|
Figure 1. Diagram of proposed ρ1 GABA receptor subunit topology and transmembrane domain sequences. (A) Endogenously charged residues are indicated by the appropriate charge sign and these residues were neutralized in this study to examine the role of endogenous charges on ionic selectivity. The TM2 numbering system used in the manuscript begins with the 0′ TM2 arginine with increasing numbers proceeding toward the C terminus. Charge-scanning mutants covered the region from −12′ (intracellular valine, V) to 20′ (extracellular alanine, A) and are indicated with gray-filled circles. This figure is to merely show the extent of the residues examined in this study and the secondary structure or positions of these residues with respect to the membrane should not be taken literally. (B) Aligned TM1–TM2 linker and TM2 sequences from several representative members of the receptor family. Anionic members are above the dashed line, cationic members are below. The GABA ρ1 receptor sequence is human, all others are rat.
|
|
Figure 2. Dose–response relationships for selected glutamate scan mutants. (A) GABA-gated currents at a range of agonist concentrations in oocytes expressing the indicated mutants are shown. (B) Maximum current amplitudes were plotted as a function of GABA concentration and fitted with the Hill equation (Eq. 1 in Materials and methods). Parameters from these fits are provided in Table I. In general, the mutations had a modest effect on agonist sensitivity.
|
|
Figure 3. A negatively charged substitution at P290 (−2′) results in cation-preferring receptors. (A) Amplitudes of GABA-induced currents from P290E were measured at a continuously varying membrane potential using the ramp protocol described in the Materials and methods. Ramps obtained in low sodium OR2 are the dashed lines and those in low-chloride OR2 are the solid lines. The normal OR2 ramp is omitted for clarity, but the reversal potential is indicated with an arrow. For the graph on the right, reversal potentials were measured at different concentrations of sodium (solid) or chloride (open) and fit with the GHK equation (Eq. 2) to determine the relative permeability coefficients. Substitution of negatively charged glutamate for the native proline at position 290 (−2′) resulted in cation-preferring receptors, but receptors were chloride selective when this site was mutated to neutral alanine (B) or positively charged lysine (C).
|
|
Figure 4. Glutamate scanning reveals a pattern that alternates between cation and nonspecific permeability. (A–H) For each indicated glutamate mutant, reversal potentials were measured in solutions with different concentrations of sodium (solid) or chloride (open). Lines are GHK fits to the data. Reversal potentials were nearly unaffected by chloride concentration in mutants that are cation preferring (A, C, and E), while for nonselective mutants the reversal potentials were shifted by changes in sodium or chloride concentration (B, D, G, and H). Systematic substitution of glutamate for each amino acid from −2′ to 5′ exhibited a nearly alternating pattern of cation-preferring receptors and nonselective receptors.
|
|
Figure 5. Alternate glutamate mutants at the intracellular end of TM2 are cation preferring. (A) Relative potassium permeability plotted against amino acid number shows the alternating pattern of cationic glutamate mutants (solid circles). Substitutions by positive lysine residues (gray circles) resulted in only small increases in potassium permeability. Substitution by neutral alanines (open circles) had no significant effect on ionic permeabilities. (B) Same as A, showing relative sodium permeability of mutant receptors.
|
|
Figure 6. Endogenous charged residues in the wild-type receptor have little effect on ionic permeability. Charged amino acids at the intracellular membrane interface were neutralized to examine the role of native charges on ionic permeability. Although small elevations in relative cation permeability were evident, none of the constructs increased relative cation permeability to more than 0.15, in stark contrast to the previously described deletion of P290 and mutation of A291 to E (ΔP290/A291E), shown for comparison (Wotring et al., 2003). RRR/QQM is R286Q/R287Q/R292M, DRRR/NQQM is D285N/R286Q/R287Q/R292M, and DRR/PAD is D285P/R286A/R287D. For the latter case, the mutations were to the corresponding sequence in nAChR rather than a simple neutralization.
|
|
Figure 7. Glutamate substitutions do not alter the apparent pore size. (A–F) Reversal potentials were measured after substitution with anions (open symbols) and cations (closed symbols) of different diameters. The dashed and dotted lines indicate the calculated reversal potential shift for an impermeant anion or cation, respectively, and were based on the determined relative permeabilities for that particular mutant. For the wild-type receptor, the reversal potential shift approaches its maximum value for anions between 4 and 6 Å. Using this approximation, the data indicate that for all cases, the pore size was between 4 and 6 Å. The cations and their diameters (in Å) were as follows: cations, sodium, 1.9; rubidium, 3.0; cesium, 3.4; imadazole, 4.8; choline, 5.6; triethanolamine, 7.0; N-methyl-d-glucamine, 9.0; anions, bicarbonate, 4.1; acetate, 4.5; propionate, 5.1; gluconate, 6.9; isethionate, 7.0. Under the particular mutation, we provide the difference in side chain volume imparted by the substitution.
|
