A Quartet of Leucine Residues in the Guanylate Kinase Domain of CaVβ Determines the Plasma Membrane Density of the CaV2.3 Channel

Ca_Vβ subunits are formed by a Src homology 3 domain and a guanylate kinase-like (GK) domain connected through a variable HOOK domain. Complete deletion of the Src homology 3 domain (75 residues) as well as deletion of the HOOK domain (47 residues) did not alter plasma membrane density of Ca_V2.3 nor its typical activation gating. In contrast, six-residue deletions in the GK domain disrupted cell surface trafficking and functional expression of Ca_V2.3. Mutations of residues known to carry nanomolar affinity binding in the GK domain of Ca_Vβ (P175A, P179A, M195A, M196A, K198A, S295A, R302G, R307A, E339G, N340G, and A345G) did not significantly alter cell surface targeting or gating modulation of Ca_V2.3. Nonetheless, mutations of a quartet of leucine residues (either single or multiple mutants) in the α3, α6, β10, and α9 regions of the GK domain were found to significantly impair cell surface density of Ca_V2.3 channels. Furthermore, the normalized protein density of Ca_V2.3 was nearly abolished with the quadruple Ca_Vβ3 Leu mutant L200G/L303G/L337G/L342G. Altogether, our observations suggest that the four leucine residues in Ca_Vβ3 form a hydrophobic pocket surrounding key residues in the α-interacting domain of Ca_V2.3. This interaction appears to play an essential role in conferring Ca_Vβ-induced modulation of the protein density of Ca_Vα1 subunits in Ca_V2 channels.     

Identification of Glycosylation Sites Essential for Surface Expression of the CaVα2δ1 Subunit and Modulation of the Cardiac CaV1.2 Channel Activity

Alteration in the L-type current density is one aspect of the electrical remodeling observed in patients suffering from cardiac arrhythmias. Changes in channel function could result from variations in the protein biogenesis, stability, post-translational modification, and/or trafficking in any of the regulatory subunits forming cardiac L-type Ca²⁺ channel complexes. Ca_Vα2δ1 is potentially the most heavily N-glycosylated subunit in the cardiac L-type Ca_V1.2 channel complex. Here, we show that enzymatic removal of N-glycans produced a 50-kDa shift in the mobility of cardiac and recombinant Ca_Vα2δ1 proteins. This change was also observed upon simultaneous mutation of the 16 Asn sites. Nonetheless, the mutation of only 6/16 sites was sufficient to significantly 1) reduce the steady-state cell surface fluorescence of Ca_Vα2δ1 as characterized by two-color flow cytometry assays and confocal imaging; 2) decrease protein stability estimated from cycloheximide chase assays; and 3) prevent the Ca_Vα2δ1-mediated increase in the peak current density and voltage-dependent gating of Ca_V1.2. Reversing the N348Q and N812Q mutations in the non-operational sextuplet Asn mutant protein partially restored Ca_Vα2δ1 function. Single mutation N663Q and double mutations N348Q/N468Q, N348Q/N812Q, and N468Q/N812Q decreased protein stability/synthesis and nearly abolished steady-state cell surface density of Ca_Vα2δ1 as well as the Ca_Vα2δ1-induced up-regulation of L-type currents. These results demonstrate that Asn-663 and to a lesser extent Asn-348, Asn-468, and Asn-812 contribute to protein stability/synthesis of Ca_Vα2δ1, and furthermore that N-glycosylation of Ca_Vα2δ1 is essential to produce functional L-type Ca²⁺ channels.     

The HOOK-Domain Between the SH3- and the GK-Domains of CaVβ Subunits Contains Key Determinants Controlling Calcium Channel Inactivation

Ca_Vβ subunits of voltage-gated calcium channels contain two conserved domains, a src-homology-3 (SH3)-domain and a guanylate kinase-like (GK)-domain. The SH3-domain is split, with its final (5th) β-strand separated from the rest of the domain by an intervening sequence termed the HOOK-domain, whose sequence varies between Ca_Vβ subunits. Here we have been guided by the recent structural studies of Ca_Vβ subunits in the design of specific truncated constructs, with the goal of investigating the role of the HOOK-domain of Ca_Vβ subunits in the modulation of inactivation of N-type calcium channels. We have co-expressed the β subunit constructs with Ca_V2.2 and α2δ-2, using the N-terminally palmitoylated β2a subunit, because it supports very little voltage-dependent inactivation, and making comparisons with β1b domains. Deletion of the variable region of the β2a HOOK-domain resulted in currents with a rapidly inactivating component, and additional mutation of the β2a palmitoylation motif further enhanced inactivation. The isolated GK-domain of β2a alone enhanced current amplitude, but the currents were rapidly and completely inactivating. When the β2a-GK-domain construct was extended proximally, by including the HOOK-domain and the ε-strand of the SH3-domain, inactivation was about 4 fold slower than in the absence of the HOOK domain. When the SH3-domain of β2a truncated prior to the HOOK-domain was co-expressed with the (HOOK+εSH3+GK)-domain of β2a, all the properties of β2a were restored, in terms of loss of inactivation. Furthermore, removal of the HOOK sequence from the (HOOK+εSH3+GK)-β2a construct increased inactivation. Together, these results provide evidence that the HOOK domain is an important determinant of inactivation.     

Leukocyte Adhesion—an Integrated Molecular Process at the Leukocyte Plasma Membrane

Leukocyte adhesion is of pivotal functional importance, because most leukocyte functions depend on cell–cell contact. It must be strictly controlled, both at the level of specificity and strength of interaction, and therefore several molecular systems are involved. The most important leukocyte adhesion molecules are the selectins, the leukocyte-specific ₂-integrins and the intercellular adhesion molecules. The selectins induce an initial weak contact between cells, whereas firm adhesion is achieved through integrin–intercellular adhesion molecular binding. Although studies during the past twenty years have revealed several important features of leukocyte adhesion much is still poorly understood, and further work dealing with several aspects of adhesion is urgently needed. In this short essay, we review some recent developments in the field.     

Is the Drug-Responsive NADH Oxidase of the Cancer Cell Plasma Membrane a Molecular Target for Adriamycin?

Enhanced growth inhibition and antitumor responses to adriamycin have been observed repeatedly from several laboratories using impermeant forms of adriamycin where entry into the cell was greatly reduced or prevented. Our laboratory has described an NADH oxidase activity at the external surface of plasma membrane vesicles from tumor cells where inhibition by an antitumor sulfonylurea, N-(4-methylphenylsulfonyl)-N-(4-chlorophenyl)urea (LY181984), and by the vanilloid, capsaicin (8-methyl-N-vanillyl-6-noneamide) correlated with inhibition of growth. Here we report that the oxidation of NADH by isolated plasma membrane vesicles was inhibited, as well, by adriamycin. An external site of inhibition was indicated from studies where impermeant adriamycin conjugates were used. The EC₅₀ for inhibition of the oxidase of rat hepatoma plasma membranes by adriamycin was several orders of magnitude less than that for rat liver. Adriamycin cross-linked to diferric transferrin and other impermeant supports also was effective in inhibition of NADH oxidation by isolated plasma membrane vesicles and in inhibition of growth of cultured cells. The findings suggest the NADH oxidase of the plasma membrane as a growth-related adriamycin target at the surface of cancer cells responsive to adriamycin. Whereas DNA intercalation remains clearly one of the principal bases for the cytotoxic action of free adriamycin, this second site, possibly related to a more specific antitumor action, may be helpful in understanding the enhanced efficacy reported previously for immobilized adriamycin forms compared to free adriamycin.     

Structural Flexibility of CaV1.2 and CaV2.2 I-II Proximal Linker Fragments in?Solution

Voltage-dependent calcium channels (Ca_V) enable the inward flow of calcium currents for a wide range of cells. Ca_V1 and Ca_V2 subtype α1 subunits form the conducting pore using four repeated membrane domains connected by intracellular linkers. The domain I-II linker connects to the membrane gate (IS6), forming an α-helix, and is bound to the Ca_Vβ subunit. Previous studies indicated that this region may or may not form a continuous helix depending on the Ca_V subtype, thereby modulating channel activation and inactivation properties. Here, we used small-angle x-ray scattering and ensemble modeling analysis to investigate the solution structure of these linkers, extending from the membrane domain and including the Ca_Vβ-binding site, called the proximal linker (PL). The results demonstrate that the Ca_V1.2 PL is more flexible than the Ca_V2.2 PL, the flexibility is intrinsic and not dependent on Ca_Vβ binding, and the flexibility can be most easily explained by the presence of conserved glycines. Our analysis also provides a robust example of investigating protein domains in which flexibility plays an essential role.     

The CaVβ Subunit Protects the I-II Loop of the Voltage-gated Calcium Channel CaV2.2 from Proteasomal Degradation but Not Oligoubiquitination

Ca_Vβ subunits interact with the voltage-gated calcium channel Ca_V2.2 on a site in the intracellular loop between domains I and II (the I-II loop). This interaction influences the biophysical properties of the channel and leads to an increase in its trafficking to the plasma membrane. We have shown previously that a mutant Ca_V2.2 channel that is unable to bind Ca_Vβ subunits (Ca_V2.2 W391A) was rapidly degraded (Waithe, D., Ferron, L., Page, K. M., Chaggar, K., and Dolphin, A. C. (2011) J. Biol. Chem. 286, 9598–9611). Here we show that, in the absence of Ca_Vβ subunits, a construct consisting of the I-II loop of Ca_V2.2 was directly ubiquitinated and degraded by the proteasome system. Ubiquitination could be prevented by mutation of all 12 lysine residues in the I-II loop to arginines. Including a palmitoylation motif at the N terminus of Ca_V2.2 I-II loop was insufficient to target it to the plasma membrane in the absence of Ca_Vβ subunits even when proteasomal degradation was inhibited with MG132 or ubiquitination was prevented by the lysine-to-arginine mutations. In the presence of Ca_Vβ subunit, the palmitoylated Ca_V2.2 I-II loop was protected from degradation, although oligoubiquitination could still occur, and was efficiently trafficked to the plasma membrane. We propose that targeting to the plasma membrane requires a conformational change in the I-II loop that is induced by binding of the Ca_Vβ subunit.     

Increased intracellular magnesium attenuates β-adrenergic stimulation of the cardiac CaV1.2 channel

Increases in intracellular Mg²⁺ (Mg²⁺_i), as observed in transient cardiac ischemia, decrease L-type Ca²⁺ current of mammalian ventricular myocytes (VMs). However, cardiac ischemia is associated with an increase in sympathetic tone, which could stimulate L-type Ca²⁺ current. Therefore, the effect of Mg²⁺_i on L-type Ca²⁺ current in the context of increased sympathetic tone was unclear. We tested the impact of increased Mg²⁺_i on the β-adrenergic stimulation of L-type Ca²⁺ current. Exposure of acutely dissociated adult VMs to higher Mg²⁺_i concentrations decreased isoproterenol stimulation of the L-type Ca²⁺ current from 75 ± 13% with 0.8 mM Mg²⁺_i to 20 ± 8% with 2.4 mM Mg²⁺_i. We activated this signaling cascade at different steps to determine the site or sites of Mg²⁺_i action. Exposure of VMs to increased Mg²⁺_i attenuated the stimulation of L-type Ca²⁺ current induced by activation of adenylyl cyclase with forskolin, inhibition of cyclic nucleotide phosphodiesterases with isobutylmethylxanthine, and inhibition of phosphoprotein phosphatases I and IIA with calyculin A. These experiments ruled out significant effects of Mg²⁺_i on these upstream steps in the signaling cascade and suggested that Mg²⁺_i acts directly on Ca_V1.2 channels. One possible site of action is the EF-hand in the proximal C-terminal domain, just downstream in the signaling cascade from the site of regulation of Ca_V1.2 channels by protein phosphorylation on the C terminus. Consistent with this hypothesis, Mg²⁺_i had no effect on enhancement of Ca_V1.2 channel activity by the dihydropyridine agonist (S)-BayK8644, which activates Ca_V1.2 channels by binding to a site formed by the transmembrane domains of the channel. Collectively, our results suggest that, in transient ischemia, increased Mg²⁺_i reduces stimulation of L-type Ca²⁺ current by the β-adrenergic receptor by directly acting on Ca_V1.2 channels in a cell-autonomous manner, effectively decreasing the metabolic stress imposed on VMs until blood flow can be reestablished.     

Fusicoccin Binding to Its Plasma Membrane Receptor and the Activation of the Plasma Membrane H+-ATPase (III. Is There a Direct Interaction between the Fusicoccin Receptor and the Plasma Membrane H+-ATPase?)

A radioimmunoassay using antibodies raised against bovine serum albumin-conjugated fusicoccin (FC) was applied to measure FC bound to the plasma membrane (PM) isolated from seedlings of radish (Raphanus sativus L.) and of Arabidopsis thaliana treated in vivo plus or minus the toxin. FC bound to the PM from seedlings treated with 5 [mu]M FC was 2-fold (radish) to 7-fold (A. thaliana) higher than the binding capacity of control PM. FC binding depended on the duration of the in vivo treatment but was unaffected by cycloheximide. When FC binding and the PM H+-ATPase activity were compared under different conditions (in vivo or in vitro treatment of different lengths or with different concentrations of FC), a strict linear relation between FC binding and the activation of the PM H+-ATPase was observed in both plant materials under all the conditions tested. Comparison between the maximum binding capacity and the amount of H+-ATPase observed in PM from the two plant materials suggest a one-to-one stoichiometry between the FC receptor and the PM H+-ATPase.     

Kir6.2ΔC26 Channel Traffics to Plasma Membrane by Constitutive Exocytosis

Adenosine triphosphate （ATP）-sensitive K^＊ （KATP） channels regulate many cellular functions by coupling the metabolic state of the cell to the changes in membrane potential. Truncation of C-terminal 26 amino acid residues of Kir6.2 protein （Kir6.2ΔC26） deletes its endoplasmic reticulum retention signal, allowing functional expression of Kit6.2 in the absence of sulfonylurea receptor subunit, pEGFP-Kir6.2ΔC26 and pKir6.2ΔC26-IRES2-EGFP expression plasmids were constructed and transfected into HEK293 cells. We identified that Kir6.2ΔC26 was localized on the plasma membrane and trafficked to the plasmalemma by means of constitutive exocytosis of Kir6.2ΔC26 transport vesicles, using epi-fluorescence and total intemal reflection fluorescence microscopy. Our electrophysiological data showed that Kir6.2ΔC26 alone expressed KATP currents, whereas EGFP-Kir6.2ΔC26 fusion protein displayed no KATP channel activity.     

Physical Determinants of β-Barrel Membrane Protein Folding in Lipid Vesicles

The spontaneous folding of two Neisseria outer membrane proteins, opacity-associated (Opa)₆₀ and Opa₅₀ into lipid vesicles was investigated by systematically varying bulk and membrane properties. Centrifugal fractionation coupled with sodium dodecyl sulfate polyacrylamide gel electrophoresis mobility assays enabled the discrimination of aggregate, unfolded membrane-associated, and folded membrane-inserted protein states as well as the influence of pH, ionic strength, membrane surface potential, lipid saturation, and urea on each. Protein aggregation was reduced with increasing lipid chain length, basic pH, low salt, the incorporation of negatively charged guest lipids, or by the addition of urea to the folding reaction. Insertion from the membrane-associated form was improved in shorter chain lipids, with more basic pH and low ionic strength; it is hindered by unsaturated or ether-linked lipids. The isolation of the physical determinants of insertion suggests that the membrane surface and dipole potentials are driving forces for outer membrane protein insertion and folding into lipid bilayers.     

Are B-type Ca2+ Channels of Cardiac Myocytes Akin to the Passive Ion Channel in the Plasma Membrane Ca2+ Pump?

The present study demonstrates that B-type Ca²⁺ channels observed in rat ventricular myocytes markedly reacted to agents known to affect the ion-motive plasma membrane Ca²⁺-ATPase (PMCA) pump. Chlorpromazine (CPZ)-activated B-type Ca²⁺ channels were completely blocked by internal application of PMCA pump inhibitors, namely La³⁺ (100 μm), eosin (10 μm) and AIF₃ (100 μm). Calmodulin (50 U/ml), the main endogenous positive regulator of PMCA, was unable to activate but significantly reduced CPZ-activated B-type channel activity. In the same manner, ATP (1 and 4 mm), the main energizing substrate of PMCA, was able to reversibly and significantly reduce this activity in a dose-dependent manner. Interestingly, anti-PMCA antibody 5F10, but not anti-Na/K ATPase antibody (used as a negative control) induced a marked Ba²⁺-conducting channel activity that shared the same characteristics with that of CPZ-activated B-type channels. 5F10-Activated channels were mostly selective towards Ba²⁺, mainly had three observed conductance levels (23, 47 and 85 pS), were observed with a frequency of about 1 out of 5 membrane patches and were completely blocked by 10 μm eosin. These results suggest that B-type Ca²⁺ channels are some form of the PMCA pump. Received: 24 July 2000/Revised: 5 October 2000     

The N-terminal Domain Tethers the Voltage-gated Calcium Channel β2e-subunit to the Plasma Membrane via Electrostatic and Hydrophobic Interactions

The β-subunit associates with the α₁ pore-forming subunit of high voltage-activated calcium channels and modulates several aspects of ion conduction. Four β-subunits are encoded by four different genes with multiple splice variants. Only two members of this family, β_2a and β_2e, associate with the plasma membrane in the absence of the α₁-subunit. Palmitoylation on a di-cysteine motif located at the N terminus of β_2a promotes membrane targeting and correlates with the unique ability of this protein to slow down inactivation. In contrast, the mechanism by which β_2e anchors to the plasma membrane remains elusive. Here, we identified an N-terminal segment in β_2e encompassing a cluster of positively charged residues, which is strictly required for membrane anchoring, and when transferred to the cytoplasmic β_1b isoform it confers membrane localization to the latter. In the presence of negatively charged phospholipid vesicles, this segment binds to acidic liposomes dependently on the ionic strength, and the intrinsic fluorescence emission maxima of its single tryptophan blue shifts considerably. Simultaneous substitution of more than two basic residues impairs membrane targeting. Coexpression of the fast inactivating R-type calcium channels with wild-type β_2e, but not with a β_2e membrane association-deficient mutant, slows down inactivation. We propose that a predicted α-helix within this domain orienting parallel to the membrane tethers the β_2e-subunit to the lipid bilayer via electrostatic interactions. Penetration of the tryptophan side chain into the lipidic core stabilizes the membrane-bound conformation. This constitutes a new mechanism for membrane anchoring among the β-subunit family that also sustains slowed inactivation.     

β-Barrel Mobility Underlies Closure of the Voltage-Dependent Anion Channel

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Highlights? The rigid N-terminal helix of VDAC stabilizes the cylindrical β-barrel ? Removal of the helix leads to elliptical, semicollapsed pore shapes ? Semicollapse can quantitatively explain conductance and selectivity of closed VDAC ? The N terminus acts as a switch controlling VDAC entry into the closed state     

Molecular dynamics investigation of the ω-current in the Kv1.2 voltage sensor domains

Voltage sensor domains (VSD) are transmembrane proteins that respond to changes in membrane voltage and modulate the activity of ion channels, enzymes, or in the case of proton channels allow permeation of protons across the cell membrane. VSDs consist of four transmembrane segments, S1-S4, forming an antiparallel helical bundle. The S4 segment contains several positively charged residues, mainly arginines, located at every third position along the helix. In the voltage-gated Shaker K(+) channel, the mutation of the first arginine of S4 to a smaller uncharged amino acid allows permeation of cations through the VSD. These currents, known as ω-currents, pass through the VSD and are distinct from K(+) currents passing through the main ion conduction pore. Here we report molecular dynamics simulations of the ω-current in the resting-state conformation for Kv1.2 and for four of its mutants. The four tested mutants exhibit various degrees of conductivity for K(+) and Cl(-) ions, with a slight selectivity for K(+) over Cl(-). Analysis of the ion permeation pathway, in the case of a highly conductive mutant, reveals a negatively charged constriction region near the center of the membrane that might act as a selectivity filter to prevent permeation of anions through the pore. The residues R1 in S4 and E1 in S2 are located at the narrowest region of the ω-pore for the resting state conformation of the VSD, in agreement with experiments showing that the largest increase in current is produced by the double mutation E1D and R1S.     

Integrin αIIbβ3 Transmembrane Domain Separation Mediates Bi-Directional Signaling across the Plasma Membrane

Integrins play an essential role in hemostasis, thrombosis, and cell migration, and they transmit bidirectional signals. Transmembrane/cytoplasmic domains are hypothesized to associate in the resting integrins; whereas, ligand binding and intracellular activating signals induce transmembrane domain separation. However, how this conformational change affects integrin outside-in signaling and whether the α subunit cytoplasmic domain is important for this signaling remain elusive. Using Chinese Hamster Ovary (CHO) cells that stably expressed different integrin α_IIbβ₃ constructs, we discovered that an α_IIb cytoplasmic domain truncation led to integrin activation but not defective outside-in signaling. In contrast, preventing transmembrane domain separation abolished both inside-out and outside-in signaling regardless of removing the α_IIb cytoplasmic tail. Truncation of the α_IIb cytoplasmic tail did not obviously affect adhesion-induced outside-in signaling. Our research revealed that transmembrane domain separation is a downstream conformational change after the cytoplasmic domain dissociation in inside-out activation and indispensable for ligand-induced outside-in signaling. The result implicates that the β TM helix rearrangement after dissociation is essential for integrin transmembrane signaling. Furthermore, we discovered that the PI3K/Akt pathway is not essential for cell spreading but spreading-induced Erk1/2 activation is PI3K dependent implicating requirement of the kinase for cell survival in outside-in signaling.     

Molecular Basis of the Membrane Interaction of the β2e Subunit of Voltage-Gated Ca2+ Channels

The auxiliary β subunit plays an important role in the regulation of voltage-gated calcium (Ca_V) channels. Recently, it was revealed that β2e associates with the plasma membrane through an electrostatic interaction between N-terminal basic residues and anionic phospholipids. However, a molecular-level understanding of β-subunit membrane recruitment in structural detail has remained elusive. In this study, using a combination of site-directed mutagenesis, liposome-binding assays, and multiscale molecular-dynamics (MD) simulation, we developed a physical model of how the β2e subunit is recruited electrostatically to the plasma membrane. In a fluorescence resonance energy transfer assay with liposomes, binding of the N-terminal peptide (23 residues) to liposome was significantly increased in the presence of phosphatidylserine (PS) and phosphatidylinositol 4,5-bisphosphate (PIP₂). A mutagenesis analysis suggested that two basic residues proximal to Met-1, Lys-2 (K2) and Trp-5 (W5), are more important for membrane binding of the β2e subunit than distal residues from the N-terminus. Our MD simulations revealed that a stretched binding mode of the N-terminus to PS is required for stable membrane attachment through polar and nonpolar interactions. This mode obtained from MD simulations is consistent with experimental results showing that K2A, W5A, and K2A/W5A mutants failed to be targeted to the plasma membrane. We also investigated the effects of a mutated β2e subunit on inactivation kinetics and regulation of Ca_V channels by PIP₂. In experiments with voltage-sensing phosphatase (VSP), a double mutation in the N-terminus of β2e (K2A/W5A) increased the PIP₂ sensitivity of Ca_V2.2 and Ca_V1.3 channels by ∼3-fold compared with wild-type β2e subunit. Together, our results suggest that membrane targeting of the β2e subunit is initiated from the nonspecific electrostatic insertion of N-terminal K2 and W5 residues into the membrane. The PS-β2e interaction observed here provides a molecular insight into general principles for protein binding to the plasma membrane, as well as the regulatory roles of phospholipids in transporters and ion channels.