Cryo-EM structure of the hyperpolarization-activated inwardly rectifying potassium channel KAT1 from Arabidopsis.
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ABSTRACT:
SUBMITTER: Li S
PROVIDER: S-EPMC7784887 | biostudies-literature | 2020 Nov
REPOSITORIES: biostudies-literature
Publications
Cryo-EM structure of the hyperpolarization-activated inwardly rectifying potassium channel KAT1 from Arabidopsis.
Li Siyu S Yang Fan F Sun Demeng D Zhang Yong Y Zhang Mengge M Liu Sanling S Zhou Peng P Shi Chaowei C Zhang Longhua L Tian Changlin C
Cell research 20200908 11
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Project description: Not available
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Project description:Voltage-gated potassium (Kv) channels coordinate electrical signalling and control cell volume by gating in response to membrane depolarization or hyperpolarization. However, although voltage-sensing domains transduce transmembrane electric field changes by a common mechanism involving the outward or inward translocation of gating charges1-3, the general determinants of channel gating polarity remain poorly understood4. Here we suggest a molecular mechanism for electromechanical coupling and gating polarity in non-domain-swapped Kv channels on the basis of the cryo-electron microscopy structure of KAT1, the hyperpolarization-activated Kv channel from Arabidopsis thaliana. KAT1 displays a depolarized voltage sensor, which interacts with a closed pore domain directly via two interfaces and indirectly via an intercalated phospholipid. Functional evaluation of KAT1 structure-guided mutants at the sensor-pore interfaces suggests a mechanism in which direct interaction between the sensor and the C-linker hairpin in the adjacent pore subunit is the primary determinant of gating polarity. We suggest that an inward motion of the S4 sensor helix of approximately 5-7 Å can underlie a direct-coupling mechanism, driving a conformational reorientation of the C-linker and ultimately opening the activation gate formed by the S6 intracellular bundle. This direct-coupling mechanism contrasts with allosteric mechanisms proposed for hyperpolarization-activated cyclic nucleotide-gated channels5, and may represent an unexpected link between depolarization- and hyperpolarization-activated channels.
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Project description:Phosphoinositides are essential signaling lipids that play a critical role in regulating ion channels, and their dysregulation often results in fatal diseases including cardiac arrhythmia and paralysis. Despite decades of intensive research, the underlying molecular mechanism of lipid agonism and specificity remains largely unknown. Here, we present a systematic study of the binding mechanism and specificity of a native agonist, phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2) and two of its variants, PI(3,4)P2 and PI(3,4,5)P3, on inwardly rectifying potassium channel Kir2.2, using molecular dynamics simulations and free energy perturbations (FEPs). Our results demonstrate that the major driving force for the PI(4,5)P2 specificity on Kir2.2 comes from the highly organized salt-bridge network formed between the charged inositol head and phosphodiester linker of PI(4,5)P2. The unsaturated arachidonic chain is also shown to contribute to the stable binding through hydrophobic interactions with nearby Kir2.2 hydrophobic residues. Consistent with previous experimental findings, our FEP results confirmed that non-native ligands, PI(3,4)P2 and PI(3,4,5)P3, show significant loss in binding affinity as a result of the substantial shift from the native binding mode and unfavorable local solvation environment. However, surprisingly, the underlying molecular pictures for the unfavorable binding of both ligands are quite distinctive: for PI(3,4)P2, it is due to a direct destabilization in the bound state, whereas for PI(3,4,5)P3, it is due to a relative stabilization in its free state. Our findings not only provide a theoretical basis for the ligand specificity, but also generate new insights into the allosteric modulation of ligand-gated ion channels.
| S-EPMC6247517 | biostudies-literature
Project description:TheKCNJ10gene encoding Kir4.1 contains numerous SNPs whose molecular effects remain unknown. We investigated the functional consequences of uncharacterized SNPs (Q212R, L166Q, and G83V) on homomeric (Kir4.1) and heteromeric (Kir4.1-Kir5.1) channel function. We compared these with previously characterized EAST/SeSAME mutants (G77R and A167V) in kidney-derived tsA201 cells and in glial cell-derived C6 glioma cells. The membrane potentials of tsA201 cells expressing G77R and G83V were significantly depolarized as compared with WTKir4.1, whereas cells expressing Q212R, L166Q, and A167V were less affected. Furthermore, macroscopic currents from cells expressing WTKir4.1 and Q212R channels did not differ, whereas currents from cells expressing L166Q, G83V, G77R, and A167V were reduced. Unexpectedly, L166Q current responses were rescued when co-expressed with Kir5.1. In addition, we observed notable differences in channel activity between C6 glioma cells and tsA201 cells expressing L166Q and A167V, suggesting that there are underlying differences between cell lines in terms of Kir4.1 protein synthesis, stability, or expression at the surface. Finally, we determined spermine (SPM) sensitivity of these uncharacterized SNPs and found that Q212R-containing channels displayed reduced block by 1 μmSPM. At 100 μmSPM, the block was equal to or greater than WT, suggesting that the greater driving force of SPM allowed achievement of steady state. In contrast, L166Q-Kir5.1 channels achieved a higher block than WT, suggesting a more stable interaction of SPM in the deep pore cavity. Overall, our data suggest that G83V, L166Q, and Q212R residues play a pivotal role in controlling Kir4.1 channel function.
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Project description:Ethanol consumption leads to a wide range of pharmacological effects by acting on the signaling proteins in the human nervous system, such as ion channels. Despite its familiarity and biological importance, very little is known about the molecular mechanisms underlying the ethanol action, due to extremely weak binding affinity and the dynamic nature of the ethanol interaction. In this research, we focused on the primary in vivo target of ethanol, G-protein-activated inwardly rectifying potassium channel (GIRK), which is responsible for the ethanol-induced analgesia. By utilizing solution NMR spectroscopy, we characterized the changes in the structure and dynamics of GIRK induced by ethanol binding. We demonstrated here that ethanol binds to GIRK with an apparent dissociation constant of 1.0 M and that the actual physiological binding site of ethanol is located on the cavity formed between the neighboring cytoplasmic regions of the GIRK tetramer. From the methyl-based NMR relaxation analyses, we revealed that ethanol activates GIRK by shifting the conformational equilibrium processes, which are responsible for the gating of GIRK, to stabilize an open conformation of the cytoplasmic ion gate. We suggest that the dynamic molecular mechanism of the ethanol-induced activation of GIRK represents a general model of the ethanol action on signaling proteins in the human nervous system.
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Project description:Honey bees are economically important pollinators of a wide variety of crops that have attracted the attention of both researchers and the public alike due to unusual declines in the numbers of managed colonies in some parts of the world. Viral infections are thought to be a significant factor contributing to these declines, but viruses have proven a challenging pathogen to study in a bee model and interactions between viruses and the bee antiviral immune response remain poorly understood. In the work described here, we have demonstrated the use of flock house virus (FHV) as a model system for virus infection in bees and revealed an important role for the regulation of the bee antiviral immune response by ATP-sensitive inwardly rectifying potassium (KATP) channels. We have shown that treatment with the KATP channel agonist pinacidil increases survival of bees while decreasing viral replication following infection with FHV, whereas treatment with the KATP channel antagonist tolbutamide decreases survival and increases viral replication. Our results suggest that KATP channels provide a significant link between cellular metabolism and the antiviral immune response in bees.
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Project description:The inwardly rectifying potassium channel (Kir) regulates resting membrane potential, K+ homeostasis, heart rate, and hormone secretion. The outward current is blocked in a voltage-dependent manner, upon the binding of intracellular polyamines or Mg2+ to the transmembrane pore domain. Meanwhile, electrophysiological studies have shown that mutations of several acidic residues in the intracellular regions affected the inward rectification. Although these acidic residues are assumed to bind polyamines, the functional role of the binding of polyamines and Mg2+ to the intracellular regions of Kirs remains unclear. Here, we report thermodynamic and structural studies of the interaction between polyamines and the cytoplasmic pore of mouse Kir3.1/GIRK1, which is gated by binding of G-protein betagamma-subunit (Gbetagamma). ITC analyses showed that two spermine molecules bind to a tetramer of Kir3.1/GIRK1 with a dissociation constant of 26 microM, which is lower than other blockers. NMR analyses revealed that the spermine binding site is Asp-260 and its surrounding area. Small but significant chemical shift perturbations upon spermine binding were observed in the subunit-subunit interface of the tetramer, suggesting that spermine binding alters the relative orientations of the four subunits. Our ITC and NMR results postulated a spermine binding mode, where one spermine molecule bridges two Asp-260 side chains from adjacent subunits, with rearrangement of the subunit orientations. This suggests the functional roles of spermine binding to the cytoplasmic pore: stabilization of the resting state conformation of the channel, and instant translocation to the transmembrane pore upon activation through the Gbetagamma-induced conformational rearrangement.
| S-EPMC2758011 | biostudies-literature
Project description:Previously, we demonstrated that the inwardly rectifying K(+) (Kir) channel subunit Kir7.1 is highly expressed in bovine and human retinal pigment epithelium (RPE). The purpose of this study was to determine whether any of the 14 other members of the Kir gene family are expressed in native human RPE. Conventional reverse transcription-polymerase chain reaction (RT-PCR) analysis indicated that in addition to Kir7.1, seven other Kir channel subunits (Kir1.1, Kir2.1, Kir2.2, Kir3.1, Kir3.4, Kir4.2 and Kir6.1) are expressed in the RPE, whereas in neural retina, all 14 of the Kir channel subunits examined are expressed. The identities of RT-PCR products in the RPE were confirmed by DNA sequencing. Real-time RT-PCR analysis showed, however, that transcripts of these channels are significantly less abundant than Kir7.1 in the RPE. Western blot analysis of the Kir channel subunits detected in the RPE by RT-PCR revealed the expression of Kir2.1, Kir3.1, Kir3.4, Kir4.2, Kir6.1, and possibly Kir2.2, but not Kir1.1, in both human RPE and neural retina. Our results indicate that human RPE expresses at least five other Kir channel subtypes in addition to Kir7.1, suggesting that multiple members of the Kir channel family may function in this epithelium.
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Project description:Polymyxin antibiotics are often used as a last-line defense to treat life-threatening Gram-negative pathogens. However, polymyxin-induced kidney toxicity is a dose-limiting factor of paramount importance and can lead to suboptimal treatment. To elucidate the mechanism and develop effective strategies to overcome polymyxin toxicity, we employed a whole-genome CRISPR screen in human kidney tubular HK-2 cells and identified 86 significant genes that upon knock-out rescued polymyxin-induced toxicity. Specifically, we discovered that knockout of the inwardly rectifying potassium channels Kir4.2 and Kir5.1 (encoded by KCNJ15 and KCNJ16, respectively) rescued polymyxin-induced toxicity in HK-2 cells. Furthermore, we found that polymyxins induced cell depolarization via Kir4.2 and Kir5.1 and a significant cellular uptake of polymyxins was evident. All-atom molecular dynamics simulations revealed that polymyxin B1 spontaneously bound to Kir4.2, thereby increasing opening of the channel, resulting in a potassium influx, and changes of the membrane potential. Consistent with these findings, small molecule inhibitors (BaCl2 and VU0134992) of Kir potassium channels reduced polymyxin-induced toxicity in cell culture and mouse explant kidney tissue. Our findings provide critical mechanistic information that will help attenuate polymyxin-induced nephrotoxicity in patients and facilitate the design of novel, safer polymyxins.
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