Human monocarboxylate transporters accept and relay protons via the bound substrate for selectivity and activity at physiological pH

MCT1 Lys 38 mutants are functional at acidic pH

Initially, we assayed uptake of ¹⁴C-l-lactate in the absence of a transmembrane proton gradient at pH 6.8 (19). As reported by others (10, 12, 15, 16), at these conditions, MCT1 mutants with altered Lys 38, Asp 309, or Arg 313 sites appeared nonfunctional (Fig. 1B, Fig. S1C), whereas MCT1 wild-type facilitated l-lactate uptake (Fig. 1B). Transport equilibrium with equal import and export velocities of l-lactate/H⁺ was reached within 16 min. At lower pH, MCT1 D309N and MCT1 R313N remained nonfunctional (Fig. S1C), whereas MCT1 K38M enabled some l-lactate uptake at pH 4.8 (20) and switched to full activity at pH 3.8 and 2.8 (Fig. 1B). MCT1 wildtype was maximally active at pH 4.8, and the inward proton gradient shifted the cytosolic l-lactate concentration to a markedly higher level (Fig. 1B).

We generated more MCT1 Lys 38 mutants, namely MCT1 K38A with a shorter neutral sidechain, MCT1 K38E to reverse the sidechain charge to negative, and MCT1 K38R carrying the two orders of magnitude stronger guanidine base to prevent potential proton release (Figs. S1A and S1B). MCT1 K38A behaved as MCT1 K38M (Fig. S1D). MCT1 K38E was inactive at pH 6.8 and 4.8; yet pH 2.8, which is about one log-unit below the pK_a of the glutamate γ-carboxyl, initiated transport (Fig. S1E). This indicates that a glutamate at position 38 is solvent accessible, and partial neutralization by protonation allows for transport similar in fashion to K38M and K38A. MCT1 K38R was nonfunctional at all tested pH conditions (Fig. S1F). Possibly, a permanent charge at position 38 prohibits transport, whereas Lys 38 in MCT1 wildtype may not be charged at all times but could be part of a proton transfer process. It cannot be excluded, though, that the larger size of the arginine sidechain may contribute to the loss of function.

Lys 38 enables MCT1 transport at physiological pH

Next, we carried out a fine-grained titration of MCT1 wildtype and K38M transport rates against pH to determine the number of accessible protonation sites relevant to l-lactate/H⁺ transport (Fig. 1C). The titration curves appeared strongly shifted, and differed in complexity. The sigmoidal curve of MCT1 K38M indicated a single transport-relevant protonation step. The inflection point of the sigmoidal function (pH₅₀) was determined to 3.97 (Fig. 1C, blue curve), which corresponded to the intrinsic pK_a of l-lactic acid (pK_a 3.86; Fig. 1D). The curve shape and pH₅₀ were highly reminiscent of the l-lactic acid permeability of the solute channel aquaporin-9, AQP9 (19, 21) supporting the view that MCT1 K38M equally uses neutral l-lactic acid as a substrate. Accordingly, at sufficiently acidic buffer, the l-lactate anion itself binds and carries the proton cosubstrate for MCT1 K38M.

With a lysine present at position 38 of MCT1 wildtype, l-lactate/H⁺ transport functionality was shifted to neutral and mildly acidic physiological pH (Fig. 1C, orange curve). Further, the curve can be sectioned: a monotonically increasing part depicting an initial raise in the transport rate from neutral pH towards mildly acidic conditions followed by a prominent boost in transport velocity at more acidic conditions. This part was fitted best with a double-sigmoidal function (r² = 0.9978) with inflection points at pH 7.43 and pH 5.61 (Fig. 1C). Transport velocity was maximal at pH 4.8, and exponentially decreased inverse in shape to the pH curve of MCT1 K38M (Fig. 1C). The opposite pH dependence in the most acidic curve section suggests that MCT1 wildtype accepts the l-lactate anion as a substrate rather than neutral l-lactic acid.

A double-sigmoidal curve shape may indicate two buffer-accessible protonation sites that influence the overall transport process. Since this part is absent in the Lys 38-less MCT1 K38M mutant, the protonation sites may be linked to the Lys 38 sidechain ɛ-amine in the substrate-unbound state and to the complex of Lys 38 with l-lactate. While the protonation status of the Lys 38 sidechain appears to be determined by the buffer pH (see also MCT1 K38E mutant; Fig. S1E), we did not notice direct protonation of Asp 309 in our titrations by external protons. This is fostered by solvation simulations based on the protein structure of MCT1 (12) in which bulk-linked water clusters appeared close to Lys 38, whereas water was absent around the Asp 309 site (Fig. S2). Together, the pH data show that MCT1 with replaced Lys 38 is functional in principle; yet, the basicity of the lysine ɛ-amine is required to enable transport at physiological pH.

Direct evidence for binding of l-lactate to the protonated Lys 38 ɛ-amine

We figured that if the observed pH₅₀ of 7.43 was derived from Lys 38, l-lactate affinity should be strongly decreased at alkaline pH due to deprotonation of the ɛ-amine. Therefore, we determined Michaelis–Menten kinetics (Fig. 2). Indeed, the l-lactate affinity at pH 7.8 appeared one order of magnitude lower (K_m 19.2 ± 4.1 mM) than at physiological pH conditions (K_m 2.32 ± 0.36 mM) (Figs. 2A–C). The decrease in affinity at pH 3.8 (K_m 7.29 ± 0.40 mM) is due to the shift of the l-lactate/lactic acid equilibrium towards the neutral species (Fig. 1D). A pH of 8 was shown to lock MCT1 in the outward-open conformation (12), and a strong decrease in l-lactate affinity at alkaline pH was seen with human erythrocytes in which MCT1 is the prominent l-lactate transporter (22). Together with our data, this provides evidence for a binding mechanism in which a proton binds first to the Lys 38 ɛ-amine and generates a positive charge for the subsequent binding of the l-lactate anion. The observed decrease in affinity at the most acidic buffer pH of 3.8 (Fig. 2C) is due to the shifted l-lactate/lactic acid equilibrium towards the neutral form (Fig. 1D).

The maximum velocity of transport, v_max, also correlated with the MCT1 Lys 38 protonation status (Fig. 2D). At pH 7.8, i.e. in the mainly uncharged state of Lys 38, transport was up to one order of magnitude faster than at lower pH conditions. We attribute this to less impedance of the substrate at the binding site due to weaker interaction with the neutral Lys 38 sidechain.

To meet the acidic pH requirements of MCT1 K38M, we measured K_m at pH 3.8 (Fig. 2E). The substrate affinity (K_m 2.13 ± 0.19 mM; Fig. 2C) was equal to MCT1 wildtype at a physiological pH, and v_max was higher than that of MCT1 wildtype at pH 6.8 and 4.8, yet not as high as at pH 7.8 (Fig. 2D). An uncharged residue at position 38 is beneficial for transport velocity, and polar interactions with the amine of Lys 38 further accelerate transport. The generally comparable K_m and v_max values of MCT1 wildtype and K38M suggest that the proton-driven alternating access transport mechanism remained unaltered in principle despite entry of the substrate in different forms, i.e. l-lactate/H⁺ (MCT1 wildtype) vs. l-lactic acid (MCT1 K38M).

MCT1 is selective for protonatable weak acid anions

Next, we tested whether MCT1 wildtype or MCT1 K38M would accept l-lactamide, a permanently neutral mimic of l-lactic acid (Fig. 3A), as a transport substrate. We chose a buffer pH of 3.8 for equal concentrations of l-lactate and l-lactic acid (Fig. 1D). At an equimolar concentration or 10-fold excess of l-lactamide, neither MCT1 wildtype nor MCT1 K38M showed a decrease in the uptake rate by competition (Fig. 3A), indicating that l-lactamide is not an MCT1 or MCT1 K38M substrate.

We employed a second method to test more directly for l-lactamide transport by MCT1 using stopped-flow light scattering (Fig. 3B) (21, 23). This assay requires a steep substrate gradient to elicit sufficiently large changes in cell volume for detection. In the initial phase, the hyperosmotic conditions rapidly drive water out of the yeast eliciting an increase in light scattering (Fig. 3B). The slower import of MCT substrates leads to partial re-swelling and a decrease in light scattering. As in the radiolabeled competition assays, l-lactamide was not transported (Fig. 3C). Testing of further similarly sized compounds fostered the mechanistic findings of this study. We noted that the transport rate of l-lactate and chloroacetate (24) decreased with decreasing pK_a, i.e. 3.86 and 2.83, respectively. This again indicates that direct substrate protonation is relevant for transport. Neutral glycerol, which is incapable of binding and transferring protons, was excluded from transport by MCT1, as was zwitterionic l-proline (Fig. 3C) (25).

Bound weak acid substrates shuttle protons from Lys 38 to Asp309

The distance between the Lys 38 ɛ-amine and the Asp 309/Arg 313 salt bridge was determined to about 8 Å (Fig. S2) (12). A substrate carboxyl group appears suited to fill the gap and may shuttle a proton from Lys 38 to Asp 309. Protonation of Asp 309 was suggested to break the salt bridge initiating the alternating access mechanism (12). To gain insight into relevant proton transfer events, we determined the effect of heavy water, D₂O, on MCT1 transport.

Deuterons have twice the mass of protons, which increases the chemical bond strength resulting in a characteristic pK_a shift of monocarboxylic acids by approx. 0.5 (26), and a decrease of the deuteron-hopping velocity (27). The degree by which a proton-dependent chemical process is retarded in heavy water, thus, is indicative of the complexity of the involved transfer chain (28).

We changed the solvent of the yeast suspensions from light water to heavy water at least 30 min prior to the transport assays for adjustment (29, 30). Other than that, the buffers were identically composed and contained the same concentrations of protons or deuterons, i.e. pH or pD. The height of the plateaus derived from the obtained or extrapolated uptake curves (Fig. 3D) indicates the total amount of intracellular substrate in the equilibrium states, termed capacity. Throughout, the capacity of l-lactate/D⁺ transport was higher than with l-lactate/H⁺ (Fig. 3D, Fig. S3A). The heavy water effect was most pronounced at pD 6.8, and higher free deuteron concentrations somewhat compensated the effect (Fig. 3E, top). The rate constants indicated slower transport of MCT1 wildtype in heavy water at all pH/pD conditions (Fig. 3E, bottom, Fig. S3B). MCT1 K38M transport, in turn, appeared slightly accelerated in heavy water. Inverse isotope effects are rare and can indicate that substrate protonation takes place outside of the protein, and other rate limiting events determine the velocity of the overall process (31).

The alternating access transition is usually a major rate-limiting process in the transporter class. Making use of the pre-protonated pool of l-lactic acid in the external buffer may explain the mild inverse isotope effect observed with MCT1 K38M. The normal isotope effect with MCT1 wildtype, in turn, indicates that proton transfers inside the protein are relevant for the overall transport velocity. With the previous observations of this study, this suggests that l-lactate binds to Lys 38 and accepts a proton from the lysine ɛ-amine. The final and common step for MCT1 wildtype and the K38M mutant would be a proton transfer from the substrate to Asp 309 that breaks the salt bridge and initiates the alternative access conformational change.

Plasmids, cloning, and mutagenesis

Human MCT1 in pDRT196 with N-terminal hemagglutinin- and C-terminal His₁₀-tags was generated previously (17). Primers for site-directed mutagenesis are listed in Table S1. All constructs were sequenced.

pH/pD-adjusted buffers used in ¹⁴C-labeled transport assays

Standard buffers were 50 mM/50 mM TRIS/HEPES (pH 8.3, 7.8, 7.5, 7.3, 7.0, 6.8, 6.3), TRIS/MES (pH 6.6, 5.8), and TRIS/citric acid (pH 5.3, 4.8, 4.3, 3.8, 3.3, 2.8). For determining isotope effects, phosphate/citric acid buffers were used (43). 0.2 M Na₂HPO₄ and 0.1 M citric acid stocks were prepared in light, H₂O, and heavy water, D₂O. The heavy water stocks were combined in different ratios for pD 6.8, 5.8, 4.8, and 3.8. pD was adjusted using a D₂O-equilibrated and calibrated glass electrode (44). For light water buffers, the stocks were combined in the same ratios as above. Due to higher dissociation in light water, pH was adjusted using NaOH. This procedure assured equal concentrations of protons and deuterons at each pH/pD condition.