Table 1. Summary of the L-lactate Km values and sensitivity to inhibition by AR-C155858 of MCT chimaeras expressed in the presence and absence of exogenous embigin.
The Km values reported were derived either by monitoring changes in intracellular pH with BCECF or using L-[14C]lactate uptake (asterisk) as described in the Experimental section. The majority of the kinetic data was obtained using the BCECF technique which was preferred over the radioactive technique since it allowed a single egg to be used for a complete Km estimation, reducing errors associated with the various levels of expression. However, this technique could not be used for MCT2 and chimaeras that were poorly expressed and have low Km values because the pH changes associated with uptake were too small to detect accurately with BCECF. In these cases, we needed to use the radioactive technique as we have done previously [2]. Results are means±S.E.M. derived from the fit of the mean data to the Michaelis–Menten equation by non-linear least-squares analysis. For L-[14C]lactate uptake, L-lactate concentrations used were 0.2, 0.5, 1, 2 and 5 mM, whereas for BCECF measurements, the concentrations were 2.5, 5, 10, 20 and 50 mM. The n values given in parentheses represent the number of separate eggs used at each substrate concentration. The expression at the plasma membrane is a qualitative indicator only, with +++ for good expression (as for MCT1 with or without embigin), ++ for modest expression (as for MCT2/1c without embigin), + for poor expression (as for MCT2 without embigin) and − for no expression (as for MCT2trn without embigin). The transporter activity represents the activity in the absence of inhibitor as monitored by L-[14C]lactate (0.5 mM, pH 6) uptake with results as means±S.E.M. (as for Figure 5). Initial sensitivity to AR-C155858 describes the decrease in rate between control and 10 nM AR-C155858 as a percentage of the control rate, whereas the inhibitor-insensitive component is the rate remaining at 100 nM AR-C155858 as a percentage of the control rate±S.E.M. Inhibitor data are taken from Figures 2, 4 and 6 and are expressed with or without (in parentheses) correction for rates of uptake in water-injected oocytes. n/a, not applicable.
| MCT chimaera | Embigin co-expressed | Expression at plasma membrane | Activity (pmol/min per oocyte) | Lactate Km (mM) | Initial sensitivity to AR-C155858 (%) | Inhibitor-insensitive component (%) |
|---|---|---|---|---|---|---|
| MCT1 | No | +++ | 85±6.4 (30) | 4.4±1.5 (4) | 28±3 (26±3) | 1.9±0.6 (8±0.6) |
| MCT1 | Yes | +++ | 119±7.8 (30) | 6.1±1.7 (4) | 34±5 (32±5) | 7.4±1.3 (12±1) |
| MCT2 | No | + | 32±3 (30) | 1.0±0.2 (8)* | 51±7 (42±6) | 12±2 (25±2) |
| MCT2 | Yes | +++ | 83±3.3 (106) | 2.3±0.3 (8)* | 31±4 (28±4) | 43±3 (47±3) |
| MCT1/2 | No | +++ | 1.8±0.3 (10) | n/a | n/a | n/a |
| MCT1/2 | Yes | +++ | 2.3±0.3 (10) | n/a | n/a | n/a |
| MCT1/2c | No | +++ | 143±2.7 (20) | 4.4±1.4 (6) | 21±2 (20±2) | 6±0.4 (10±0.4) |
| MCT1/2c | Yes | +++ | 127±3.7 (20) | 4.3±1.2 (6) | 6±6 (6±6) | 44±4 (46±4) |
| MCT1trn | No | +++ | 149±10.2 (20) | 4.5±2.1 (7) | 25±7 (24±7) | 10±2 (13±1) |
| MCT1trn | Yes | +++ | 139±5.4 (20) | 4.5±1.0 (5) | 27±5 (26±5) | 12±1 (15±1) |
| MCT2/1 | No | +++ | 40±2.2 (10) | 5.04±0.60 (4) | 40±5 (34±4) | 0±3 (8±2) |
| MCT2/1 | Yes | +++ | 54±4.4 (10) | 4.70±0.96 (4) | 34±13 (30±11) | 20±4 (28±4) |
| MCT2/1c | No | ++ | 74±6.1 (29) | 2.4±2.2 (4) | 33±6 (30±6) | 21±2 (26±2) |
| MCT2/1c | Yes | +++ | 103±7 (28) | 1.5±0.5 (7) | 10±8 (9±8) | 73±6 (74±6) |
| MCT2trn | No | − | 2.4±1.3 (15) | n/a | n/a | n/a |
| MCT2trn | Yes | + | 44±2.7 (28) | 1.4±0.2 (8)* | 46±3 (39±2) | 37±3 (44±2) |