Shozeb Haider

Functional analysis of a structural model of the ATP‐binding site of the KATP channel Kir6.2 subunit

ATP‐sensitive potassium (KATP) channels couple cell metabolism to electrical activity by regulating K+ flux across the plasma membrane. Channel closure is mediated by ATP, which binds to the pore‐forming subunit (Kir6.2). Here we use homology modelling and ligand docking to construct a model of the Kir6.2 tetramer and identify the ATP‐binding site. The model is consistent with a large amount of functional data and was further tested by mutagenesis. Ligand binding occurs at the interface between two subunits. The phosphate tail of ATP interacts with R201 and K185 in the C‐terminus of one subunit, and with R50 in the N‐terminus of another; the N6 atom of the adenine ring interacts with E179 and R301 in the same subunit. Mutation of residues lining the binding pocket reduced ATP‐dependent channel inhibition. The model also suggests that interactions between the C‐terminus of one subunit and the ‘slide helix’ of the adjacent subunit may be involved in ATP‐dependent gating. Consistent with a role in gating, mutations in the slide helix bias the intrinsic channel conformation towards the open state.

A gating mutation at the internal mouth of the Kir6.2 pore is associated with DEND syndrome

Inwardly rectifying potassium (Kir) channels control cell membrane K+ fluxes and electrical signalling in diverse cell types. Heterozygous mutations in the human Kir6.2 gene (KCNJ11), the pore‐forming subunit of the ATP‐sensitive (KATP) channel, cause permanent neonatal diabetes mellitus. However, the I296L mutation also results in developmental delay, muscle weakness and epilepsy. We investigated the functional effects of the I296L mutation by expressing wild‐type or mutant Kir6.2/SUR1 channels in Xenopus oocytes. The mutation caused a marked increase in resting whole‐cell KATP currents by reducing channel inhibition by ATP, in both homomeric and simulated heterozygous states. Kinetic analysis showed that the mutation impaired ATP sensitivity indirectly, by stabilizing the open state of the channel and possibly also by means of an allosteric effect on ATP binding and/or transduction. The results implicate a new region in Kir‐channel gating and suggest that disease severity is correlated with the extent of reduction in ATP sensitivity.

Identification of residues contributing to the ATP binding site of Kir6.2.

The ATP‐sensitive potassium (KATP) channel links cell metabolism to membrane excitability. Intracellular ATP inhibits channel activity by binding to the Kir6.2 subunit of the channel, but the ATP binding site is unknown. Using cysteine‐scanning mutagenesis and charged thiol‐modifying reagents, we identified two amino acids in Kir6.2 that appear to interact directly with ATP: R50 in the N‐terminus, and K185 in the C‐terminus. The ATP sensitivity of the R50C and K185C mutant channels was increased by a positively charged thiol reagent (MTSEA), and was reduced by the negatively charged reagent MTSES. Comparison of the inhibitory effects of ATP, ADP and AMP after thiol modification suggests that K185 interacts primarily with the β‐phosphate, and R50 with the γ‐phosphate, of ATP. A molecular model of the C‐terminus of Kir6.2 (based on the crystal structure of Kir3.1) was constructed and automated docking was used to identify residues interacting with ATP. These results support the idea that K185 interacts with the β‐phosphate of ATP. Thus both N‐ and C‐termini may contribute to the ATP binding site.

Identification of the PIP2‐binding site on Kir6.2 by molecular modelling and functional analysis

ATP‐sensitive potassium (KATP) channels couple cell metabolism to electrical activity by regulating K+ fluxes across the plasma membrane. Channel closure is facilitated by ATP, which binds to the pore‐forming subunit (Kir6.2). Conversely, channel opening is potentiated by phosphoinositol bisphosphate (PIP2), which binds to Kir6.2 and reduces channel inhibition by ATP. Here, we use homology modelling and ligand docking to identify the PIP2‐binding site on Kir6.2. The model is consistent with a large amount of functional data and was further tested by mutagenesis. The fatty acyl tails of PIP2 lie within the membrane and the head group extends downwards to interact with residues in the N terminus (K39, N41, R54), transmembrane domains (K67) and C terminus (R176, R177, E179, R301) of Kir6.2. Our model suggests how PIP2 increases channel opening and decreases ATP binding and channel inhibition. It is likely to be applicable to the PIP2‐binding site of other Kir channels, as the residues identified are conserved and influence PIP2 sensitivity in other Kir channel family members.

Structural and functional analysis of the putative pH sensor in the Kir1.1 (ROMK) potassium channel

The pH‐sensitive renal potassium channel Kir1.1 is important for K+ homeostasis. Disruption of the pH‐sensing mechanism causes type II Bartter syndrome. The pH sensor is thought to be an anomalously titrated lysine residue (K80) that interacts with two arginine residues as part of an ‘RKR triad’. We show that a Kir1.1 orthologue from Fugu rubripes lacks this lysine and yet is still highly pH sensitive, indicating that K80 is not the H+ sensor. Instead, K80 functionally interacts with A177 on transmembrane domain 2 at the ‘helix‐bundle crossing’ and controls the ability of pH‐dependent conformational changes to induce pore closure. Although not required for pH inhibition, K80 is indispensable for the coupling of pH gating to the extracellular K+ concentration, explaining its conservation in most Kir1.1 orthologues. Furthermore, we demonstrate that instead of interacting with K80, the RKR arginine residues form highly conserved inter‐ and intra‐subunit interactions that are important for Kir channel gating and influence pH sensitivity indirectly.

Molecular simulations and lipid–protein interactions: potassium channels and other membrane proteins

Molecular dynamics simulations may be used to probe the interactions of membrane proteins with lipids and with detergents at atomic resolution. Examples of such simulations for ion channels and for bacterial outer membrane proteins are described. Comparison of simulations of KcsA (an alpha-helical bundle) and OmpA (a beta-barrel) reveals the importance of two classes of side chains in stabilizing interactions with the head groups of lipid molecules: (i) tryptophan and tyrosine; and (ii) arginine and lysine. Arginine residues interacting with lipid phosphate groups play an important role in stabilizing the voltage-sensor domain of the KvAP channel within a bilayer. Simulations of the bacterial potassium channel KcsA reveal specific interactions of phosphatidylglycerol with an acidic lipid-binding site at the interface between adjacent protein monomers. A combination of molecular modelling and simulation reveals a potential phosphatidylinositol 4,5-bisphosphate-binding site on the surface of Kir6.2.