A role for the 2' residue in the second transmembrane helix of the GABA A receptor gamma2S subunit in channel conductance and gating.
GABA(A) receptors composed of alpha, beta and gamma subunits display a significantly higher single-channel conductance than receptors comprised of only alpha and beta subunits. The pore of GABA(A) receptors is lined by the second transmembrane region from each of its five subunits and includes conserved threonines at the 6', 10' and 13' positions. At the 2' position, however, a polar residue is present in the gamma subunit but not the alpha or beta subunits. As residues at the 2', 6' and 10' positions are exposed in the open channel and as such polar channel-lining residues may interact with permeant ions by substituting for water interactions, we compared both the single-channel conductance and the kinetic properties of wild-type alpha1beta1 and alpha1beta1gamma2S receptors with two mutant receptors, alphabetagamma(S2'A) and alphabetagamma(S2'V). We found that the single-channel conductance of both mutant alphabetagamma receptors was significantly decreased with respect to wild-type alphabetagamma, with the presence of the larger valine side chain having the greatest effect. However, the conductance of the mutant alphabetagamma receptors remained larger than wild-type alphabeta channels. This reduction in the conductance of mutant alphabetagamma receptors was observed at depolarized potentials only (E(Cl) = -1.8 mV), which revealed an asymmetry in the ion conduction pathway mediated by the gamma2' residue. The substitutions at the gamma2' serine residue also altered the gating properties of the channel in addition to the effects on the conductance with the open probability of the mutant channels being decreased while the mean open time increased. The data presented in this study show that residues at the 2' position in M2 of the gamma subunit affects both single-channel conductance and receptor kinetics.
Luu
T
T
Division of Molecular Bioscience, The John Curtin School of Medical Research, The Australian National University, Canberra 2601, Australia.
Cromer
B
B
Gage
P W
PW
Tierney
M L
ML
D019943:Amino Acid Substitution / Q000235:genetics*; D000818:Animals; D002460:Cell Line; D004553:Electric Conductivity; D006801:Humans; D015640:Ion Channel Gating / Q000502:physiology*; D008564:Membrane Potentials / Q000502:physiology; D051379:Mice; D017433:Protein Structure, Secondary / Q000235:genetics; D011963:Receptors, GABA-A / Q000235:genetics / Q000378:metabolism*
D011963:Receptors, GABA-A