Table 2.
Measurement solutions employed in SSME assays.
| Assay | Main Solution (M) | Non-Activating Solution (NA) | Activating Solution (A) | Resting Solution (R) |
|---|---|---|---|---|
| Standard Assay (Figure 1G,K) |
30 mM HEPES, 30 mM MES, 5 mM MgCl2, 90 mM NaCl, pH 7.6 (NaOH) | M + 50 mM NaCl | M + 50 mM KCl | -- |
| Ion selectivity (Figure 2) |
30 mM HEPES, 30 mM MES, 5 mM MgCl2, 90 mM NMDG-Cl, pH 7.6 (NMDG) | M + 50 mM NMDG-Cl | M + 50 mM Cl- salt (Li, Na, K, Rb, Cs, choline) |
-- |
| K+ translocation (I/c curve, Figure 3) |
30 mM HEPES, 30 mM MES, 5 mM MgCl2, 90 mM NaCl, pH 7.6 (NaOH) | M + 300 mM NaCl | M + x mM KCl + (300 − x) mM NaCl (1 ≤ x ≤ 300 mM) |
-- |
| pH dependence, K+ flux (no ΔpH, Figure 4A–D) | 30 mM HEPES, 30 mM MES, 5 mM MgCl2, 90 mM NaCl | M + 50 mM NaCl titrated to pH 3.0–10.0 (NaOH, HCl) |
M + 50 mM KCl titrated to pH 3.0–10.0 (NaOH, HCl) |
-- |
| pH dependence, K+ flux (with ΔpH, Figure 4E–G) | 30 mM HEPES, 30 mM MES, 5 mM MgCl2, 90 mM NaCl | M + 50 mM NaCl titrated to pH 7.5 (NaOH) |
M + 50 mM KCl titrated to pH 7.5 (NaOH) |
M + 50 mM NaCl titrated to pH 3.0–10.0 (NaOH, HCl) |
| H+ translocation (influx, Figure 5A–E) |
30 mM HEPES, 30 mM MES, 5 mM MgCl2, 140 mM NaCl | M titrated to pH 7.6 (NaOH) | M titrated to pH 7.4–4.6 (NaOH) | -- |
| H+ translocation (efflux, Figure 5F–J) |
30 mM HEPES, 30 mM MES, 5 mM MgCl2, 140 mM NaCl | M titrated to pH 4.6 (NaOH) | M titrated to pH 4.8–7.6 (NaOH) | -- |
| Compound IC50/EC50 (Figure 6) | 30 mM HEPES, 30 mM MES, 5 mM MgCl2, 90 mM NaCl, pH 7.6 (NaOH) | M + 50 mM NaCl + x µM compound (0.1% DMSO) |
M + 50 mM KCl + x µM compound (0.1% DMSO) |
-- |