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The Molecular Mechanism of Opening the Helix Bundle Crossing (HBC) Gate of a Kir Channel.

ABSTRACT: Inwardly rectifying K(+) (Kir) channels, serving as natural molecular nanomachines, transport potassium ions across the plasma membrane of the cell. Along the ion permeation pathway, three relatively narrow regions (the selectivity filter (SF), the inner helix bundle crossing (HBC), and the cytosolic G loop) may serve as gates to control ion permeation. Our previous molecular dynamics simulations based on the crystal structure of a Kir3.1 chimera revealed the possible gating mechanism of the G loop gate. Here, we introduced a proline mutation in the inner helix and obtained a channel model of the open HBC gate. The open HBC gate reaches 0.6?nm in diameter, which allows partial hydrated K(+) ions to pass through. During the gating process, both the transmembrane helices TM1 and TM2 cooperatively rotate in a counterclockwise direction (viewed from the extracellular side) with the aid of the phospholipid PIP2. Only when all the transmembrane helices adopt a counterclockwise rotation, the HBC gate can be stabilized in the open state. We estimate that introduction of the proline mutation decreases the energy required to open the HBC gate by about 1.4?kcal/mol (??G).

SUBMITTER: Meng XY

PROVIDER: S-EPMC4954981 | biostudies-literature | 2016 Jul

REPOSITORIES: biostudies-literature

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The Molecular Mechanism of Opening the Helix Bundle Crossing (HBC) Gate of a Kir Channel.

Meng Xuan-Yu XY Liu Shengtang S Cui Meng M Zhou Ruhong R Logothetis Diomedes E DE

Scientific reports 20160721

Inwardly rectifying K(+) (Kir) channels, serving as natural molecular nanomachines, transport potassium ions across the plasma membrane of the cell. Along the ion permeation pathway, three relatively narrow regions (the selectivity filter (SF), the inner helix bundle crossing (HBC), and the cytosolic G loop) may serve as gates to control ion permeation. Our previous molecular dynamics simulations based on the crystal structure of a Kir3.1 chimera revealed the possible gating mechanism of the G l ...[more]

PMID: 27439597

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Project description:BACKGROUND: In-frame deletion mutation (Del-L955) in NaV1.7 sodium channel from a kindred with erythromelalgia hyperpolarizes activation. RESULTS: Del-L955 twists the S6 helix, displacing the Phe960 activation gate. Replacement of Phe960 at the correct helical position depolarizes activation. CONCLUSION: Radial tuning of the activation gate is critical to the activation of NaV1.7 channel. SIGNIFICANCE: Structural modeling guided electrophysiology reveals the functional importance of radial tuning of the S6 segment. Voltage-gated sodium (NaV) channels are membrane proteins that consist of 24 transmembrane segments organized into four homologous domains and are essential for action potential generation and propagation. Although the S6 helices of NaV channels line the ion-conducting pore and participate in channel activation, their functional architecture is incompletely understood. Our recent studies show that a naturally occurring in-frame deletion mutation (Del-L955) of NaV1.7 channel, identified in individuals with a severe inherited pain syndrome (inherited erythromelalgia) causes a substantial hyperpolarizing shift of channel activation. Here we took advantage of this deletion mutation to understand the role of the S6 helix in the channel activation. Based on the recently published structure of a bacterial NaV channel (NaVAb), we modeled the WT and Del-L955 channel. Our structural model showed that Del-L955 twists the DII/S6 helix, shifting location and radial orientation of the activation gate residue (Phe(960)). Hypothesizing that these structural changes produce the shift of channel activation of Del-L955 channels, we restored a phenylalanine in wild-type orientation by mutating Ser(961) (Del-L955/S961F), correcting activation by ?10 mV. Correction of the displaced Phe(960) (F960S) together with introduction of the rescuing activation gate residue (S961F) produced an additional ?6-mV restoration of activation of the mutant channel. A simple point mutation in the absence of a twist (L955A) did not produce a radial shift and did not hyperpolarize activation. Our results demonstrate the functional importance of radial tuning of the sodium channel S6 helix for the channel activation.

Project description:Kainate receptors are ligand-gated ion channels that have two major roles in the central nervous system: they mediate a postsynaptic component of excitatory neurotransmission at some glutamatergic synapses and modulate transmitter release at both excitatory and inhibitory synapses. Accumulating evidence implicates kainate receptors in a variety of neuropathologies, including epilepsy, psychiatric disorders, developmental delay, and cognitive impairment. Here, to gain a deeper understanding of the conformational changes associated with agonist binding and channel opening, we generate a series of Cys substitutions in the GluK2 kainate receptor subunit, focusing on the M3 helices that line the ion pore and form the bundle-crossing gate at the extracellular mouth of the channel. Exposure to 50 µM Cd produces direct activation of homomeric mutant channels bearing Cys substitutions in (A657C), or adjacent to (L659C), the conserved SYTANLAAF motif. Activation by Cd is occluded by modification with 2-aminoethyl MTS (MTSEA), indicating that Cd binds directly and specifically to the substituted cysteines. Cd potency for the A657C mutation (EC50 = 10 µM) suggests that binding involves at least two coordinating residues, whereas weaker Cd potency for L659C (EC50 = 2 mM) implies that activation does not require tight coordination by multiple side chains for this substitution. Experiments with heteromeric and chimeric channels indicate that activation by Cd requires Cys substitution at only two of the four subunits within a tetrameric receptor and that activation is similar for substitution within subunits in either the A/C or B/D conformations. We develop simple kinetic models for the A657C substitution that reproduce several features of Cd activation as well as the low-affinity inhibition observed at higher Cd concentrations (5-20 mM). Together, these results demonstrate rapid and reversible channel activation, independent of agonist site occupancy, upon Cd binding to Cys side chains at two specific locations along the GluK2 inner helix.

Dataset Information

The Molecular Mechanism of Opening the Helix Bundle Crossing (HBC) Gate of a Kir Channel.

Publications

The Molecular Mechanism of Opening the Helix Bundle Crossing (HBC) Gate of a Kir Channel.

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