-containing PLs for 1-min periods in assay buffer composed of 200 mM Tris–Gly-Gly, pH 7. The PLs were preloaded with 200 mM Tris–Gly-Gly, pH 6.5, pH 8.0, or pH 6.5, and 100 mM NaCl (equimolar replacement with Tris–Gly-Gly) as indicated in the scheme. D, 1-min uptakes were measured with pendrin-containing PLs preloaded with 100 mM Tris–Gly-Gly, pH 6.5 and 100 mM KCl (100 mM Cl^-_i), 100 mM Tris–Gly-Gly, pH 6.5 and 1 mM NaCl/99 mM KCl (100 mM Cl^-_i), or 100 mM Tris–Gly-Gly, pH 6.5 and 99 mM K-gluconate/1 mM KCl (1 mM Cl^-_i) and diluted into assay buffer composed of 100 mM Tris–Gly-Gly, pH 7.0/100 mM K-gluconate or 199 mM Tris–Gly-Gly, pH 7.0/1 mM K-gluconate in the presence or absence of the K⁺ ionophore valinomycin (1 μM) plus 10 μM ¹²⁵I^-. The scheme illustrates the experimental conditions. Data are means ± SD (n = 6 independent biological replicates).∗p < 0.05, ∗∗∗p < 0.001 versus pH 6.5 l^-, #p < 0.05 versus pH 7.5. Statistical tests and exact p values are provided in Table S1.

Figure 4

Measurements in proteoliposomes (PLs) containing PfCRT. A, representative SSM recordings of PLs containing indicated PfCRT variants upon the addition of 10 μM of CQ (7G8 in red, 7G8_F145I in green, and 7G8_C350R in orange) or PPQ (7G8 in dark blue, 7G8_F145I in purple, and 7G8_C350R in blue). CQ and PPQ were added to the assay buffer as indicated by the arrow. B, ttransport of 10 μM CQ or PPQ using PLs containing PfCRT with a K⁺ diffusion potential–driven membrane potential and inwardly directed pH gradient (pH_in = 7.5; pH_out = 5.5). Data are mean ± SD (n = 6–9 independent biological replicates) of the area under the peak. Currents were integrated over time to determine the transfer of charges (Coulombs) associated with protein-specific transport by determining the area under the curve using GraphPad Prism 10. C, transport of 100 nM ³H-CQ and ³H-PPQ was measured for 30 s using PLs containing PfCRT with a K⁺ diffusion potential–driven membrane potential and inwardly directed pH gradient (pH_in = 7.5; pH_out = 5.5). Data are mean ± SD (n = 6 independent biological replicates). Uptake by PfCRT-containing PLs was corrected for unspecific uptake in control liposomes and data. D, proton flux measurements. PfCRT PLs were loaded with 10 μM Oregon Green 514 carboxylic acid, and the fluorescence was monitored. In the presence of 0.03 M HCl, the addition of the ionophore CCCP, that disrupts the pH gradient, rapidly reduces the fluorescence to the baseline level. Inset, polar lipid liposomes under the same conditions. E and F, 100 μM CQ or PPQ. For consistency with previous measurements (48), a K⁺ diffusion potential–driven membrane potential was generated with valinomycin, and an inwardly directed pH gradient (pH_in = 7.5; pH_out = 5.5) was present in A–F. Data are mean ± SD (n = 5–6 independent biological replicates). ∗p < 0.05, ∗∗p < 0.01, and ∗∗∗p < 0.001. Statistical tests and exact p values are provided in Table S1. CQ, chloroquine; PfCRT, Plasmodium falciparum chloroquine resistance transporter; PPQ, piperaquine; SSM, solid-supported membrane.