Structure of the Shaker Kv channel and mechanism of slow C-type inactivation.

Voltage-activated potassium (Kv) channels open upon membrane depolarization and proceed to spontaneously inactivate. Inactivation controls neuronal firing rates and serves as a form of short-term memory and is implicated in various human neurological disorders. Here, we use high-resolution cryo-electron microscopy and computer simulations to determine one of the molecular mechanisms underlying this physiologically crucial process. Structures of the activated Shaker Kv channel and of its W434F mutant in lipid bilayers demonstrate that C-type inactivation entails the dilation of the ion selectivity filter and the repositioning of neighboring residues known to be functionally critical. Microsecond-scale molecular dynamics trajectories confirm that these changes inhibit rapid ion permeation through the channel. This long-sought breakthrough establishes how eukaryotic K+ channels self-regulate their functional state through the plasticity of their selectivity filters.

TMEM266 is a functional voltage sensor regulated by extracellular Zn2.

Voltage-activated ion channels contain S1-S4 domains that sense membrane voltage and control opening of ion-selective pores, a mechanism that is crucial for electrical signaling. Related S1-S4 domains have been identified in voltage-sensitive phosphatases and voltage-activated proton channels, both of which lack associated pore domains. hTMEM266 is a protein of unknown function that is predicted to contain an S1-S4 domain, along with partially structured cytoplasmic termini. Here we show that hTMEM266 forms oligomers, undergoes both rapid (µs) and slow (ms) structural rearrangements in response to changes in voltage, and contains a Zn2+ binding site that can regulate the slow conformational transition. Our results demonstrate that the S1-S4 domain in hTMEM266 is a functional voltage sensor, motivating future studies to identify cellular processes that may be regulated by the protein. The ability of hTMEM266 to respond to voltage on the µs timescale may be advantageous for designing new genetically encoded voltage indicators.

Heat activation is intrinsic to the pore domain of TRPV1.

The TRPV1 channel is a sensitive detector of pain-producing stimuli, including noxious heat, acid, inflammatory mediators, and vanilloid compounds. Although binding sites for some activators have been identified, the location of the temperature sensor remains elusive. Using available structures of TRPV1 and voltage-activated potassium channels, we engineered chimeras wherein transmembrane regions of TRPV1 were transplanted into the Shaker Kv channel. Here we show that transplanting the pore domain of TRPV1 into Shaker gives rise to functional channels that can be activated by a TRPV1-selective tarantula toxin that binds to the outer pore of the channel. This pore-domain chimera is permeable to Na+, K+, and Ca2+ ions, and remarkably, is also robustly activated by noxious heat. Our results demonstrate that the pore of TRPV1 is a transportable domain that contains the structural elements sufficient for activation by noxious heat.

Gating the pore of P2X receptor channels.

Three families of ligand-activated ion channels mediate synaptic communication between excitable cells in mammals. For pentameric channels related to nicotinic acetylcholine receptors and tetrameric channels such as glutamate receptors, the pore-forming and gate regions have been studied extensively. In contrast, little is known about the structure of trimeric P2X receptor channels, a family of channels that are activated by ATP and are important in neuronal signaling, pain transmission and inflammation. To identify the pore-forming and gate regions in P2X receptor channels, we introduced cysteine residues throughout the two transmembrane (TM) segments and studied their accessibility to thiol-reactive compounds and ions. Our results show that TM2 lines the central ion-conduction pore, TM1 is positioned peripheral to TM2 and the flow of ions is minimized in the closed state by a gate formed by the external region of TM2.

Functional interactions at the interface between voltage-sensing and pore domains in the Shaker K(v) channel.

Voltage-activated potassium (K(v)) channels contain a central pore domain that is partially surrounded by four voltage-sensing domains. Recent X-ray structures suggest that the two domains lack extensive protein-protein contacts within presumed transmembrane regions, but whether this is the case for functional channels embedded in lipid membranes remains to be tested. We investigated domain interactions in the Shaker K(v) channel by systematically mutating the pore domain and assessing tolerance by examining channel maturation, S4 gating charge movement, and channel opening. When mapped onto the X-ray structure of the K(v)1.2 channel the large number of permissive mutations support the notion of relatively independent domains, consistent with crystallographic studies. Inspection of the maps also identifies portions of the interface where residues are sensitive to mutation, an external cluster where mutations hinder voltage sensor activation, and an internal cluster where domain interactions between S4 and S5 helices from adjacent subunits appear crucial for the concerted opening transition.

Secondary structure and gating rearrangements of transmembrane segments in rat P2X4 receptor channels.

P2X receptors are cation selective channels that are activated by extracellular nucleotides. These channels are likely formed by three identical or related subunits, each having two transmembrane segments (TM1 and TM2). To identify regions that undergo rearrangement during gating and to probe their secondary structure, we performed tryptophan scanning mutagenesis on the two putative TMs of the rat P2X4 receptor channel. Mutant channels were expressed in Xenopus oocytes, concentration-response relationships constructed for ATP, and the EC50 estimated by fitting the Hill equation to the data. Of the 22 mutations in TM1 and 24 in TM2, all but one in TM1 and seven in TM2 result in functional channels. Interestingly, the majority of the functional mutants display an increased sensitivity to ATP, and in general these perturbations are more pronounced for TM2 when compared with TM1. For TM1 and for the outer half of TM2, the perturbations are consistent with these regions adopting alpha-helical secondary structures. In addition, the greatest perturbations in the gating equilibrium occur for mutations near the outer ends of both TM1 and TM2. Surface biotinylation experiments reveal that all the nonfunctional mutants traffic to the surface membrane at levels comparable to the WT channel, suggesting that these mutations likely disrupt ion conduction or gating. Taken together, these results suggest that the outer parts of TM1 and TM2 are helical and that they move during activation. The observation that the majority of nonconducting mutations are clustered toward the inner end of TM2 suggests a critical functional role for this region.

Scanning the intracellular S6 activation gate in the shaker K+ channel.

In Kv channels, an activation gate is thought to be located near the intracellular entrance to the ion conduction pore. Although the COOH terminus of the S6 segment has been implicated in forming the gate structure, the residues positioned at the occluding part of the gate remain undetermined. We use a mutagenic scanning approach in the Shaker Kv channel, mutating each residue in the S6 gate region (T469-Y485) to alanine, tryptophan, and aspartate to identify positions that are insensitive to mutation and to find mutants that disrupt the gate. Most mutants open in a steeply voltage-dependent manner and close effectively at negative voltages, indicating that the gate structure can both support ion flux when open and prevent it when closed. We find several mutant channels where macroscopic ionic currents are either very small or undetectable, and one mutant that displays constitutive currents at negative voltages. Collective examination of the three types of substitutions support the notion that the intracellular portion of S6 forms an activation gate and identifies V478 and F481 as candidates for occlusion of the pore in the closed state.

Enhanced growth inhibition by combination differentiation therapy with ligands of peroxisome proliferator-activated receptor-gamma and inhibitors of histone deacetylase in adenocarcinoma of the lung.

PURPOSE: Histone deacetylase (HDAC) inhibitors and ligands of the peroxisome proliferator-activated receptor gamma (PPARgamma) have been shown previously to induce growth arrest and differentiation in a variety of cancer cell lines. The purpose of this study was to determine whether HDAC inhibitors function similarly in non-small cell lung cancer (NSCLC) and whether combination treatment with HDAC inhibitors and PPARgamma ligands is more efficacious than either agent alone. EXPERIMENTAL DESIGN AND RESULTS: Nanomolar concentrations of trichostatin A induced growth arrest in five of seven NSCLC cell lines, whereas sodium phenylbutyrate (PB) was markedly less potent. In adenocarcinomas, trichostatin A up-regulated general differentiation markers (gelsolin, Mad, and p21/WAF1) and down-regulated markers of the type II pneumocyte progenitor cell lineage (MUC1 and SP-A), indicative of a more mature phenotype. PB had a similar effect. Simultaneous treatment with a PPARgamma ligand and PB enhanced the growth inhibition in adenocarcinomas but not in nonadenocarcinomas. Growth arrest was accompanied by markedly decreased cyclin D1 expression but not enhanced differentiation. CONCLUSIONS: The present study demonstrates potent growth-inhibitory and differentiation-inducing activity of HDAC inhibitors in NSCLC and suggests that combination differentiation therapy should be explored further for the treatment of lung adenocarcinomas.