Control of Inward Rectifier K Channel Activity by Lipid Tethering of Cytoplasmic Domains

Interactions between nontransmembrane domains and the lipid membrane are proposed to modulate activity of many ion channels. In Kir channels, the so-called “slide-helix” is proposed to interact with the lipid headgroups and control channel gating. We examined this possibility directly in a cell-free system consisting of KirBac1.1 reconstituted into pure lipid vesicles. Cysteine substitution of positively charged slide-helix residues (R49C and K57C) leads to loss of channel activity that is rescued by in situ restoration of charge following modification by MTSET+ or MTSEA+, but not MTSES− or neutral MMTS. Strikingly, activity is also rescued by modification with long-chain alkyl-MTS reagents. Such reagents are expected to partition into, and hence tether the side chain to, the membrane. Systematic scanning reveals additional slide-helix residues that are activated or inhibited following alkyl-MTS modification. A pattern emerges whereby lipid tethering of the N terminus, or C terminus, of the slide-helix, respectively inhibits, or activates, channel activity. This study establishes a critical role of the slide-helix in Kir channel gating, and directly demonstrates that physical interaction of soluble domains with the membrane can control ion channel activity

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 PIP(2)-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

KirBac1.1: It's an Inward Rectifying Potassium Channel

KirBac1.1 is a prokaryotic homologue of eukaryotic inward rectifier potassium (Kir) channels. The crystal structure of KirBac1.1 and related KirBac3.1 have now been used extensively to generate in silico models of eukaryotic Kir channels, but functional analysis has been limited to 86Rb+ flux experiments and bacteria or yeast complementation screens, and no voltage clamp analysis has been available. We have expressed pure full-length His-tagged KirBac1.1 protein in Escherichia coli and obtained voltage clamp recordings of recombinant channel activity in excised membrane patches from giant liposomes. Macroscopic currents of wild-type KirBac1.1 are K+ selective and spermine insensitive, but blocked by Ba2+, similar to “weakly rectifying” eukaryotic Kir1.1 and Kir6.2 channels. The introduction of a negative charge at a pore-lining residue, I138D, generates high spermine sensitivity, similar to that resulting from the introduction of a negative charge at the equivalent position in Kir1.1 or Kir6.2. KirBac1.1 currents are also inhibited by PIP2, consistent with 86Rb+ flux experiments, and reversibly inhibited by short-chain di-c8-PIP2. At the single-channel level, KirBac1.1 channels show numerous conductance states with two predominant conductances (15 pS and 32 pS at −100 mV) and marked variability in gating kinetics, similar to the behavior of KcsA in recombinant liposomes. The successful patch clamping of KirBac1.1 confirms that this prokaryotic channel behaves as a bona fide Kir channel and opens the way for combined biochemical, structural, and electrophysiological analysis of a tractable model Kir channel, as has been successfully achieved for the archetypal K+ channel KcsA

Lipids driving protein structure? Evolutionary adaptations in Kir channels

Many eukaryotic channels, transporters and receptors are activated by phosphatidyl inositol bisphosphate (PIP(2)) in the membrane, and every member of the eukaryotic inward rectifier potassium (Kir) channel family requires membrane PIP(2) for activity. In contrast, a bacterial homolog (KirBac1.1) is specifically inhibited by PIP(2). We speculate that a key evolutionary adaptation in eukaryotic channels is the insertion of additional linkers between trans-membrane and cytoplasmic domains, revealed by new crystal structures, that convert PIP(2) inhibition to activation. Such an adaptation may reflect a novel evolutionary drive to protein structure,; one that was necessary to permit channel function within the highly negatively charged membranes that evolved in the eukaryotic lineage

Molecular Basis of Inward Rectification: Polyamine Interaction Sites Located by Combined Channel and Ligand Mutagenesis

Polyamines cause inward rectification of (Kir) K+ channels, but the mechanism is controversial. We employed scanning mutagenesis of Kir6.2, and a structural series of blocking diamines, to combinatorially examine the role of both channel and blocker charges. We find that introduced glutamates at any pore-facing residue in the inner cavity, up to and including the entrance to the selectivity filter, can confer strong rectification. As these negative charges are moved higher (toward the selectivity filter), or lower (toward the cytoplasm), they preferentially enhance the potency of block by shorter, or longer, diamines, respectively. MTSEA+ modification of engineered cysteines in the inner cavity reduces rectification, but modification below the inner cavity slows spermine entry and exit, without changing steady-state rectification. The data provide a coherent explanation of classical strong rectification as the result of polyamine block in the inner cavity and selectivity filter

Structural basis of control of inward rectifier Kir2 channel gating by bulk anionic phospholipids

Inward rectifier potassium (Kir) channel activity is controlled by plasma membrane lipids. Phosphatidylinositol-4,5-bisphosphate (PIP(2)) binding to a primary site is required for opening of classic inward rectifier Kir2.1 and Kir2.2 channels, but interaction of bulk anionic phospholipid (PL(−)) with a distinct second site is required for high PIP(2) sensitivity. Here we show that introduction of a lipid-partitioning tryptophan at the second site (K62W) generates high PIP(2) sensitivity, even in the absence of PL(−). Furthermore, high-resolution x-ray crystal structures of Kir2.2[K62W], with or without added PIP(2) (2.8- and 2.0-Å resolution, respectively), reveal tight tethering of the C-terminal domain (CTD) to the transmembrane domain (TMD) in each condition. Our results suggest a refined model for phospholipid gating in which PL(−) binding at the second site pulls the CTD toward the membrane, inducing the formation of the high-affinity primary PIP(2) site and explaining the positive allostery between PL(−) binding and PIP(2) sensitivity