EMC chaperone-CaV structure reveals an ion channel assembly intermediate.

Voltage-gated ion channels (VGICs) comprise multiple structural units, the assembly of which is required for function1,2. Structural understanding of how VGIC subunits assemble and whether chaperone proteins are required is lacking. High-voltage-activated calcium channels (CaVs)3,4 are paradigmatic multisubunit VGICs whose function and trafficking are powerfully shaped by interactions between pore-forming CaV1 or CaV2 CaVα1 (ref. 3), and the auxiliary CaVß5 and CaVα2δ subunits6,7. Here we present cryo-electron microscopy structures of human brain and cardiac CaV1.2 bound with CaVß3 to a chaperone-the endoplasmic reticulum membrane protein complex (EMC)8,9-and of the assembled CaV1.2-CaVß3-CaVα2δ-1 channel. These structures provide a view of an EMC-client complex and define EMC sites-the transmembrane (TM) and cytoplasmic (Cyto) docks; interaction between these sites and the client channel causes partial extraction of a pore subunit and splays open the CaVα2δ-interaction site. The structures identify the CaVα2δ-binding site for gabapentinoid anti-pain and anti-anxiety drugs6, show that EMC and CaVα2δ interactions with the channel are mutually exclusive, and indicate that EMC-to-CaVα2δ hand-off involves a divalent ion-dependent step and CaV1.2 element ordering. Disruption of the EMC-CaV complex compromises CaV function, suggesting that the EMC functions as a channel holdase that facilitates channel assembly. Together, the structures reveal a CaV assembly intermediate and EMC client-binding sites that could have wide-ranging implications for the biogenesis of VGICs and other membrane proteins.

Long-chain fatty dihydropyridines: Docking calcium channel studies and antihypertensive activity.

AIMS: From the synthesis of 43 lipophilic dihydropyridines, the aim of this study was to verify whether the new dihydropyridines have calcium channel affinity using coupling studies and to determine antihypertensive and antioxidant properties, as well as toxicology and toxicity nifedipine and three new compounds, were chosen from the previous results. MATERIALS AND METHODS: The animals were treated for 56 days, 28 days with N (ω) -nitro-L-arginine methyl ester to induce hypertension, and then treated for another 28 days with the new di- hydropyridine and the standard drug nifedipine. Throughout the treatment the animals had their blood pressure measured and their heart rate checked by pletysmography. After treatment the animals were euthanised, blood samples were collected for creatine kinase and urea analysis, and the brain, heart and liver were collected for oxidative status analysis (quantification of reactive oxygen species, total antioxidant capacity, and lipid peroxidation). KEY FINDINGS: Compounds 2c, and 9a, and nifedipine significantly reduced blood pressure to control group levels. The tachycardia caused by the induction of hypertension was reversed by 2c and 9a compounds. Regarding oxidative stress analyzes, the compounds that had the best performances were also 2c and 9a. Overall the results demonstrate that two of the three new dihydropyridines tested demonstrated performance equal to or superior to the standard drug nifedipine. SIGNIFICANCE: In this study, for the first time, docking was applied to analyse 43 fatty dihydropyridines regarding their calcium channel binding. Afterwards, three fatty dihydropyridines were chosen and their antihypertensive and antioxidant properties.

Molecular Basis for Ligand Modulation of a Mammalian Voltage-Gated Ca2+ Channel.

The L-type voltage-gated Ca2+ (Cav) channels are modulated by various compounds exemplified by 1,4-dihydropyridines (DHP), benzothiazepines (BTZ), and phenylalkylamines (PAA), many of which have been used for characterizing channel properties and for treatment of hypertension and other disorders. Here, we report the cryoelectron microscopy (cryo-EM) structures of Cav1.1 in complex with archetypal antagonistic drugs, nifedipine, diltiazem, and verapamil, at resolutions of 2.9 Å, 3.0 Å, and 2.7 Å, respectively, and with a DHP agonist Bay K 8644 at 2.8 Å. Diltiazem and verapamil traverse the central cavity of the pore domain, directly blocking ion permeation. Although nifedipine and Bay K 8644 occupy the same fenestration site at the interface of repeats III and IV, the coordination details support previous functional observations that Bay K 8644 is less favored in the inactivated state. These structures elucidate the modes of action of different Cav ligands and establish a framework for structure-guided drug discovery.

Clustering and Functional Coupling of Diverse Ion Channels and Signaling Proteins Revealed by Super-resolution STORM Microscopy in Neurons.

The fidelity of neuronal signaling requires organization of signaling molecules into macromolecular complexes, whose components are in intimate proximity. The intrinsic diffraction limit of light makes visualization of individual signaling complexes using visible light extremely difficult. However, using super-resolution stochastic optical reconstruction microscopy (STORM), we observed intimate association of individual molecules within signaling complexes containing ion channels (M-type K+, L-type Ca2+, or TRPV1 channels) and G protein-coupled receptors coupled by the scaffolding protein A-kinase-anchoring protein (AKAP)79/150. Some channels assembled as multi-channel supercomplexes. Surprisingly, we identified novel layers of interplay within macromolecular complexes containing diverse channel types at the single-complex level in sensory neurons, dependent on AKAP79/150. Electrophysiological studies revealed that such ion channels are functionally coupled as well. Our findings illustrate the novel role of AKAP79/150 as a molecular coupler of different channels that conveys crosstalk between channel activities within single microdomains in tuning the physiological response of neurons.

Structure of the voltage-gated calcium channel Ca(v)1.1 at 3.6 Å resolution.

The voltage-gated calcium (Cav) channels convert membrane electrical signals to intracellular Ca2+-mediated events. Among the ten subtypes of Cav channel in mammals, Cav1.1 is specified for the excitation-contraction coupling of skeletal muscles. Here we present the cryo-electron microscopy structure of the rabbit Cav1.1 complex at a nominal resolution of 3.6 Å. The inner gate of the ion-conducting α1-subunit is closed and all four voltage-sensing domains adopt an 'up' conformation, suggesting a potentially inactivated state. The extended extracellular loops of the pore domain, which are stabilized by multiple disulfide bonds, form a windowed dome above the selectivity filter. One side of the dome provides the docking site for the α2δ-1-subunit, while the other side may attract cations through its negative surface potential. The intracellular I-II and III-IV linker helices interact with the ß1a-subunit and the carboxy-terminal domain of α1, respectively. Classification of the particles yielded two additional reconstructions that reveal pronounced displacement of ß1a and adjacent elements in α1. The atomic model of the Cav1.1 complex establishes a foundation for mechanistic understanding of excitation-contraction coupling and provides a three-dimensional template for molecular interpretations of the functions and disease mechanisms of Cav and Nav channels.

Three-dimensional localization of the α and ß subunits and of the II-III loop in the skeletal muscle L-type Ca2+ channel.

The L-type Ca(2+) channel (dihydropyridine receptor (DHPR) in skeletal muscle acts as the voltage sensor for excitation-contraction coupling. To better resolve the spatial organization of the DHPR subunits (α(1s) or Ca(V)1.1, α(2), ß(1a), δ1, and γ), we created transgenic mice expressing a recombinant ß(1a) subunit with YFP and a biotin acceptor domain attached to its N- and C- termini, respectively. DHPR complexes were purified from skeletal muscle, negatively stained, imaged by electron microscopy, and subjected to single-particle image analysis. The resulting 19.1-Å resolution, three-dimensional reconstruction shows a main body of 17 × 11 × 8 nm with five corners along its perimeter. Two protrusions emerge from either face of the main body: the larger one attributed to the α(2)-δ1 subunit that forms a flexible hook-shaped feature and a smaller protrusion on the opposite side that corresponds to the II-III loop of Ca(V)1.1 as revealed by antibody labeling. Novel features discernible in the electron density accommodate the atomic coordinates of a voltage-gated sodium channel and of the ß subunit in a single docking possibility that defines the α1-ß interaction. The ß subunit appears more closely associated to the membrane than expected, which may better account for both its role in localizing the α(1s) subunit to the membrane and its suggested role in excitation-contraction coupling.

Polymodal activation of the endocannabinoid system in the extended amygdala.

The reason why neurons synthesize more than one endocannabinoid (eCB) and how this is involved in the regulation of synaptic plasticity in a single neuron is not known. We found that 2-arachidonoylglycerol (2-AG) and anandamide mediate different forms of plasticity in the extended amygdala of rats. Dendritic L-type Ca(2+) channels and the subsequent release of 2-AG acting on presynaptic CB1 receptors triggered retrograde short-term depression. Long-term depression was mediated by postsynaptic mGluR5-dependent release of anandamide acting on postsynaptic TRPV1 receptors. In contrast, 2-AG/CB1R-mediated retrograde signaling mediated both forms of plasticity in the striatum. These data illustrate how the eCB system can function as a polymodal signal integrator to allow the diversification of synaptic plasticity in a single neuron.

Structural modeling of calcium binding in the selectivity filter of the L-type calcium channel.

Calcium channels play crucial physiological roles. In the absence of high-resolution structures of the channels, the mechanism of ion permeation is unknown. Here we used a method proposed in an accompanying paper (Cheng and Zhorov in Eur Biophys J, 2009) to predict possible chelation patterns of calcium ions in a structural model of the L-type calcium channel. We compared three models in which two or three calcium ions interact with the four selectivity filter glutamates and a conserved aspartate adjacent to the glutamate in repeat II. Monte Carlo energy minimizations yielded many complexes with calcium ions bound to at least two selectivity filter carboxylates. In these complexes calcium-carboxylate attractions are counterbalanced by calcium-calcium and carboxylate-carboxylate repulsions. Superposition of the complexes suggests a high degree of mobility of calcium ions and carboxylate groups of the glutamates. We used the predicted complexes to propose a permeation mechanism that involves single-file movement of calcium ions. The key feature of this mechanism is the presence of bridging glutamates that coordinate two calcium ions and enable their transitions between different chelating patterns involving four to six oxygen atoms from the channel protein. The conserved aspartate is proposed to coordinate a calcium ion incoming to the selectivity filter from the extracellular side. Glutamates in repeats III and IV, which are most distant from the repeat II aspartate, are proposed to coordinate the calcium ion that leaves the selectivity filter to the inner pore. Published experimental data and earlier proposed permeation models are discussed in view of our model.

Three-dimensional structure of the alpha1-beta complex in the skeletal muscle dihydropyridine receptor by single-particle electron microscopy.

The dihydropyridine receptor (DHPR) is a protein complex that consists of five distinct subunits of alpha(1), alpha(2), beta, gamma and delta and functions as a voltage-dependent L-type Ca(2+) channel. Here we purified the alpha(1)-beta complex (approximately 250 kDa) from the rabbit skeletal muscle DHPR and reconstructed its three-dimensional (3D) structure to 38 A resolution by single particle analysis of negative staining electron microscopy. The alpha(1)-beta structure exhibited two unique regions: a pseudo-4-fold petaloid region and an elongated region. X-ray crystallographic models of a homologous voltage-dependent K(+) channel and the beta subunit fit well into the individual regions of the alpha(1)-beta structure, revealing that the two regions correspond to the transmembrane alpha(1) and the cytoplasmic beta subunits, respectively. In addition, 3D reconstruction and immuno-electron microscopic analysis performed on the independently purified DHPR demonstrated that the alpha(1)-beta complex was located in the large globular portion of the DHPR, and the N-terminal region of the beta subunit was extended to the leg-shaped protrusion of the DHPR, which includes the alpha(2)delta subunits. Our results propose a model in which the beta subunit may regulate ion channel function by acting as a hinge between alpha(1) and alpha(2)delta subunits of the DHPR.

Axial tubules of rat ventricular myocytes form multiple junctions with the sarcoplasmic reticulum.

Ryanodine receptors (RyRs) are located primarily on the junctional sarcoplasmic reticulum (SR), adjacent to the transverse tubules and on the cell surface near the Z-lines, but some RyRs are on junctional SR adjacent to axial tubules. Neither the size of the axial junctions nor the numbers of RyRs that they contain have been determined. RyRs may also be located on the corbular SR and on the free or network SR. Because determining and quantifying the distribution of RyRs is critical for both understanding and modeling calcium dynamics, we investigated the distribution of RyRs in healthy adult rat ventricular myocytes, using electron microscopy, electron tomography, and immunofluorescence. We found RyRs in only three regions: in couplons on the surface and on transverse tubules, both of which are near the Z-line, and in junctions on most of the axial tubules--axial junctions. The axial junctions averaged 510 nm in length, but they occasionally spanned an entire sarcomere. Numerical analysis showed that they contain as much as 19% of a cell's RyRs. Tomographic analysis confirmed the axial junction's architecture, which is indistinguishable from junctions on transverse tubules or on the surface, and revealed a complexly structured tubule whose lumen was only 26 nm at its narrowest point. RyRs on axial junctions colocalize with Ca(v)1.2, suggesting that they play a role in excitation-contraction coupling.

[Molecular architecture of the sarcoplasmic reticulum and its role in the ECC]. / Architecture moléculaire du réticulum sarcoplasmique et son rôle dans le couplage excitation-contraction.

The sarcoplasmic reticulum (SR) plays a fundamental role in excitation-contraction coupling, which propagates the electric signal conversion along the muscle fiber's plasmic membrane to a mechanical event manifested as a muscle contraction. It plays a crucial role in calcium homeostasis and intracellular calcium storage control (storage, liberation and uptake) necessary for fiber muscle contraction and then relaxation. These functions take place at the triad, made up of individualized SR subdomains where the protein-specific organization provides efficient and fast coupling. Ryanodine receptors (RyR) and dihydropyridine receptors (DHPR) mainly act in calcium exchanges in the SR. This particular structural and molecular architecture must be correlated to its functional specificity.

Single-channel L-type Ca2+ currents in chicken embryo semicircular canal type I and type II hair cells.

Few data are available concerning single Ca channel properties in inner ear hair cells and particularly none in vestibular type I hair cells. By using the cell-attached configuration of the patch-clamp technique in combination with the semicircular canal crista slice preparation, we determined the elementary properties of voltage-dependent Ca channels in chicken embryo type I and type II hair cells. The pipette solutions included Bay K 8644. With 70 mM Ba(2+) in the patch pipette, Ca channel activity appeared as very brief openings at -60 mV. Ca channel properties were found to be similar in type I and type II hair cells; therefore data were pooled. The mean inward current amplitude was -1.3 +/- 0.1 (SD) pA at - 30 mV (n = 16). The average slope conductance was 21 pS (n = 20). With 5 mM Ba(2+) in the patch pipette, very brief openings were already detectable at -80 mV. The mean inward current amplitude was -0.7 +/- 0.2 pA at -40 mV (n = 9). The average slope conductance was 11 pS (n = 9). The mean open time and the open probability increased significantly with depolarization. Ca channel activity was still present and unaffected when omega-agatoxin IVA (2 microM) and omega-conotoxin GVIA (3.2 microM) were added to the pipette solution. Our results show that types I and II hair cells express L-type Ca channels with similar properties. Moreover, they suggest that in vivo Ca(2+) influx might occur at membrane voltages more negative than -60 mV.

Near-field scanning fluorescence microscopy study of ion channel clusters in cardiac myocyte membranes.

Near-field scanning optical microscopy (NSOM) has been used to study the nanoscale distribution of voltage-gated L-type Ca2+ ion channels, which play an important role in cardiac function. NSOM fluorescence imaging of immunostained cardiac myocytes (H9C2 cells) demonstrates that the ion channel is localized in small clusters with an average diameter of 100 nm. The clusters are randomly distributed throughout the cell membrane, with some larger fluorescent patches that high-resolution images show to consist of many small closely-spaced clusters. We have imaged unstained cells to assess the contribution of topography-induced artifacts and find that the topography-induced signal is <10% of the NSOM fluorescence intensity. We have also examined the dependence of the NSOM signal intensity on the tip-sample separation to assess the contributions from fluorophores that are significantly below the cell surface. This indicates that chromophores > approximately 200 nm below the probe will have negligible contributions to the observed signal. The ability to quantitatively measure small clusters of ion channels will facilitate future studies that examine changes in protein localization in stimulated cells and during cardiac development. Our work illustrates the potential of NSOM for studying membrane domains and protein localization/colocalization on a length scale which exceeds that available with optical microscopy.

Evidence for conformational coupling between two calcium channels.

Ryanodine receptor 1 (RyR1, the sarcoplasmic reticulum Ca(2+) release channel) and alpha(1S)dihydropyridine receptor (DHPR, the surface membrane voltage sensor) of skeletal muscle belong to separate membrane systems but are functionally and structurally linked. Four alpha(1S)DHPRs associated with the four identical subunits of a RyR form a tetrad. We treated skeletal muscle cell lines with ryanodine, at concentrations that block RyRs, and determined whether this treatment affects the distance between DHPRs in the tetrad. We find a substantial ( approximately 2-nm) shift in the alpha(1S)DHPR positions, indicating that ryanodine induces large conformational changes in the RyR1 cytoplasmic domain and that the alpha(1S)DHPR-RyR complex acts as a unit.

Altered localization of Cav1.2 (L-type) calcium channels in nerve fibers, Schwann cells, odontoblasts, and fibroblasts of tooth pulp after tooth injury.

We have determined the localization of Cav1.2 (L-Type) Ca2+ channels in the cells and nerve fibers in molars of normal or injured rats. We observed high levels of immunostaining of L-type Ca2+ channels in odontoblast cell bodies and their processes, in fibroblast cell bodies and in Schwann cells. Many Cav1.2-containing unmyelinated and myelinated axons were also present in root nerves and proximal branches in coronal pulp, but were usually missing from nerve fibers in dentin. Labeling in the larger fibers was present along the axonal membrane, localized in axonal vesicles, and in nodal regions. After focal tooth injury, there is a marked loss of Cav1.2 channels in injured teeth. Immunostaining of Cav1.2 channels was lost selectively in nerve fibers and local cells of the tooth pulp within 10 min of the lesion, without loss of other Cav channel or pulpal labels. By 60 min, Cav1.2 channels in odontoblasts were detected again but at levels below controls, whereas fibroblasts were labeled well above control levels, similar to upregulation of Cav1.2 channels in astrocytes after injury. By 3 days after the injury, Cav1.2 channels were again detected in nerve fibers and immunostaining of fibroblasts and odontoblasts had returned to control levels. These findings provide new insight into the localization of Cav1.2 channels in dental pulp and sensory fibers, and demonstrate unexpected plasticity of channel distribution in response to nerve injury.

Structural insights into excitation-contraction coupling by electron cryomicroscopy.

In muscle, excitation-contraction coupling is defined as the process linking depolarization of the surface membrane with Ca2+ release from cytoplasmic stores, which activates contraction of striated muscle. This process is primarily controlled by interplay between two Ca2+ channels--the voltage-gated L-type Ca2+ channel (dihydropyridine receptor, DHPR) localized in the t-tubule membrane and the Ca2+-release channel (ryanodine receptor, RyR) of the sarcoplasmic reticulum membrane. The structures of both channels have been extensively studied by several groups using electron cryomicroscopy and single particle reconstruction techniques. The structures of RyR, determined at resolutions of 22-30 A, reveal a characteristic mushroom shape with a bulky cytoplasmic region and the membrane-spanning stem. While the cytoplasmic region exhibits a complex structure comprising a multitude of distinctive domains with numerous intervening cavities, at this resolution no definitive statement can be made about the location of the actual pore within the transmembrane region. Conformational changes associated with functional transitions of the Ca2+ release channel from closed to open states have been characterized. Further experiments determined localization of binding sites for various channel ligands. The structural studies of the DHPR are less developed. Although four 3D maps of the DHPR were reported recently at 24-30 A resolution from studies of frozen-hydrated and negatively stained receptors, there are some discrepancies between reported structures with respect to the overall appearance and dimensions of the channel structure. Future structural studies at higher resolution are needed to refine the structures of both channels and to substantiate a proposed molecular model for their interaction.

Localization and function of the Na+/Ca2+-exchanger in normal and detubulated rat cardiomyocytes.

It is controversial whether the Na+/Ca2+-exchanger (NCX) can induce cardiomyocyte contraction through reverse-mode exchange and Ca2+-induced Ca2+ release (CICR). Information about the spatial distribution and functional activity within different sarcolemmal (SL) regions could shed light on this potential role. We raised a new antibody to the NCX and showed by confocal laser scanning microscopy (CLSM) that immunoreactivity is strongly expressed throughout the surface SL and intercalated disk regions with punctate labeling of the vertical transverse (T)-tubules but not the longitudinal T-tubules. Immuno-electron microscopy confirmed CLSM observations. Gold particles associated with the exchanger were within nanometer range of particles signaling ryanodine receptors. A similar close association was found between the L-type Ca2+ channel (known to be concentrated in the dyad) and ryanodine receptors. In whole-cell patch-clamped cardiomyocytes, peak I(NCX) (measured at 90 mV) decreased by approximately 40% (497 +/- 32 vs. 304 +/- 12 pA, P < 0.001) after detubulation, while membrane capacitance decreased by 27% (204 +/- 11 vs. 150 +/- 7 pF, P < 0.01) thus giving a small but significant 16% reduction in current density. Thus, the density and/or functional activity of the NCX is greater in the vertical T-tubules than in the longitudinal T-tubules, surface SL or disk regions, pointing to important functional differences between these plasma membrane domains. Our combined co-immunolocalization and physiological data suggest that the NCX has multiple functions depending upon membrane location. We suggest the possibility that NCX modulates CICR, sarcoplasmic reticulum Ca2+ load, and that it also serves to regulate Ca2+ handling in neighboring cells.

Enrichment of triadic and terminal cisternae vesicles from rabbit skeletal muscle.

An enriched triad and terminal cisternae preparation was achieved from skeletal muscle through alterations of the differential centrifugation and muscle homogenization protocols. Both yield and specific activity (pmoles of radioligand binding per mg protein) were optimized for (3)H-PN200-110 (transverse tubule marker) and (3)H-ryanodine (terminal cisternae marker) binding sites. By pelleting crude microsomes between 2,000 an 12,000 x g without any rehomogenizations, we improved both the yield and specific activity of transverse tubule and terminal cisternae markers in crude microsomes by approximately 4-fold to 1000-3000 pmoles binding sites (starting material: approximately 400 grams wet weight fast twitch skeletal muscle), with 10-15 pmoles/mg. Rehomogenization of the 1,000 x g pellet, which is typically discarded, allowed recovery of an additional 5000 pmoles PN200-110 binding sites and an additional 8000 pmoles ryanodine binding sites. Crude microsomes from the rehomogenized 1,000 x g pellets typically displayed specific activities of 20-25 pmoles binding/mg for both (3)H-PN200-110 and (3)H-ryanodine. Separation of crude microsomes on a sucrose gradient increased specific activity up to a maximum of 50 pmoles/mg in a specific fraction, a five- to ten-fold increase over standard triadic or terminal cisternae preparations. The mean specific activity for enriched triads was 30-40 pmoles/mg for both PN200-110 and ryanodine in pooled fractions, while pooled fractions of enriched terminal cisternae displayed low (3)H-PN200-110 binding (3-5 pmoles/mg) and high (3)H-ryanodine-specific activity (30-40 pmoles/mg).

Visualization of the domain structure of an L-type Ca2+ channel using electron cryo-microscopy.

The three-dimensional structure of the skeletal muscle voltage-gated L-type calcium channel (Ca(v)1.1; dihydropyridine receptor, DHPR) was determined using electron cryo-microscopy and single-particle averaging. The structure shows a single channel complex with an approximate total molecular mass of 550 kDa, corresponding to the five known subunits of the DHPR, and bound detergent and lipid. Features visible in our structure together with antibody labeling of the beta and alpha(2) subunits allowed us to assign locations for four of the five subunits within the structure. The most striking feature of the structure is the extra-cellular alpha(2) subunit that protrudes from the membrane domain in close proximity to the alpha(1) subunit. The cytosolic beta subunit is located close to the membrane and adjacent to subunits alpha(1), gamma and delta. Our structure correlates well with the functional and biochemical data available for this channel and suggests a three-dimensional model for the excitation-contraction coupling complex consisting of DHPR tetrads and the calcium release channel.