Figure 1: Electrostatic interactions restore potassium selectivity in molecular evolution experiments. A: Yeast growth selection from mutant libraries of the mammalian G protein–activated inwardly rectifying potassium channel GIRK2 (Kir3.2) recovers the constitutively active, potassium-selective, S177T mutant channel. On the left is a homology model with S177T (blue) on the M2 inner helix, behind the selectivity filter. On the right, representative current traces obtained fromXenopus oocytes expressing S177T channels bathed in 90-mM Na+ or 90-mM K+ solution, showing that these channels carry K+ current but not Na+ current.
B: Substitution of S177 with all 19 other natural amino acids leads to identification of the S177W mutant channel as a nonselective, constitutively active mutant GIRK2 channel that cannot support yeast growth. The S177W (red) substitution that fills the space behind the selectivity filter (left) results in large, inward fluxes for both K+ and Na+ ions (right).
C: Yeast growth selection in molecular evolution experiments identifies the N184D mutation as a second-site suppressor that restores potassium selectivity to the S177W-N184D double mutant. The N184D mutation (green) in the center of the central cavity near a K+ ion (blue) compensates for the S177W mutation (red; left) so that the double-mutant channels permeate K+ but not Na+ions (right). Currents were recorded at membrane potentials ranging from –150 mV to +40 mV in 20-mV increments from a holding potential of 0 mV.
D: Aspartate scan along the M2 inner helix bearing the S177W mutation (red), for three residues on face 1 (green) lining the central cavity (G180D, N184D, and V188D) and three residues on face 2 (red; S181D, A185D, and G189D).
E: Aspartate substitution of residues on face 1 (green) but not on face 2 (red) restores K+selectivity, as shown by the ratio of Na+ permeability and K+ permeability deduced from the difference in reversal potential measured in 90-mM Na+ or 90-mM K+ solution. *p < 0.001.
F: Molecular surface representation of the cavity of the GIRK2 channel homology model viewed from the extracellular side. Residues on face 1 (green) are closer to the cavity ion (blue) and have greater solvent exposure than the residues on face 2 (red). This surprising finding, that nonselective electrostatic stabilization of cations in the channel cavity can amplify ion selectivity independent of the selectivity filter, can be accounted for in a kinetic model of the multi-ion long pore of the potassium channel.
From Bichet, D., Grabe, M., Jan, Y.N., and Jan, L.Y. 2006. Proceedings of the National Academy of Sciences USA103:14355–14360. © 2006 by The National Academy of Sciences.
See also Bichet, D., Lin, Y.F., Ibarra, C.A., Huang, C.S., Yi, B.A., Jan, Y.N., and Jan, L.Y. 2004. Proc Natl Acad Sci USA 101:4441–4446; Grabe, M., Bichet, D., Qian, X., Jan, Y.N., and Jan, L.Y. 2006. Proc Natl Acad Sci USA103:14361–14366; and Yi, B.A., Lin, Y.F., Jan, Y.N., and Jan, L.Y. 2001. Neuron 29:657–667.
Figure 2: Seven evolutionarily conserved yeast proteins in the early secretory pathway for membrane traffic that are important for functional expression of Kir3.2 channels. ER: endoplasmic reticulum.
Figure generated by Maya Schuldiner in Jonathan Weissman's laboratory, University of California, San Francisco. See also Haass, F. A., Jonikas, M., Walter, P., Weissman, J. S., Jan, Y. N., Jan, L. Y., and Schuldiner, M. 2007. Proceedings of the National Academy of Sciences U.S.A. 104:18079–18084.
Figure 3: GIRK2 in dendritic spines of hippocampal neurons. The GIRK2 (Kir3.2) channel protein (red immunofluorescence) resides in dendritic spines as well as shafts of hippocampal neurons expressing enhanced green fluorescence protein (EGFP), in culture for 4 weeks. Scale bar, 10mm. The presence of the machinery for slow synaptic inhibition in dendritic spines, home for the signaling pathway for long-term potentiation of fast excitation, prompted the study showing that the same signaling pathway also induces long-term potentiation of slow synaptic inhibition mediated by GABAB receptors and GIRK channels.
From Huang, C.S., Shi, S.-H., Ule, J., Ruggiu, M., Barker, L.A., Darnell, R.B., Jan, Y.N., and Jan, L.Y. 2005.Cell 123:105–118. © 2005, with permission from Elsevier.
Figure 4: Large-scale mutant screens in yeast yield reliable structural information. Model of Kir channels based on the KirBac1.1 structure, showing 10 M1 residues per subunit predicted to face lipid (yellow), 4 M2 residues predicted to line the pore (cyan), and 11 M1 and M2 residues predicted to be buried within the channel protein (red), based on analyses of IRK1 (Kir2.1) mutant channels that rescue potassium-transport-deficient yeast for growth in low-potassium medium. The outer pair (pink and blue) and inner pair (orange and brown) yielding GIRK2 (Kir3.2) gating mutants isolated from unbiased yeast screens are shown on the green subunit on the left.
The amino acids in IRK1 (and corresponding residues given in parentheses for KirBac1.1) are I87(S66), L90(A69), A91(L70), L94(V73), L97(T76), F98(L77), C101(L80), W104(Q83), L105(L84), and L108(A87) for lipid-facing; S165(I131), C169(M135), D172(I138), and I176(T142) for pore-lining; and F92(F71), S95(N74), W96(N75), F99(F78), G100(A79), A107(D86), A157(A123), V158(H124), V161(A127), Q164(E130), and G168(G134) for buried residues. The outer and inner pair of GIRK2 residues important for holding the channel in the closed conformation (and corresponding residues given in parentheses for KirBac1.1) are E152(L108) in pink and S177(I131) in blue; N94(F63) in orange and V188(T142) in brown.
See also Minor, D.L., Jr., Masseling, S.J., Jan, Y.N., and Jan, L.Y. 1999. Cell 96:879–891; Yi, B.A., Lin, Y.F., Jan, Y.N., and Jan, L.Y. 2001. Neuron 29:657–667; and Kuo, A., Gulbis, J.M., Antcliff, J.F., Rahman, T., Lowe, E.D., Zimmer, J., Cuthbertson, J., Ashcroft, F.M., Ezaki, T., and Doyle, D.A. 2003. Science 300:1922–1926.
Figure 5: Three fluorescent mutants of the bacterial protein G antibody–binding domain with the environment-sensitive fluorescent amino acid Aladan in a buried position (that of phenylalanine 30, center), a partially exposed position (that of tryptophan 43, left), and an exposed position (that of alanine 24, right) show three different colors, as depicted by the colors of Aladan in the three ribbon diagrams.
Figure generated by Tim B. McAnaney in Steven G. Boxer's laboratory, Stanford University. See also Cohen, B.E., McAnaney, T.B., Park, E.S., Jan, Y.N., Boxer, S.G., and Jan, L.Y. 2002. Science 296:1700–1703.
Figure 6: Different traffic patterns for Kir3 channels of different subunit composition. G protein–gated inwardly rectifying potassium (GIRK) channels have different subunit compositions in the brain (Kir3.1/Kir3.2) and the heart (Kir3.1/Kir3.4). They have similar electrophysiological properties but different traffic patterns. Kir3.2 and Kir3.4, but not Kir3.3, contain endoplasmic reticulum (ER) export signals and promote the ER exit of Kir3.1. In addition, Kir3.4 and the alternatively spliced variants of Kir3.2 (Kir3.2A–D) contain traffic motifs that act in the endocytic pathway; differences in these trafficking signals allow Kir3.1/Kir3.4 channels to reside primarily on the cell surface but cause Kir3.1/Kir3.2A channels to show prominent endosomal localization. In contrast, Kir3.3 contains a lysosomal targeting signal and diverts the heterotetrameric channels for degradation. The ER, Golgi, TGN (trans-Golgi network), endosomes, and lysosomes are intracellular compartments in the secretary pathway for membrane protein synthesis, maturation, and trafficking.
From Ma, D., Zerangue, N., Raab-Graham, K., Fried, S.R., Jan, Y.N., and Jan, L.Y. 2002. Neuron 33:715–729. © 2002, with permission from Elsevier Science.
