Interactions between selectivity filter and pore helix control filter gating in the MthK channel.

K+ channel activity can be limited by C-type inactivation, which is likely initiated in part by dissociation of K+ ions from the selectivity filter and modulated by the side chains that surround it. While crystallographic and computational studies have linked inactivation to a "collapsed" selectivity filter conformation in the KcsA channel, the structural basis for selectivity filter gating in other K+ channels is less clear. Here, we combined electrophysiological recordings with molecular dynamics simulations, to study selectivity filter gating in the model potassium channel MthK and its V55E mutant (analogous to KcsA E71) in the pore-helix. We found that MthK V55E has a lower open probability than the WT channel, due to decreased stability of the open state, as well as a lower unitary conductance. Simulations account for both of these variables on the atomistic scale, showing that ion permeation in V55E is altered by two distinct orientations of the E55 side chain. In the "vertical" orientation, in which E55 forms a hydrogen bond with D64 (as in KcsA WT channels), the filter displays reduced conductance compared to MthK WT. In contrast, in the "horizontal" orientation, K+ conductance is closer to that of MthK WT; although selectivity filter stability is lowered, resulting in more frequent inactivation. Surprisingly, inactivation in MthK WT and V55E is associated with a widening of the selectivity filter, unlike what is observed for KcsA and reminisces recent structures of inactivated channels, suggesting a conserved inactivation pathway across the potassium channel family.

A novel NEDD4L-TXNIP-CHOP axis in the pathogenesis of nonalcoholic steatohepatitis.

Background: Nonalcoholic steatohepatitis (NASH) is a leading cause of chronic liver diseases worldwide. There is a pressing clinical need to identify potential therapeutic targets for NASH treatment. Thioredoxin interacting protein (Txnip) is a stress responsive gene that has been implicated in the pathogenesis of NASH, but its exact role is not fully understood. Here, we investigated the liver- and gene-specific role of Txnip and its upstream/downstream signaling in the pathogenesis of NASH. Methods and Results: Using four independent NASH mouse models, we found that TXNIP protein abnormally accumulated in NASH mouse livers. A decrease in E3 ubiquitin ligase NEDD4L resulted in impaired TXNIP ubiquitination and its accumulation in the liver. TXNIP protein levels were positively correlated with that of CHOP, a major regulator of ER stress-mediated apoptosis, in NASH mouse liver. Moreover, gain- and loss-of-function studies showed that TXNIP increased protein not mRNA levels of Chop both in vitro and in vivo. Mechanistically, the C-terminus of TXNIP associated with the N-terminus of the α-helix domain of CHOP and decreased CHOP ubiquitination, thus increasing the stability of CHOP protein. Lastly, selective knockdown of Txnip by adenovirus-mediated shRNA (not targets Txnip antisense lncRNA) delivery in the livers of both young and aged NASH mice suppressed the expression of CHOP and its downstream apoptotic pathway, and ameliorated NASH by reducing hepatic apoptosis, inflammation, and fibrosis. Conclusions: Our study revealed a pathogenic role of hepatic TXNIP in NASH and identified a novel NEDD4L-TXNIP-CHOP axis in the pathogenesis of NASH.

Interrogating the gating motions of the NaK channel.

Previous crystallographic studies depicted a physical gate of the NaK channel localized at a bundle crossing of pore-lining helices, but solution NMR studies in the current issue of JGP suggest otherwise.

Knockdown of lncRNA TP53TG1 Enhances the Efficacy of Sorafenib in Human Hepatocellular Carcinoma Cells.

The multikinase inhibitor, sorafenib, is a first-line treatment for hepatocellular carcinoma (HCC), but its limited efficacy, drug resistance and toxicity are a concern. In this study, we investigated the role of lncRNA TP53TG1 in the efficacy of sorafenib in HCC cells. We found that treatment with sorafenib increased the expression of TP53TG1 in HCC cells. Knockdown of TP53TG1 sensitized tumor cells to the antiproliferative effects of sorafenib. Furthermore, TP53TG1 knockdown had an additive inhibitory effect on HCC cell proliferation and migration in the presence of sorafenib. The combination of TP53TG1 knockdown and sorafenib drastically inhibited the activation of the ERK pathway. This work demonstrates that TP53TG1 deficiency enhances the efficacy of sorafenib in HCC. Combining TP53TG1 knockdown with sorafenib may be an optimal form of therapy for HCC treatment.

State-dependent inhibition of BK channels by the opioid agonist loperamide.

Large-conductance Ca2+-activated K+ (BK) channels control a range of physiological functions, and their dysfunction is linked to human disease. We have found that the widely used drug loperamide (LOP) can inhibit activity of BK channels composed of either α-subunits (BKα channels) or α-subunits plus the auxiliary γ1-subunit (BKα/γ1 channels), and here we analyze the molecular mechanism of LOP action. LOP applied at the cytosolic side of the membrane rapidly and reversibly inhibited BK current, an effect that appeared as a decay in voltage-activated BK currents. The apparent affinity for LOP decreased with hyperpolarization in a manner consistent with LOP behaving as an inhibitor of open, activated channels. Increasing LOP concentration reduced the half-maximal activation voltage, consistent with relative stabilization of the LOP-inhibited open state. Single-channel recordings revealed that LOP did not reduce unitary BK channel current, but instead decreased BK channel open probability and mean open times. LOP elicited use-dependent inhibition, in which trains of brief depolarizing steps lead to accumulated reduction of BK current, whereas single brief depolarizing steps do not. The principal effects of LOP on BK channel gating are described by a mechanism in which LOP acts as a state-dependent pore blocker. Our results suggest that therapeutic doses of LOP may act in part by inhibiting K+ efflux through intestinal BK channels.

Gating and calcium-sensing mechanisms of TRPA1 channels revealed.

Novel structures of the human TRPA1 channel were determined in the presence of the agonist iodoacetamide and the antagonist A-967079, to reveal the open and closed states of the channel, respectively. The structures further revealed the location of Ca2+ modulatory site that is likely conserved among several TRP subgroups.

Polyamine blockade and binding energetics in the MthK potassium channel.

Polyamines such as spermidine and spermine are found in nearly all cells, at concentrations ranging up to 0.5 mM. These cations are endogenous regulators of cellular K+ efflux, binding tightly in the pores of inwardly rectifying K+ (Kir) channels in a voltage-dependent manner. Although the voltage dependence of Kir channel polyamine blockade is thought to arise at least partially from the energetically coupled movements of polyamine and K+ ions through the pore, the nature of physical interactions between these molecules is unclear. Here we analyze the polyamine-blocking mechanism in the model K+ channel MthK, using a combination of electrophysiology and computation. Spermidine (SPD3+) and spermine (SPM4+) each blocked current through MthK channels in a voltage-dependent manner, and blockade by these polyamines was described by a three-state kinetic scheme over a wide range of polyamine concentrations. In the context of the scheme, both SPD3+ and SPM4+ access a blocking site with similar effective gating valences (0.84 ± 0.03 e0 for SPD3+ and 0.99 ± 0.04 e0 for SPM4+), whereas SPM4+ binds in the blocked state with an ∼20-fold higher affinity than SPD3+ (Kd = 28.1 ± 3.1 µM for SPD3+ and 1.28 ± 0.20 µM for SPM4+), consistent with a free energy difference of 1.8 kcal/mol. Molecular simulations of the MthK pore in complex with either SPD3+ or SPM4+ are consistent with the leading amine interacting with the hydroxyl groups of T59, at the selectivity filter threshold, with access to this site governed by outward movement of K+ ions. These coupled movements can account for a large fraction of the voltage dependence of blockade. In contrast, differences in binding energetics between SPD3+ and SPM4+ may arise from distinct electrostatic interactions between the polyamines and carboxylate oxygens on the side chains of E92 and E96, located in the pore-lining helix.

Molecular mechanism of BK channel activation by the smooth muscle relaxant NS11021.

Large-conductance Ca2+-activated K+ channels (BK channels) are activated by cytosolic calcium and depolarized membrane potential under physiological conditions. Thus, these channels control electrical excitability in neurons and smooth muscle by gating K+ efflux and hyperpolarizing the membrane in response to Ca2+ signaling. Altered BK channel function has been linked to epilepsy, dyskinesia, and other neurological deficits in humans, making these channels a key target for drug therapies. To gain insight into mechanisms underlying pharmacological modulation of BK channel gating, here we studied mechanisms underlying activation of BK channels by the biarylthiourea derivative, NS11021, which acts as a smooth muscle relaxant. We observe that increasing NS11021 shifts the half-maximal activation voltage for BK channels toward more hyperpolarized voltages, in both the presence and nominal absence of Ca2+, suggesting that NS11021 facilitates BK channel activation primarily by a mechanism that is distinct from Ca2+ activation. 30 µM NS11021 slows the time course of BK channel deactivation at -200 mV by ∼10-fold compared with 0 µM NS11021, while having little effect on the time course of activation. This action is most pronounced at negative voltages, at which the BK channel voltage sensors are at rest. Single-channel kinetic analysis further shows that 30 µM NS11021 increases open probability by 62-fold and increases mean open time from 0.15 to 0.52 ms in the nominal absence of Ca2+ at voltages less than -60 mV, conditions in which BK voltage sensors are largely in the resting state. We could therefore account for the major activating effects of NS11021 by a scheme in which the drug primarily shifts the pore-gate equilibrium toward the open state.

Molecular mechanism of a potassium channel gating through activation gate-selectivity filter coupling.

Potassium channels are presumed to have two allosterically coupled gates, the activation gate and the selectivity filter gate, that control channel opening, closing, and inactivation. However, the molecular mechanism of how these gates regulate K+ ion flow through the channel remains poorly understood. An activation process, occurring at the selectivity filter, has been recently proposed for several potassium channels. Here, we use X-ray crystallography and extensive molecular dynamics simulations, to study ion permeation through a potassium channel MthK, for various opening levels of both gates. We find that the channel conductance is controlled at the selectivity filter, whose conformation depends on the activation gate. The crosstalk between the gates is mediated through a collective motion of channel helices, involving hydrophobic contacts between an isoleucine and a conserved threonine in the selectivity filter. We propose a gating model of selectivity filter-activated potassium channels, including pharmacologically relevant two-pore domain (K2P) and big potassium (BK) channels.

Molecular determinants of Ca2+ sensitivity at the intersubunit interface of the BK channel gating ring.

The large-conductance calcium-activated K+ (BK) channel contains two intracellular tandem Ca2+-sensing RCK domains (RCK1 and RCK2), which tetramerize into a Ca2+ gating ring that regulates channel opening by conformational expansion in response to Ca2+ binding. Interestingly, the gating ring's intersubunit assembly interface harbors the RCK2 Ca2+-binding site, known as the Ca2+ bowl. The gating ring's assembly interface is made in part by intersubunit coordination of a Ca2+ ion between the Ca2+ bowl and an RCK1 Asn residue, N449, and by apparent intersubunit electrostatic interactions between E955 in RCK2 and R786 and R790 in the RCK2 of the adjacent subunit. To understand the role of the intersubunit assembly interface in Ca2+ gating, we performed mutational analyses of these putative interacting residues in human BK channels. We found that N449, despite its role in Ca2+ coordination, does not set the channel's Ca2+ sensitivity, whereas E955 is a determinant of Ca2+ sensitivity, likely through intersubunit electrostatic interactions. Our findings provide evidence that the intersubunit assembly interface contains molecular determinants of Ca2+-sensitivity in BK channels.

Permeating disciplines: Overcoming barriers between molecular simulations and classical structure-function approaches in biological ion transport.

Ion translocation across biological barriers is a fundamental requirement for life. In many cases, controlling this process-for example with neuroactive drugs-demands an understanding of rapid and reversible structural changes in membrane-embedded proteins, including ion channels and transporters. Classical approaches to electrophysiology and structural biology have provided valuable insights into several such proteins over macroscopic, often discontinuous scales of space and time. Integrating these observations into meaningful mechanistic models now relies increasingly on computational methods, particularly molecular dynamics simulations, while surfacing important challenges in data management and conceptual alignment. Here, we seek to provide contemporary context, concrete examples, and a look to the future for bridging disciplinary gaps in biological ion transport. This article is part of a Special Issue entitled: Beyond the Structure-Function Horizon of Membrane Proteins edited by Ute Hellmich, Rupak Doshi and Benjamin McIlwain.

Understanding the conformational motions of RCK gating rings.

Regulator of conduction of K+ (RCK) domains are ubiquitous regulators of channel and transporter activity in prokaryotes and eukaryotes. In humans, RCK domains form an integral component of large-conductance calcium-activated K channels (BK channels), key modulators of nerve, muscle, and endocrine cell function. In this review, we explore how the study of RCK domains in bacterial and human channels has contributed to our understanding of the structural basis of channel function. This knowledge will be critical in identifying mechanisms that underlie BK channelopathies that lead to epilepsy and other diseases, as well as regions of the channel that might be successfully targeted to treat such diseases.

The STIM1-binding site nexus remotely controls Orai1 channel gating.

The ubiquitously expressed Orai Ca2+ channels are gated through a unique process of intermembrane coupling with the Ca2+-sensing STIM proteins. Despite the significance of Orai1-mediated Ca2+ signals, how gating of Orai1 is triggered by STIM1 remains unknown. A widely held gating model invokes STIM1 binding directly to Orai1 pore-forming helix. Here we report that an Orai1 C-terminal STIM1-binding site, situated far from the N-terminal pore helix, alone provides the trigger that is necessary and sufficient for channel gating. We identify a critical 'nexus' within Orai1 connecting the peripheral C-terminal STIM1-binding site to the Orai1 core helices. Mutation of the nexus transforms Orai1 into a persistently open state exactly mimicking the action of STIM1. We suggest that the Orai1 nexus transduces the STIM1-binding signal through a conformational change in the inner core helices, and that STIM1 remotely gates the Orai1 channel without the necessity for direct STIM1 contact with the pore-forming helix.

Sterol hindrance of Orai activation.

Orai channels at the plasma membrane mediate store-operated Ca(2+) entry in response to Ca(2+) depletion of the endoplasmic reticulum. Orai channels are gated by stromal-interacting molecule proteins, which act as Ca(2+) sensors, and the association of these proteins is enhanced in cholesterol-rich lipid rafts. In research published in Science Signaling, Derler et al. report that cholesterol inhibits Orai function through direct association with the channel amino terminus.

STIM1 dimers undergo unimolecular coupling to activate Orai1 channels.

The endoplasmic reticulum (ER) Ca(2+) sensor, STIM1, becomes activated when ER-stored Ca(2+) is depleted and translocates into ER-plasma membrane junctions where it tethers and activates Orai1 Ca(2+) entry channels. The dimeric STIM1 protein contains a small STIM-Orai-activating region (SOAR)--the minimal sequence sufficient to activate Orai1 channels. Since SOAR itself is a dimer, we constructed SOAR concatemer-dimers and introduced mutations at F394, which is critical for Orai1 coupling and activation. The F394H mutation in both SOAR monomers completely blocks dimer function, but F394H introduced in only one of the dimeric SOAR monomers has no effect on Orai1 binding or activation. This reveals an unexpected unimolecular coupling between STIM1 and Orai1 and argues against recent evidence suggesting dimeric interaction between STIM1 and two adjacent Orai1 channel subunits. The model predicts that STIM1 dimers may be involved in crosslinking between Orai1 channels with implications for the kinetics and localization of Orai1 channel opening.

Neuronal STIMulation at rest.

Almost a decade has passed since first STIM, and later Orai, proteins were identified as the molecular constituents of store-operated calcium entry (SOCE). Their roles in immune function have been intensely investigated, but the roles of STIM and Orai in neuronal cells have been much less clear. Lalonde et al. show that when neurons are hyperpolarized or "at rest," constitutive endoplasmic reticulum (ER) Ca(2+) release leads to SOCE-mediated activation of neuronal transcription factors. Precisely why ER Ca(2+) release is constitutive in neurons remains an important question. Irrespective of the answer, this observation provides an intriguing new perspective on why a relatively low-abundance, small-conductance channel such as Orai1 would be important in neurons, which contain a relative abundance of voltage-operated Ca(2+) channels.