Renouncing electroneutrality is not free of charge: switching on electrogenicity in a Na+-coupled phosphate cotransporter

Abstract

Renal type IIa Na+-coupled inorganic phosphate (Pi) cotransporters (NaPi-IIa) mediate divalent Pi transport in an electrogenic manner, whereas the renal type IIc isoform (NaPi-IIc) is electroneutral, yet it shows high sequence identity with NaPi-IIa. Dual uptake (32Pi/22Na) assays confirmed that NaPi-IIc displayed Na+-coupled Pi cotransport with a 2:1 (Na+:Pi) stoichiometry compared with 3:1 established for NaPi-IIa. This finding suggested that the electrogenicity of NaPi-IIa arises from the interaction of an additional Na+ ion compared with NaPi-IIc. To identify the molecular elements responsible for the functional difference between isoforms, we used chimera and amino acid replacement approaches. Transport activity of chimeras constructed with NaPi-IIa and NaPi-IIc indicated that residues within the first six transmembrane domains were essential for the electrogenicity of NaPi-IIa. Sequence comparison between electrogenic and electroneutral isoforms revealed differences in the charge and polarity of residues clustered in three areas, one of which included part of the predicted third transmembrane domain. Here, substitution of three residues with their NaPi-IIa equivalents in NaPi-IIc (S189A, S191A, and G195D) resulted in a transporter that displayed a 1:1 charge/Pi coupling, a 3:1 Na+:Pi stoichiometry, and transient currents that resembled pre-steady-state relaxations. The mutant's weaker voltage dependency and 10-fold lower apparent Pi affinity compared with NaPi-IIa indicated that other residues important for the NaPi-IIa kinetic fingerprint exist. Our findings demonstrate that, through a minimal number of side chain substitutions, we can effect a switch from electroneutral to electrogenic cotransporter function, concomitant with the appearance of a cosubstrate interaction site.

Fig. 1.

NaPi-IIc has a 2:1 cotransport stoichiometry. Dual ²²Na/³²P_i uptake assay data for oocytes expressing NaPi-IIc (□) and control oocytes (○) from the same donor frog. Slope of linear regression line: 2.0 ± 0.2.

Fig. 2.

Chimeras of NaPi-IIc and NaPi-IIa localize elements critical for the NaPi-IIa electrogenic phenotype to TMDs 1-6. (A) Topology of NaPi-II protein based on hydrophobicity analysis. (B) ³²P_i uptake of chimeras compared with WT NaPi-IIc and NaPi-IIa. (Inset) Example of chimera composition for A6C2 and C2A6.

Fig. 3.

Sequence comparison of NaPi-IIa/c. Multiple sequence alignment indicates position of residues that are 100% identical in all 21 candidate sequences (Ident), changed between putative electrogenic and electroneutral sequences with 100% identity at the respective site (Δ-Ident) or changed with conservative substitutions in either or both groups (Δ-Cons). Shaded areas (I, II, and III) indicate clusters of changed residues. Residue numbering is according to mouse NaPi-IIa sequence. Topological designation shows position and relative lengths of TMDs and linker stretches as predicted by hydrophobicity analysis. NT, N-terminal; CT, C-terminal.

Fig. 4.

Steady-state properties of the triple mutant AAD-IIc. (A) Representative current tracings of oocytes from the same batch, comparing NI, NaPi-IIc, NaPi-IIa, and AAD-IIc under the superfusion conditions indicated. Na, ND100; P_i, ND100 plus 1 mM P_i; PFA, ND100 plus 1 mM PFA. Oocytes were voltage-clamped at -50 mV. (B) Net charge translocated plotted as a function of P_i uptake for individual oocytes expressing AAD-IIc (▪) and control oocytes from the same donor frog (•). Slope of linear regression line: 0.9 ± 0.1. (C) Dual uptake for AAD-IIc (▪) and control oocytes (□) from the same donor frog. Slope of linear regression line: 3.0 ± 0.2. For comparison purposes, the data of Fig. 1 for the WT NaPi-IIc have been replotted with the respective mean of control oocytes subtracted (○). (D) Normalized I-V plots that compare NaPi-IIa (▪, n = 5) and AAD-IIc (•, n = 6). Each data point is the difference between the currents recorded in ND100 plus 1 mM P_i and ND100, respectively, at a given V. Data for each cell were normalized to the P_i-induced current at -100 mV.

Fig. 5.

Substrate dependency of AAD-IIc. (A) I-V plots for P_i activation. Data for individual oocytes were determined for the P_i concentrations indicated (in mM) and data for each cell were normalized to the P_i-dependent current at 1 mM P_i and -100 mV (n = 4). (B) Apparent affinity for P_i-activation () for NaPi-IIa (□, n = 3) and AAD-IIc (▪, n = 4) as found by fitting a form of the Michaelis-Menten equation, , to the data of A at each membrane potential, where [P_i] is the P_i concentration, is the apparent affinity for P_i, is the maximum cotransport rate, and K is a variable offset (7). Note the logarithmic ordinate scale. (C) I-V plots for Na⁺ activation. Data for individual oocytes were determined for the Na⁺ concentrations indicated (in mM) with 1 mM P_i, and data for each cell were normalized to the P_i-dependent current at 1 mM P_i and -100 mV (n = 4). (D) Apparent affinities for Na⁺ activation () for NaPi-IIa (□, n = 4) and AAD-IIc (▪, n = 4) as reported from fitting a form of the modified Hill equation: , to the data of panel C at each membrane potential, where [Na] is the concentration of Na⁺, nH is the Hill coefficient, is the apparent affinity for Na⁺. (E) Proton dependency for NaPi-IIa (circles, continuous lines) and AAD-IIc (squares, dotted lines) at 0 mV (open symbols) and -120 mV (filled symbols). Data for NaPi-IIa (n=5) and AAD-IIc (n=5) were normalized to the response to 1 mM at -100mV, pH7.4. Current reversal at low substrate concentrations in A and C results from the subtraction of the uncoupled leak current (7, 9).

Fig. 6.

Voltage steps induce transient currents. (A) Currents recorded in response to a series of voltage steps in the range -140 mV to +60 mV from a -60-mV holding potential for a representative WT NaPi-IIc- and AAD-IIc-expressing oocyte from the same donor frog (superfusion in ND100). (B) Correlation of charge movement for AAD-IIc induced by a voltage step from -60 mV to -100 mV for superfusion in ND0, with the steady-state current induced by 1 mM P_i at -100 mV (superfusion in ND100). Line is a linear regression, with r² = 0.74, n = 9. (C) Normalized charge-voltage (Q-V) data for superfusion in ND0 and ND100 for the same batch of AAD-IIc-expressing oocytes (n = 8). The charge induced by a voltage step from -60 mV to -100 mV (in ND0) was used to normalize data for each cell before pooling. Error bars smaller than symbols are not shown. □ indicates ON charge movement from -60 mV to target potential. ▪ indicates OFF charge movement from target potential to -60 mV. Continuous lines are fits to data using a Boltzmann equation of the form: Q = Q_hyp + Q_max/[1 + exp(-ze(V - V_0.5)/kT)], where Q_max is the maximum charge translocated, Q_hyp, is the steady-state charge at the hyperpolarizing limit and depends on V_h, V_0.5 is the voltage at which the charge is distributed equally between two hypothetical states, z is the apparent valency per cotransporter, e is the elementary charge, k is Boltzmann's constant, and T is the absolute temperature. The dashed line indicates the hyperpolarizing limit for ON charge movement with ND0 superfusion.