A unique sorting nexin regulates trafficking of potassium channels via a PDZ domain interaction.

G protein-gated potassium (Kir3) channels are important for controlling neuronal excitability in the brain. Using a proteomics approach, we have identified a unique rodent intracellular protein, sorting nexin 27 (SNX27), which regulates the trafficking of Kir3 channels. Like most sorting nexins, SNX27 possesses a functional PX domain that selectively binds the membrane phospholipid phosphatidylinositol-3-phosphate (PI3P) and is important for trafficking to the early endosome. SNX27, however, is the only sorting nexin to contain a PDZ domain. This PDZ domain discriminates between channels with similar class I PDZ-binding motifs, associating with the C-terminal end of Kir3.3 and Kir3.2c (-ESKV), but not with that of Kir2.1 (-ESEI) or Kv1.4 (-ETDV). SNX27 promotes the endosomal movement of Kir3 channels, leading to reduced surface expression, increased degradation and smaller Kir3 potassium currents. The regulation of endosomal trafficking via sorting nexins reveals a previously unknown mechanism for controlling potassium channel surface expression.

Cytoplasmic domain structures of Kir2.1 and Kir3.1 show sites for modulating gating and rectification.

N- and C-terminal cytoplasmic domains of inwardly rectifying K (Kir) channels control the ion-permeation pathway through diverse interactions with small molecules and protein ligands in the cytoplasm. Two new crystal structures of the cytoplasmic domains of Kir2.1 (Kir2.1(L)) and the G protein-sensitive Kir3.1 (Kir3.1(S)) channels in the absence of PIP(2) show the cytoplasmic ion-permeation pathways occluded by four cytoplasmic loops that form a girdle around the central pore (G-loop). Significant flexibility of the pore-facing G-loop of Kir2.1(L) and Kir3.1(S) suggests a possible role as a diffusion barrier between cytoplasmic and transmembrane pores. Consistent with this, mutations of the G-loop disrupted gating or inward rectification. Structural comparison shows a di-aspartate cluster on the distal end of the cytoplasmic pore of Kir2.1(L) that is important for modulating inward rectification. Taken together, these results suggest the cytoplasmic domains of Kir channels undergo structural changes to modulate gating and inward rectification.

The role of the cytoplasmic pore in inward rectification of Kir2.1 channels.

Steeply voltage-dependent block by intracellular polyamines underlies the strong inward rectification properties of Kir2.1 and other Kir channels. Mutagenesis studies have identified several negatively charged pore-lining residues (D172, E224, and E299, in Kir2.1) in the inner cavity and cytoplasmic domain as determinants of the properties of spermine block. Recent crystallographic determination of the structure of the cytoplasmic domains of Kir2.1 identified additional negatively charged residues (D255 and D259) that influence inward rectification. In this study, we have characterized the kinetic and steady-state properties of spermine block in WT Kir2.1 and in mutations of the D255 residue (D255E, A, K, R). Despite minimal effects on steady-state blockade by spermine, D255 mutations have profound effects on the blocking kinetics, with D255A marginally, and D255R dramatically, slowing the rate of block. In addition, these mutations result in the appearance of a sustained current (in the presence of spermine) at depolarized voltages. These features are reproduced with a kinetic model consisting of a single open state, two sequentially linked blocked states, and a slow spermine permeation step, with residue D255 influencing the spermine affinity and rate of entry into the shallow blocked state. The data highlight a "long-pore" effect in Kir channels, and emphasize the importance of considering blocker permeation when assessing the effects of mutations on apparent blocker affinity.

Evidence for a centrally located gate in the pore of a serotonin-gated ion channel.

Serotonin-gated ion channels (5-HT3) are members of the ligand-gated channel family, which includes channels that are opened directly by the neurotransmitter acetylcholine, GABA, glycine, or glutamate. Although there is general agreement that the second transmembrane domain (M2) lines the pore, the position of the gate in the M2 is less certain. Here, we used substituted cysteine accessibility method (SCAM) to provide new evidence for a centrally located gate that moves during channel activation. In the closed state, three cysteine substitutions, located on the extracellular side of M2, were modified by methanethiosulfonate (MTS) reagents. In contrast, 13 cysteine substitutions were modified in the open state with MTS reagents. The pattern of inhibition (every three to four substitutions) was consistent with an alpha helical structure for the middle and cytoplasmic segments of the M2 transmembrane domain. Unexpectedly, open-state modification of two amino acids in the center of M2 with three different MTS reagents prevented channels from fully closing in the absence of neurotransmitter. Our results are consistent with a model in which the central region of the M2 transmembrane domain is inaccessible in the closed state and moves during channel activation.

Andersen's syndrome mutation effects on the structure and assembly of the cytoplasmic domains of Kir2.1.

Kir2.1 channels play a key role in maintaining the correct resting potential in eukaryotic cells. Recently, specific amino acid mutations in the Kir2.1 inwardly rectifying potassium channel have been found to cause Andersen's Syndrome in humans. Here, we have characterized individual Andersen's Syndrome mutants R218Q, G300V, E303K, and delta314-315 and have found multiple effects on the ability of the cytoplasmic domains in Kir2.1 channels to form proper tetrameric assemblies. For the R218Q mutation, we identified a second site mutation (T309K) that restored tetrameric assembly but not function. We successfully crystallized and solved the structure (at 2.0 A) of the N- and C-terminal cytoplasmic domains of Kir2.1-R218Q/T309K(S). This new structure revealed multiple conformations of the G-loop and CD loop, providing an explanation for channels that assemble but do not conduct ions. Interestingly, Glu303 forms both intra- and intersubunit salt bridges, depending on the conformation of the G-loop, suggesting that the E303K mutant stabilizes both closed and open G-loop conformations. In the Kir2.1-R218Q/T309K(S) structure, we discovered that the DE loop forms a hydrophobic pocket that binds 2-methyl-2,4-pentanediol, which is located near the putative G(betagamma)-activation site of Kir3 channels. Finally, we observed a potassium ion bound to the cytoplasmic domain for this class of K+ channels.

Pertussis-toxin-sensitive Galpha subunits selectively bind to C-terminal domain of neuronal GIRK channels: evidence for a heterotrimeric G-protein-channel complex.

Neuronal G-protein-gated inwardly rectifying potassium (Kir3; GIRK) channels are activated by G-protein-coupled receptors that selectively interact with PTX-sensitive (Galphai/o) G proteins. Although the Gbetagamma dimer is known to activate GIRK channels, the role of the Galphai/o subunit remains unclear. Here, we established that Galphao subunits co-immunoprecipitate with neuronal GIRK channels. In vitro binding studies led to the identification of six amino acids in the GIRK2 C-terminal domain essential for Galphao binding. Further studies suggested that the Galphai/obetagamma heterotrimer binds to the GIRK2 C-terminal domain via Galpha and not Gbetagamma. Galphai/o binding-impaired GIRK2 channels exhibited reduced receptor-activated currents, but retained normal ethanol- and Gbetagamma-activated currents. Finally, PTX-insensitive Galphaq or Galphas subunits did not bind to the GIRK2 C-terminus. Together, these results suggest that the interaction of PTX-sensitive Galphai/o subunit with the GIRK2 C-terminal domain regulates G-protein receptor coupling, and may be important for establishing specific Galphai/o signaling pathways.

betaL-betaM loop in the C-terminal domain of G protein-activated inwardly rectifying K(+) channels is important for G(betagamma) subunit activation.

The activity of G protein-activated inwardly rectifying K(+) channels (GIRK or Kir3) is important for regulating membrane excitability in neuronal, cardiac and endocrine cells. Although G(betagamma) subunits are known to bind the N- and C-termini of GIRK channels, the mechanism underlying G(betagamma) activation of GIRK is not well understood. Here, we used chimeras and point mutants constructed from GIRK2 and IRK1, a G protein-insensitive inward rectifier, to determine the region within GIRK2 important for G(betagamma) binding and activation. An analysis of mutant channels expressed in Xenopus oocytes revealed two amino acid substitutions in the C-terminal domain of GIRK2, GIRK2(L344E) and GIRK2(G347H), that exhibited decreased carbachol-activated currents but significantly enhanced basal currents with coexpression of G(betagamma) subunits. Combining the two mutations (GIRK2(EH)) led to a more severe reduction in carbachol-activated and G(betagamma)-stimulated currents. Ethanol-activated currents were normal, however, suggesting that G protein-independent gating was unaffected by the mutations. Both GIRK2(L344E) and GIRK2(EH) also showed reduced carbachol activation and normal ethanol activation when expressed in HEK-293T cells. Using epitope-tagged channels expressed in HEK-293T cells, immunocytochemistry showed that G(betagamma)-impaired mutants were expressed on the plasma membrane, although to varying extents, and could not account completely for the reduced G(betagamma) activation. In vitro G(betagamma) binding assays revealed an approximately 60% decrease in G(betagamma) binding to the C-terminal domain of GIRK2(L344E) but no statistical change with GIRK2(EH) or GIRK2(G347H), though both mutants exhibited G(betagamma)-impaired activation. Together, these results suggest that L344, and to a lesser extent, G347 play an important functional role in G(betagamma) activation of GIRK2 channels. Based on the 1.8 A structure of GIRK1 cytoplasmic domains, L344 and G347 are positioned in the betaL-betaM loop, which is situated away from the pore and near the N-terminal domain. The results are discussed in terms of a model for activation in which G(betagamma) alters the interaction between the betaL-betaM loop and the N-terminal domain.

Minimal structural rearrangement of the cytoplasmic pore during activation of the 5-HT3A receptor.

Ligand-gated ion channel receptors mediate the response of fast neurotransmitters by opening in less than a millisecond. Here, we investigated the activation mechanism of a serotonin-gated receptor (5-HT(3A)) by systematically introducing cysteine substitutions throughout the pore-lining M1-M2 loop and M2 transmembrane domain. We hypothesized that multiple cysteines in the narrowest region of the pore, which together can form a high affinity binding site for metal cations, would reveal changes in pore structure during gating. Using cadmium (Cd2+) as a probe, two cysteine substitutions in the cytoplasmic selectivity filter, S2'C and, to a lesser extent, G-2'C, showed high affinity inhibition with Cd2+ when applied extracellularly in the open state. Cd2+ inhibition in S2'C was attenuated if applied in the presence of an open-channel inhibitor and showed voltage-dependent recovery, indicating a direct effect of Cd2+ in the pore. When applied intracellularly, Cd2+ appeared to bind S2'C receptors in the closed state. The ability of cysteine side chains at the 2' and -2' positions to coordinate Cd2+ in both the native open and closed states of the channel suggests that the cytoplasmic selectivity filter of 5-HT(3A) receptors maintains a narrow pore during channel gating.