Christine Arrabit

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 (Kir2.1L) and the G protein–sensitive Kir3.1 (Kir3.1S) channels in the absence of PIP2 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 Kir2.1L and Kir3.1S 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 Kir2.1L 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.

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.

Pertussis-toxin-sensitive Gα 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.

Mechanism underlying bupivacaine inhibition of G protein-gated inwardly rectifying K+ channels

Local anesthetics, commonly used for treating cardiac arrhythmias, pain, and seizures, are best known for their inhibitory effects on voltage-gated Na+ channels. Cardiovascular and central nervous system toxicity are unwanted side-effects from local anesthetics that cannot be attributed to the inhibition of only Na+ channels. Here, we report that extracellular application of the membrane-permeant local anesthetic bupivacaine selectively inhibited G protein-gated inwardly rectifying K+ channels (GIRK:Kir3) but not other families of inwardly rectifying K+ channels (ROMK:Kir1 and IRK:Kir2). Bupivacaine inhibited GIRK channels within seconds of application, regardless of whether channels were activated through the muscarinic receptor or directly via coexpressed G protein Gβγ subunits. Bupivacaine also inhibited alcohol-induced GIRK currents in the absence of functional pertussis toxin-sensitive G proteins. The mutated GIRK1 and GIRK2 (GIRK1/2) channels containing the high-affinity phosphatidylinositol 4,5-bisphosphate (PIP2) domain from IRK1, on the other hand, showed dramatically less inhibition with bupivacaine. Surprisingly, GIRK1/2 channels with high affinity for PIP2 were inhibited by ethanol, like IRK1 channels. We propose that membrane-permeant local anesthetics inhibit GIRK channels by antagonizing the interaction of PIP2 with the channel, which is essential for Gβγ and ethanol activation of GIRK channels.

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.

βL–βM loop in the C‐terminal domain of G protein‐activated inwardly rectifying K+ channels is important for Gβγ 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 Gβγ subunits are known to bind the N‐ and C‐termini of GIRK channels, the mechanism underlying Gβγ 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 Gβγ binding and activation. An analysis of mutant channels expressed in Xenopus oocytes revealed two amino acid substitutions in the C‐terminal domain of GIRK2, GIRK2L344E and GIRK2G347H, that exhibited decreased carbachol‐activated currents but significantly enhanced basal currents with coexpression of Gβγ subunits. Combining the two mutations (GIRK2EH) led to a more severe reduction in carbachol‐activated and Gβγ‐stimulated currents. Ethanol‐activated currents were normal, however, suggesting that G protein‐independent gating was unaffected by the mutations. Both GIRK2L344E and GIRK2EH 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 Gβγ‐impaired mutants were expressed on the plasma membrane, although to varying extents, and could not account completely for the reduced Gβγ activation. In vitro Gβγ binding assays revealed an ∼60% decrease in Gβγ binding to the C‐terminal domain of GIRK2L344E but no statistical change with GIRK2EH or GIRK2G347H, though both mutants exhibited Gβγ‐impaired activation. Together, these results suggest that L344, and to a lesser extent, G347 play an important functional role in Gβγ activation of GIRK2 channels. Based on the 1.8 Å structure of GIRK1 cytoplasmic domains, L344 and G347 are positioned in the βL–βM 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 Gβγ alters the interaction between the βL–βM 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-HT3A) 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-HT3A receptors maintains a narrow pore during channel gating.

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 Gβγ subunits are known to bind the N‐ and C‐termini of GIRK channels, the mechanism underlying Gβγ 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 Gβγ binding and activation. An analysis of mutant channels expressed in Xenopus oocytes revealed two amino acid substitutions in the C‐terminal domain of GIRK2, GIRK2L344E and GIRK2G347H, that exhibited decreased carbachol‐activated currents but significantly enhanced basal currents with coexpression of Gβγ subunits. Combining the two mutations (GIRK2EH) led to a more severe reduction in carbachol‐activated and Gβγ‐stimulated currents. Ethanol‐activated currents were normal, however, suggesting that G protein‐independent gating was unaffected by the mutations. Both GIRK2L344E and GIRK2EH 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 Gβγ‐impaired mutants were expressed on the plasma membrane, although to varying extents, and could not account completely for the reduced Gβγ activation. In vitro Gβγ binding assays revealed an ∼60% decrease in Gβγ binding to the C‐terminal domain of GIRK2L344E but no statistical change with GIRK2EH or GIRK2G347H, though both mutants exhibited Gβγ‐impaired activation. Together, these results suggest that L344, and to a lesser extent, G347 play an important functional role in Gβγ activation of GIRK2 channels. Based on the 1.8 Å structure of GIRK1 cytoplasmic domains, L344 and G347 are positioned in the βL–βM 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 Gβγ alters the interaction between the βL–βM loop and the N‐terminal domain.

βL-βM loop in the C-terminal domain of G protein-activated inwardly rectifying K+channels is important for Gβγsubunit activation: Gβγactivation domain in GIRK channels

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 Gβγ subunits are known to bind the N‐ and C‐termini of GIRK channels, the mechanism underlying Gβγ 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 Gβγ binding and activation. An analysis of mutant channels expressed in Xenopus oocytes revealed two amino acid substitutions in the C‐terminal domain of GIRK2, GIRK2L344E and GIRK2G347H, that exhibited decreased carbachol‐activated currents but significantly enhanced basal currents with coexpression of Gβγ subunits. Combining the two mutations (GIRK2EH) led to a more severe reduction in carbachol‐activated and Gβγ‐stimulated currents. Ethanol‐activated currents were normal, however, suggesting that G protein‐independent gating was unaffected by the mutations. Both GIRK2L344E and GIRK2EH 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 Gβγ‐impaired mutants were expressed on the plasma membrane, although to varying extents, and could not account completely for the reduced Gβγ activation. In vitro Gβγ binding assays revealed an ∼60% decrease in Gβγ binding to the C‐terminal domain of GIRK2L344E but no statistical change with GIRK2EH or GIRK2G347H, though both mutants exhibited Gβγ‐impaired activation. Together, these results suggest that L344, and to a lesser extent, G347 play an important functional role in Gβγ activation of GIRK2 channels. Based on the 1.8 Å structure of GIRK1 cytoplasmic domains, L344 and G347 are positioned in the βL–βM 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 Gβγ alters the interaction between the βL–βM loop and the N‐terminal domain.