Resting state of the human proton channel dimer in a lipid bilayer.

The voltage-gated proton channel Hv1 plays a critical role in the fast proton translocation that underlies a wide range of physiological functions, including the phagocytic respiratory burst, sperm motility, apoptosis, and metastatic cancer. Both voltage activation and proton conduction are carried out by a voltage-sensing domain (VSD) with strong similarity to canonical VSDs in voltage-dependent cation channels and enzymes. We set out to determine the structural properties of membrane-reconstituted human proton channel (hHv1) in its resting conformation using electron paramagnetic resonance spectroscopy together with biochemical and computational methods. We evaluated existing structural templates and generated a spectroscopically constrained model of the hHv1 dimer based on the Ci-VSD structure at resting state. Mapped accessibility data revealed deep water penetration through hHv1, suggesting a highly focused electric field, comprising two turns of helix along the fourth transmembrane segment. This region likely contains the H(+) selectivity filter and the conduction pore. Our 3D model offers plausible explanations for existing electrophysiological and biochemical data, offering an explicit mechanism for voltage activation based on a one-click sliding helix conformational rearrangement.

Structural mechanism of voltage-dependent gating in an isolated voltage-sensing domain.

The transduction of transmembrane electric fields into protein motion has an essential role in the generation and propagation of cellular signals. Voltage-sensing domains (VSDs) carry out these functions through reorientations of positive charges in the S4 helix. Here, we determined crystal structures of the Ciona intestinalis VSD (Ci-VSD) in putatively active and resting conformations. S4 undergoes an ~5-Å displacement along its main axis, accompanied by an ~60° rotation. This movement is stabilized by an exchange in countercharge partners in helices S1 and S3 that generates an estimated net charge transfer of ~1 eo. Gating charges move relative to a ''hydrophobic gasket' that electrically divides intra- and extracellular compartments. EPR spectroscopy confirms the limited nature of S4 movement in a membrane environment. These results provide an explicit mechanism for voltage sensing and set the basis for electromechanical coupling in voltage-dependent enzymes and ion channels.

Biochemical and structural analysis of the hyperpolarization-activated K(+) channel MVP.

In contrast to the majority of voltage-gated ion channels, hyperpolarization-activated channels remain closed at depolarizing potentials and are activated at hyperpolarizing potentials. The basis for this reverse polarity is thought to be a result of differences in the way the voltage-sensing domain (VSD) couples to the pore domain. In the absence of structural data, the molecular mechanism of this reverse polarity coupling remains poorly characterized. Here we report the characterization of the structure and local dynamics of the closed activation gate (lower S6 region) of MVP, a hyperpolarization-activated potassium channel from Methanococcus jannaschii, by electron paramagnetic resonance (EPR) spectroscopy. We show that a codon-optimized version of MVP has high expression levels in Escherichia coli, is purified as a stable tetramer, and exhibits expected voltage-dependent activity when reconstituted in liposomes. EPR analysis of the mid to lower S6 region revealed positions exhibiting strong spin-spin coupling, indicating that the activation gate of MVP is closed at 0 mV. A comparison of local environmental parameters along the activation gate for MVP and KcsA indicates that MVP adopts a different closed conformation. These structural details set the stage for future evaluations of reverse electromechanical coupling in MVP.

Structural basis of lipid-driven conformational transitions in the KvAP voltage-sensing domain.

Voltage-gated ion channels respond to transmembrane electric fields through reorientations of the positively charged S4 helix within the voltage-sensing domain (VSD). Despite a wealth of structural and functional data, the details of this conformational change remain controversial. Recent electrophysiological evidence showed that equilibrium between the resting ('down') and activated ('up') conformations of the KvAP VSD from Aeropyrum pernix can be biased through reconstitution in lipids with or without phosphate groups. We investigated the structural transition between these functional states, using site-directed spin-labeling and EPR spectroscopic methods. Solvent accessibility and interhelical distance determinations suggest that KvAP gates through S4 movements involving an ∼3-Šupward tilt and simultaneous ∼2-Šaxial shift. This motion leads to large accessibly changes in the intracellular water-filled crevice and supports a new model of gating that combines structural rearrangements and electric-field remodeling.

Expression, purification, and reconstitution of the voltage-sensing domain from Ci-VSP.

The voltage-sensing domain (VSD) is the common scaffold responsible for the functional behavior of voltage-gated ion channels, voltage sensitive enzymes, and proton channels. Because of the position of the voltage dependence of the available VSD structures, at present, they all represent the activated state of the sensor. Yet in the absence of a consensus resting state structure, the mechanistic details of voltage sensing remain controversial. The voltage dependence of the VSD from Ci-VSP (Ci-VSD) is dramatically right shifted, so that at 0 mV it presumably populates the putative resting state. Appropriate biochemical methods are an essential prerequisite for generating sufficient amounts of Ci-VSD protein for high-resolution structural studies. Here, we present a simple and robust protocol for the expression of eukaryotic Ci-VSD in Escherichia coli at milligram levels. The protein is pure, homogeneous, monodisperse, and well-folded after solubilization in Anzergent 3-14 at the analyzed concentration (~0.3 mg/mL). Ci-VSD can be reconstituted into liposomes of various compositions, and initial site-directed spin labeling and electron paramagnetic resonance (EPR) spectroscopic measurements indicate its first transmembrane segment folds into an α-helix, in agreement with the homologous region of other VSDs. On the basis of our results and enhanced relaxation EPR spectroscopy measurement, Ci-VSD reconstitutes essentially randomly in proteoliposomes, precluding straightforward application of transmembrane voltages in combination with spectroscopic methods. Nevertheless, these results represent an initial step that makes the resting state of a VSD accessible to a variety of biophysical and structural approaches, including X-ray crystallography, spectroscopic methods, and electrophysiology in lipid bilayers.

The membrane protein MiRP3 regulates Kv4.2 channels in a KChIP-dependent manner.

MiRP3, the single-span membrane protein encoded by KCNE4, is localized by immunofluorescence microscopy to the transverse tubules of murine cardiac myocytes. MiRP3 is found to co-localize with Kv4.2 subunits that contribute to cardiac transient outward potassium currents (I(to)). Whole-cell, voltage-clamp recordings of human MiRP3 and Kv4.2 expressed in a clonal cell line (tsA201) reveal MiRP3 to modulate Kv4.2 current activation, inactivation and recovery from inactivation. MiRP3 shifts the half-maximal voltage for activation (V(1/2)) approximately 20 mV and slows time to peak approximately 100%. In addition, MiRP3 slows inactivation approximately 100%, speeds recovery from inactivation approximately 30%, and enhances restored currents so they 'overshoot' baseline levels. The cytoplasmic accessory subunit KChIP2 also assembles with Kv4.2 in tsA201 cells to increase peak current, shift V(1/2) approximately 5 mV, slow time to peak approximately 10%, slow inactivation approximately 100%, and speed recovery from inactivation approximately 250% without overshoot. Simultaneous expression of all three subunits yields a biophysical profile unlike either accessory subunit alone, abolishes MiRP3-induced overshoot, and allows biochemical isolation of the ternary complex. Thus, regional heterogeneity in cardiac expression of MiRP3, Kv4.2 and KChIP2 in health and disease may establish the local attributes and magnitude of cardiac I(to).

MiRP3 acts as an accessory subunit with the BK potassium channel.

MinK-related peptides (MiRPs) are single-span membrane proteins that assemble with specific voltage-gated K+ (Kv) channel alpha-subunits to establish gating kinetics, unitary conductance, expression level, and pharmacology of the mixed complex. MiRP3 (encoded by the KCNE4 gene) has been shown to alter the behavior of some Kv alpha-subunits in vitro but its natural partners and physiologic functions are unknown. Seeking in vivo partners for MiRP3, immunohistochemistry was used to localize its expression to a unique subcellular site, the apical membrane of renal intercalated cells, where one potassium channel type has been recorded, the calcium- and voltage-gated channel BK. Overlapping staining of these two proteins was found in rabbit intercalated cells, and MiRP3 and BK subunits expressed in tissue culture cells were found to form detergent-stable complexes. Electrophysiologic and biochemical evaluation showed MiRP3 to act on BK to reduce current density in two fashions: shifting the current-voltage relationship to more depolarized voltages in a calcium-dependent fashion ( approximately 10 mV at normal intracellular calcium levels) and accelerating degradation of MiRP3-BK complexes. The findings suggest a role for MiRP3 modulation of BK-dependent urinary potassium excretion.

GRP94 is essential for mesoderm induction and muscle development because it regulates insulin-like growth factor secretion.

Because only few of its client proteins are known, the physiological roles of the endoplasmic reticulum chaperone glucose-regulated protein 94 (GRP94) are poorly understood. Using targeted disruption of the murine GRP94 gene, we show that it has essential functions in embryonic development. grp94-/- embryos die on day 7 of gestation, fail to develop mesoderm, primitive streak, or proamniotic cavity. grp94-/- ES cells grow in culture and are capable of differentiation into cells representing all three germ layers. However, these cells do not differentiate into cardiac, smooth, or skeletal muscle. Differentiation cultures of mutant ES cells are deficient in secretion of insulin-like growth factor II and their defect can be complemented with exogenous insulin-like growth factors I or II. The data identify insulin-like growth factor II as one developmentally important protein whose production depends on the activity of GRP94.

The peptide-binding activity of GRP94 is regulated by calcium.

GRP94 (glucose-regulated protein of 94 kDa) is a major luminal constituent of the endoplasmic reticulum with known high capacity for calcium in vivo and a peptide-binding activity in vitro. In the present study, we show that Ca2+ regulates the ability of GRP94 to bind peptides. This effect is due to a Ca2+-binding site located in the charged linker domain of GRP94, which, when occupied, enhances the association of peptides with the peptide-binding site in the N-terminal domain of the protein. We further show that grp94-/- cells are hypersensitive to perturbation of intracellular calcium and thus GRP94 is important for cellular Ca2+ storage.

Attenuation of vascular permeability by methylnaltrexone: role of mOP-R and S1P3 transactivation.

Endothelial cell (EC) barrier dysfunction (i.e., increased vascular permeability) is observed in inflammatory states, tumor angiogenesis, atherosclerosis, and both sepsis and acute lung injury. Therefore, agents that preserve vascular integrity have important clinical therapeutic implications. We examined the effects of methylnaltrexone (MNTX), a mu opioid receptor (mOP-R) antagonist, on human pulmonary EC barrier disruption produced by edemagenic agents including morphine, the endogenous mOP-R agonist DAMGO, thrombin, and LPS. Pretreatment of EC with MNTX (0.1 muM, 1 h) or the uncharged mOP-R antagonist naloxone attenuated morphine- and DAMGO-induced barrier disruption in vitro. However, MNTX, but not naloxone, pretreatment of EC inhibited thrombin- and LPS-induced barrier disruption, indicating potential mOP-R-independent effects of MNTX. In addition, intravenously delivered MNTX attenuated LPS-induced vascular hyperpermeability in the murine lung. We next examined the mechanistic basis for this MNTX barrier protection and observed that silencing of mOP-R attenuated the morphine- and DAMGO-induced EC barrier disruption, but not the permeability response to either thrombin or LPS. Because activation of the sphingosine 1-phosphate receptor, S1P(3), is key to a number of barrier-disruptive responses, we examined the role of this receptor in the permeability response to mOP-R ligation. Morphine, DAMGO, thrombin, and LPS induced RhoA/ROCK-mediated threonine phosphorylation of S1P(3), which was blocked by MNTX, suggesting S1P(3) transactivation. In addition, silencing of S1P(3) receptor expression (siRNA) abolished the permeability response to each edemagenic agonist. These results indicate that MNTX provides barrier protection against edemagenic agonists via inhibition of S1P(3) receptor activation and represents a potentially useful therapeutic agent for syndromes of increased vascular permeability.