Shizhen Wang

Secondary anionic phospholipid binding site and gating mechanism in Kir2.1 inward rectifier channels

Inwardly rectifying potassium (Kir) channels regulate multiple tissues. All Kir channels require interaction of phosphatidyl-4,5-bisphosphate (PIP2) at a crystallographically identified binding site, but an additional nonspecific secondary anionic phospholipid (PL(−)) is required to generate high PIP2 sensitivity of Kir2 channel gating. The PL(−)-binding site and mechanism are yet to be elucidated. Here we report docking simulations that identify a putative PL(−)-binding site, adjacent to the PIP2-binding site, generated by two lysine residues from neighbouring subunits. When either lysine is mutated to cysteine (K64C and K219C), channel activity is significantly decreased in cells and in reconstituted liposomes. Directly tethering K64C to the membrane by modification with decyl-MTS generates high PIP2 sensitivity in liposomes, even in the complete absence of PL(−)s. The results provide a coherent molecular mechanism whereby PL(−) interaction with a discrete binding site results in a conformational change that stabilizes the high-affinity PIP2 activatory site.

Structural dynamics of potassium-channel gating revealed by single-molecule FRET

Crystallography has provided invaluable insights regarding ion-channel selectivity and gating, but to advance understanding to a new level, dynamic views of channel structures within membranes are essential. We labeled tetrameric KirBac1.1 potassium channels with single donor and acceptor fluorophores at different sites and then examined structural dynamics within lipid membranes by single-molecule fluorescence resonance energy transfer (FRET). We found that the extracellular region is structurally rigid in both closed and open states, whereas the N-terminal slide helix undergoes marked conformational fluctuations. The cytoplasmic C-terminal domain fluctuates between two major structural states, both of which become less dynamic and move away from the pore axis and away from the membrane in closed channels. Our results reveal mobile and rigid conformations of functionally relevant KirBac1.1 channel motifs, implying similar dynamics for similar motifs in eukaryotic Kir channels and in cation channels in general.

Structural rearrangements underlying ligand-gating in Kir channels

Inward rectifier potassium (Kir) channels are physiologically regulated by a wide range of ligands that all act on a common gate, although structural details of gating are unclear. Here we show, using small molecule fluorescent probes attached to introduced cysteines, the molecular motions associated with gating of KirBac1.1 channels. The accessibility of the probes indicates a major barrier to fluorophore entry to the inner cavity. Changes in FRET between fluorophores attached to KirBac1.1 tetramers show that PIP2-induced closure involves tilting and rotational motions of secondary structural elements of the cytoplasmic domain that couple ligand binding to a narrowing of the cytoplasmic vestibule. The observed ligand-dependent conformational changes in KirBac1.1 provide a general model for ligand-induced Kir channel gating at the molecular level.

On Potential Interactions between Non-selective Cation Channel TRPM4 and Sulfonylurea Receptor SUR1

Background: SUR1, the regulatory subunit of KATP channels, was hypothesized to associate with TRPM4 to form novel channels, implicated in cell death following neurovascular trauma. Results: The properties of heterologously expressed TRPM4 channels are not modified by SUR1. Conclusion: The coupling between SUR1 and TRPM4 is unlikely. Significance: The roles of TRPM4 and KATP channels in the pathogenesis of brain edema and hemorrhage should be reassessed. The sulfonylurea receptor SUR1 associates with Kir6.2 or Kir6.1 to form KATP channels, which link metabolism to excitability in multiple cell types. The strong physical coupling of SUR1 with Kir6 subunits appears exclusive, but recent studies argue that SUR1 also modulates TRPM4, a member of the transient receptor potential family of non-selective cation channels. It has been reported that, following stroke, brain, or spinal cord injury, SUR1 is increased in neurovascular cells at the site of injury. This is accompanied by up-regulation of a non-selective cation conductance with TRPM4-like properties and apparently sensitive to sulfonylureas, leading to the postulation that post-traumatic non-selective cation currents are determined by TRPM4/SUR1 channels. To investigate the mechanistic hypothesis for the coupling between TRPM4 and SUR1, we performed electrophysiological and FRET studies in COSm6 cells expressing TRPM4 channels with or without SUR1. TRPM4-mediated currents were Ca2+-activated, voltage-dependent, underwent desensitization, and were inhibited by ATP but were insensitive to glibenclamide and tolbutamide. These properties were not affected by cotransfection with SUR1. When the same SUR1 was cotransfected with Kir6.2, functional KATP channels were formed. In cells cotransfected with Kir6.2, SUR1, and TRPM4, we measured KATP-mediated K+ currents and Ca2+-activated, sulfonylurea-insensitive Na+ currents in the same patch, further showing that SUR1 controls KATP channel activity but not TRPM4 channels. FRET signal between fluorophore-tagged TRPM4 subunits was similar to that between Kir6.2 and SUR1, whereas there was no detectable FRET efficiency between TRPM4 and SUR1. Our data suggest that functional or structural association of TRPM4 and SUR1 is unlikely.

Differential Roles of Blocking Ions in KirBac1.1 Tetramer Stability

Potassium channels are tetrameric proteins that mediate K+-selective transmembrane diffusion. For KcsA, tetramer stability depends on interactions between permeant ions and the channel pore. We have examined the role of pore blockers on the tetramer stability of KirBac1.1. In 150 mm KCl, purified KirBac1.1 protein migrates as a monomer (∼40 kDa) on SDS-PAGE. Addition of Ba2+ (K1/2 ∼ 50 μm) prior to loading results in an additional tetramer band (∼160 kDa). Mutation A109C, at a residue located near the expected Ba2+-binding site, decreased tetramer stabilization by Ba2+ (K1/2 ∼ 300 μm), whereas I131C, located nearby, stabilized tetramers in the absence of Ba2+. Neither mutation affected Ba2+ block of channel activity (using 86Rb+ flux assay). In contrast to Ba2+, Mg2+ had no effect on tetramer stability (even though Mg2+ was a potent blocker). Many studies have shown Cd2+ block of K+ channels as a result of cysteine substitution of cavity-lining M2 (S6) residues, with the implicit interpretation that coordination of a single ion by cysteine side chains along the central axis effectively blocks the pore. We examined blocking and tetramer-stabilizing effects of Cd2+ on KirBac1.1 with cysteine substitutions in M2. Cd2+ block potency followed an α-helical pattern consistent with the crystal structure. Significantly, Cd2+ strongly stabilized tetramers of I138C, located in the center of the inner cavity. This stabilization was additive with the effect of Ba2+, consistent with both ions simultaneously occupying the channel: Ba2+ at the selectivity filter entrance and Cd2+ coordinated by I138C side chains in the inner cavity.

Lipids driving protein structure? Evolutionary adaptations in Kir channels.

Many eukaryotic channels, transporters and receptors are activated by phosphatidyl inositol bisphosphate (PIP2) in the membrane, and every member of the eukaryotic inward rectifier potassium (Kir) channel family requires membrane PIP2 for activity. In contrast, a bacterial homolog (KirBac1.1) is specifically inhibited by PIP2. We speculate that a key evolutionary adaptation in eukaryotic channels is the insertion of additional linkers between transmembrane and cytoplasmic domains, revealed by new crystal structures, that convert PIP2 inhibition to activation. Such an adaptation may reflect a novel evolutionary drive to protein structure, and that was necessary to permit channel function within the highly negatively charged membranes that evolved in the eukaryotic lineage.

Control of KirBac3.1 Potassium Channel Gating at the Interface between Cytoplasmic Domains

Background: KirBac3.1 is a prokaryotic homolog of eukaryotic Kir channels. Results: A high-resolution crystal structure of a mutant channel reveals a novel open conformation. Conclusion: The intersubunit interface between the cytoplasmic domains controls channel gating. Significance: These findings help define the structures important for gating in prokaryotic and eukaryotic Kir channels. KirBac channels are prokaryotic homologs of mammalian inwardly rectifying potassium (Kir) channels, and recent structures of KirBac3.1 have provided important insights into the structural basis of gating in Kir channels. In this study, we demonstrate that KirBac3.1 channel activity is strongly pH-dependent, and we used x-ray crystallography to determine the structural changes that arise from an activatory mutation (S205L) located in the cytoplasmic domain (CTD). This mutation stabilizes a novel energetically favorable open conformation in which changes at the intersubunit interface in the CTD also alter the electrostatic potential of the inner cytoplasmic cavity. These results provide a structural explanation for the activatory effect of this mutation and provide a greater insight into the role of the CTD in Kir channel gating.

Domain Organization of the ATP-sensitive Potassium Channel Complex Examined by Fluorescence Resonance Energy Transfer

Background: We examined FRET between Kir6.2 and SUR1 domains of KATP channels, in various combinations. Results: FRET was detected within and between Kir6.2 subunits and between Kir6.2 and split SUR1 N-terminal constructs. Conclusion: Kir6.2 C termini are centrally located. SUR1 domains can self-associate and are close to Kir6.2 termini in the full complex. Significance: The results indicate domain architecture of this unique channel complex. KATP channels link cell metabolism to excitability in many cells. They are formed as tetramers of Kir6.2 subunits, each associated with a SUR1 subunit. We used mutant GFP-based FRET to assess domain organization in channel complexes. Full-length Kir6.2 subunits were linked to YFP or cyan fluorescent protein (CFP) at N or C termini, and all such constructs, including double-tagged YFP-Kir6.2-CFP (Y6.2C), formed functional KATP channels. In intact COSm6 cells, background emission of YFP excited by 430-nm light was ∼6%, but the Y6.2C construct expressed alone exhibited an apparent FRET efficiency of ∼25%, confirmed by trypsin digestion, with or without SUR1 co-expression. Similar FRET efficiency was detected in mixtures of CFP- and YFP-tagged full-length Kir6.2 subunits and transmembrane domain only constructs, when tagged at the C termini but not at the N termini. The FRET-reported Kir6.2 tetramer domain organization was qualitatively consistent with Kir channel crystal structures: C termini and M2 domains are centrally located relative to N termini and M1 domains, respectively. Additional FRET analyses were performed on cells in which tagged full-length Kir6.2 and tagged SUR1 constructs were co-expressed. These analyses further revealed that 1) NBD1 of SUR1 is closer to the C terminus of Kir6.2 than to the N terminus; 2) the Kir6.2 cytoplasmic domain is not essential for complexation with SUR1; and 3) the N-terminal half of SUR1 can complex with itself in the absence of either the C-terminal half or Kir6.2.

Molecular Dynamics Simulations of KirBac1.1 Mutants Reveal Global Gating Changes of Kir Channels

Prokaryotic inwardly rectifying (KirBac) potassium channels are homologous to mammalian Kir channels. Their activity is controlled by dynamical conformational changes that regulate ion flow through a central pore. Understanding the dynamical rearrangements of Kir channels during gating requires high-resolution structure information from channels crystallized in different conformations and insight into the transition steps, which are difficult to access experimentally. In this study, we use MD simulations on wild type KirBac1.1 and an activatory mutant to investigate activation gating of KirBac channels. Full atomistic MD simulations revealed that introducing glutamate in position 143 causes significant widening at the helix bundle crossing gate, enabling water flux into the cavity. Further, global rearrangements including a twisting motion as well as local rearrangements at the subunit interface in the cytoplasmic domain were observed. These structural rearrangements are similar to recently reported KirBac3.1 crystal structures in closed and open conformation, suggesting that our simulations capture major conformational changes during KirBac1.1 opening. In addition, an important role of protein–lipid interactions during gating was observed. Slide-helix and C-linker interactions with lipids were strengthened during activation gating.

Control of Kir channel gating by cytoplasmic domain interface interactions

Inward rectifier potassium (Kir) channels are expressed in almost all mammalian tissues and play critical roles in the control of excitability. Pancreatic ATP-sensitive K (KATP) channels are key regulators of insulin secretion and comprise Kir6.2 subunits coupled to sulfonylurea receptors. Because these channels are reversibly inhibited by cytoplasmic ATP, they link cellular metabolism with membrane excitability. Loss-of-function mutations in the pore-forming Kir6.2 subunit cause congenital hyperinsulinism as a result of diminished channel activity. Here, we show that several disease mutations, which disrupt intersubunit salt bridges at the interface of the cytoplasmic domains (CD-I) of adjacent subunits, induce loss of channel activity via a novel channel behavior: after ATP removal, channels open but then rapidly inactivate. Re-exposure to inhibitory ATP causes recovery from this inactivation. Inactivation can be abolished by application of phosphatidylinositol-4,5-bisphosphate (PIP2) to the cytoplasmic face of the membrane, an effect that can be explained by a simple kinetic model in which PIP2 binding competes with the inactivation process. Kir2.1 channels contain homologous salt bridges, and we find that mutations that disrupt CD-I interactions in Kir2.1 also reduce channel activity and PIP2 sensitivity. Kir2.1 channels also contain an additional CD-I salt bridge that is not present in Kir6.2 channels. Introduction of this salt bridge into Kir6.2 partially rescues inactivating mutants from the phenotype. These results indicate that the stability of the intersubunit CD-I is a major determinant of the inactivation process in Kir6.2 and may control gating in other Kir channels.