Steen E. Pedersen

Apocalmodulin and Ca2+Calmodulin-Binding Sites on the CaV1.2 Channel

The cardiac L-type voltage-dependent calcium channel is responsible for initiating excitation-contraction coupling. Three sequences (amino acids 1609-1628, 1627-1652, and 1665-1685, designated A, C, and IQ, respectively) of its alpha(1) subunit contribute to calmodulin (CaM) binding and Ca(2+)-dependent inactivation. Peptides matching the A, C, and IQ sequences all bind Ca(2+)CaM. Longer peptides representing A plus C (A-C) or C plus IQ (C-IQ) bind only a single molecule of Ca(2+)CaM. Apocalmodulin (ApoCaM) binds with low affinity to the IQ peptide and with higher affinity to the C-IQ peptide. Binding to the IQ and C peptides increases the Ca(2+) affinity of the C-lobe of CaM, but only the IQ peptide alters the Ca(2+) affinity of the N-lobe. Conversion of the isoleucine and glutamine residues of the IQ motif to alanines in the channel destroys inactivation (Zühlke et al., 2000). The double mutation in the peptide reduces the interaction with apoCaM. A mutant CaM unable to bind Ca(2+) at sites 3 and 4 (which abolishes the ability of CaM to inactivate the channel) binds to the IQ, but not to the C or A peptide. Our data are consistent with a model in which apoCaM binding to the region around the IQ motif is necessary for the rapid binding of Ca(2+) to the C-lobe of CaM. Upon Ca(2+) binding, this lobe is likely to engage the A-C region.

Oxidative Stress Promotes Ligand-independent and Enhanced Ligand-dependent Tumor Necrosis Factor Receptor Signaling

Tumor necrosis factor (TNF) receptor 1 (TNFR1, p55) and 2 (TNFR2, p75) are characterized by several cysteine-rich modules in the extracellular domain, raising the possibility that redox-induced modifications of these cysteine residues might alter TNFR function. To test this possibility, we examined fluorescence resonance energy transfer (FRET) in 293T cells transfected with CFP- and YFP-tagged TNFRs exposed to the thiol oxidant diamide. Treatment with high concentrations of diamide (1 mm) resulted in an increase in the FRET signal that was sensitive to inhibition with the reducing agent dithiothreitol, suggesting that oxidative stress resulted in TNFR self-association. Treatment of cells with low concentrations of diamide (1 μm) that was not sufficient to provoke TNFR self-association resulted in increased TNF-induced FRET signals relative to the untreated cells, suggesting that oxidative stress enhanced ligand-dependent TNFR signaling. Similar findings were obtained when the TNFR1- and TNFR2-transfected cells were pretreated with a cell-impermeable oxidase, DsbA, that catalyzes disulfide bond formation between thiol groups on cysteine residues. The changes in TNFR self-association were functionally significant, because pretreating the HeLa cells and 293T cells resulted in increased TNF-induced NF-κB activation and TNF-induced expression of IκB and syndecan-4 mRNA levels. Although pretreatment with DsbA did not result in an increase in TNF binding to TNFRs, it resulted in increased TNF-induced activation of NF-κB, consistent with an allosteric modification of the TNFRs. Taken together, these results suggest that oxidative stress promotes TNFR receptor self-interaction and ligand-independent and enhanced ligand-dependent TNF signaling.

Determinants in CaV1 Channels That Regulate the Ca2+ Sensitivity of Bound Calmodulin

Calmodulin binds to IQ motifs in the α1 subunit of CaV1.1 and CaV1.2, but the affinities of calmodulin for the motif and for Ca2+ are higher when bound to CaV1.2 IQ. The CaV1.1 IQ and CaV1.2 IQ sequences differ by four amino acids. We determined the structure of calmodulin bound to CaV1.1 IQ and compared it with that of calmodulin bound to CaV1.2 IQ. Four methionines in Ca2+-calmodulin form a hydrophobic binding pocket for the peptide, but only one of the four nonconserved amino acids (His-1532 of CaV1.1 and Tyr-1675 of CaV1.2) contacts this calmodulin pocket. However, Tyr-1675 in CaV1.2 contributes only modestly to the higher affinity of this peptide for calmodulin; the other three amino acids in CaV1.2 contribute significantly to the difference in the Ca2+ affinity of the bound calmodulin despite having no direct contact with calmodulin. Those residues appear to allow an interaction with calmodulin with one lobe Ca2+-bound and one lobe Ca2+-free. Our data also provide evidence for lobe-lobe interactions in calmodulin bound to CaV1.2.

The Nicotinic Acetylcholine Receptor and the Na,K-ATPase α2 Isoform Interact to Regulate Membrane Electrogenesis in Skeletal Muscle

The nicotinic acetylcholine receptor (nAChR) and the Na,K-ATPase functionally interact in skeletal muscle (Krivoi, I. I., Drabkina, T. M., Kravtsova, V. V., Vasiliev, A. N., Eaton, M. J., Skatchkov, S. N., and Mandel, F. (2006) Pflugers Arch. 452, 756–765; Krivoi, I., Vasiliev, A., Kravtsova, V., Dobretsov, M., and Mandel, F. (2003) Ann. N.Y. Acad. Sci. 986, 639–641). In this interaction, the specific binding of nanomolar concentrations of nicotinic agonists to the nAChR stimulates electrogenic transport by the Na,K-ATPase α2 isozyme, causing membrane hyperpolarization. This study examines the molecular nature and membrane localization of this interaction. Stimulation of Na,K-ATPase activity by the nAChR does not require ion flow through open nAChRs. It can be induced by nAChR desensitization alone, in the absence of nicotinic agonist, and saturates when the nAChR is fully desensitized. It is enhanced by noncompetitive blockers of the nAChR (proadifen, QX-222), which promote non-conducting or desensitized states; and retarded by tetracaine, which stabilizes the resting nAChR conformation. The interaction operates at the neuromuscular junction as well as on extrajunctional sarcolemma. The Na,K-ATPase α2 isozyme is enriched at the postsynaptic neuromuscular junction and co-localizes with nAChRs. The nAChR and Na,K-ATPase α subunits specifically coimmunoprecipitate with each other, phospholemman, and caveolin-3. In a purified membrane preparation from Torpedo californica enriched in nAChRs and the Na,K-ATPase, a ouabain-induced conformational change of the Na,K-ATPase enhances a conformational transition of the nAChR to a desensitized state. These results suggest a mechanism by which the nAChR in a desensitized state with high apparent affinity for agonist interacts with the Na,K-ATPase to stimulate active transport. The interaction utilizes a membrane-delimited complex involving protein-protein interactions, either directly or through additional protein partners. This interaction is expected to enhance neuromuscular transmission and muscle excitation.

Orientation of d-Tubocurarine in the Muscle Nicotinic Acetylcholine Receptor-binding Site

Ligand modification and receptor site-directed mutagenesis were used to examine binding of the competitive antagonist,d-tubocurarine (dTC), to the muscle-type nicotinic acetylcholine receptor (AChR). By using various dTC analogs, we measured the interactions of specific dTC functional groups with amino acid positions in the AChR γ-subunit. Because data for mutations at residue γTyr117 were the most consistent with direct interaction with dTC, we focused on that residue. Double mutant thermodynamic cycle analysis showed apparent interactions of γTyr117 with both the 2-N and the 13′-positions of dTC. Examination of a dTC analog with a negative charge at the 13′-position failed to reveal electrostatic interaction with charged side-chain substitutions at γ117, but the effects of side-chain substitutions remained consistent with proximity of Tyr117 to the cationic 2-N of dTC. The apparent interaction of γTyr117with the 13′-position of dTC was likely mediated by allosteric changes in either dTC or the receptor. The data also show that cation-π electron stabilization of the 2-N position is not required for high affinity binding. Molecular modeling of dTC within the binding pocket of the acetylcholine-binding protein places the 2-N in proximity to the residue homologous to γTyr117. This model provides a plausible structural basis for binding of dTC within the acetylcholine-binding site of the AChR family that appears consistent with findings from photoaffinity labeling studies and with site-directed mutagenesis studies of the AChR.

Electrostatic Interactions Regulate Desensitization of the Nicotinic Acetylcholine Receptor

To determine the importance of electrostatic interactions for agonist binding to the nicotinic acetylcholine receptor (AChR), we examined the affinity of the fluorescent agonist dansyl-C6-choline for the AChR. Increasing ionic strength decreased the binding affinity in a noncompetitive manner and increased the Hill coefficient of binding. Small cations did not compete directly for dansyl-C6-choline binding. The sensitivity to ionic strength was reduced in the presence of proadifen, a noncompetitive antagonist that desensitizes the receptor. Moreover, at low ionic strength, the dansyl-C6-choline affinities were similar in the absence or presence of proadifen, a result consistent with the receptor being desensitized at low ionic strength. Similar ionic strength effects were observed for the binding of the noncompetitive antagonist [(3)H]ethidium when examined in the presence and absence of agonist to desensitize the AChR. Therefore, ionic strength modulates binding affinity through at least two mechanisms: by influencing the conformation of the AChR and by electrostatic effects at the binding sites. The results show that charge-charge interactions regulate the desensitization of the receptor. Analysis of dansyl-C6-choline binding to the desensitized conformation using the Debye-Hückel equation was consistent with the presence of five to nine negative charges within 20 A of the acetylcholine binding sites.

Interaction of d-Tubocurarine Analogs with the Mouse Nicotinic Acetylcholine Receptor LIGAND ORIENTATION AT THE BINDING SITE

The binding of d-tubocurarine and several of its analogs to the mouse nicotinic acetylcholine receptor (AChR) was measured by competition against the initial rate125I-α-bungarotoxin binding to BC3H-1 cells. The changes in affinity due to methylation or halogenation at various functional groups on d-tubocurarine was measured to both the high affinity (αγ-site) and the low affinity site (αδ-site). We show that quaternization by methylation of the 2′-N ammonium group enhances the affinity for both the acetylcholine binding sites of mouse AChR, whereas this change does not affect affinity for the Torpedo AChR sites. The effect ofN-methylation suggests the presence of interactions with the ammonium moiety that cannot be readily attributed to the known conserved residues thought to stabilize this functional group. Methylation of both the 7′- and 12′-phenols produced net affinity changes at both sites. The changes resulted from contributions at both the 7′- and the 12′-positions; however, these effects were dependent on whether the ammoniums were also methylated. Substitution of bromine or iodine at the 13′-position decreased the affinity considerably to the high affinity αγ-site of mouse AChR, whereas the affinity for theTorpedo αγ-site was slightly increased. Furthermore, binding to the mouse AChR was unaffected by the conformational state, whereas these ligands strongly preferred the desensitized conformation of the Torpedo AChR. Comparison of binding changes upon 13′-halogenation to the changes in amino acid residues at the ACh binding sites of the mouse and Torpedo AChR shows mouse residue Ile-γ116 as likely to be involved in interacting with the 13′-position of d-tubocurarine. It is predicted that this residue is involved in the conformational equilibrium between the resting and desensitized conformations.

Site-specific charge interactions of alpha-conotoxin MI with the nicotinic acetylcholine receptor.

We have tested the importance of charge interactions for α-conotoxin MI binding to the nicotinic acetylcholine receptor (AChR). Ionic residues on α-conotoxin MI were altered by site-directed mutagenesis or by chemical modification. In physiological buffer, removal of charges at the N terminus, His-5, and Lys-10 had small (2–4-fold) effects on binding affinity to the mouse muscle AChR and the Torpedo AChR. It was also demonstrated that conotoxin had no effect on the conformational equilibrium of either receptor, as assessed by the effects of the noncompetitive antagonist proadifen on conotoxin binding and, conversely, the effect of conotoxin on the affinity of phencyclidine, proadifen, and ethidium. Conotoxin displayed higher binding affinity in low ionic strength buffer; neutralization of Lys-10 and the N terminus by acetylation blocked this affinity shift at the αδ site but not at the αγ site. It is concluded that Ctx residues Lys-10 and the N terminal interact with oppositely charged receptor residues only at the αδ site, and the two sites have distinct arrangements of charged residues. Ethidium fluorescence experiments demonstrated that conotoxin is formally competitive with a small cholinergic ligand, tetramethylammonium. Thus, α-conotoxin MI appears to interact with the portion of the binding site responsible for stabilizing agonist cations but does not do so with a cationic residue and is, consequently, incapable of inducing a conformational change.

Ligand binding methods for analysis of ion channel structure and function.

Publisher Summary The nicotinic acetylcholine receptor (AChR) is an ion channel that is opened by the binding of two molecules of acetylcholine on its extracellular surface. On prolonged exposure to acetylcholine, the channel desensitizes and acquires high affinity for acetylcholine. The equilibrium binding to the two sites appears weakly cooperative, but the two sites are distinct, as shown by the binding of various antagonists that preferentially bind one site versus the other. The sites are allosterically linked to a binding site located within the channel pore itself, the noncompetitive antagonist site. Binding to this site can alter the conformation in favor of either the resting conformation or the desensitized conformation, depending on the ligand. Desensitization, as induced by noncompetitive antagonists, is also marked by increased affinity for agonist binding to the acetylcholine sites. The ability of many cholinergic ligands to bind all three sites further complicates analyses of the linkage. An increased awareness of these phenomena has permitted binding to the AChR to be understood in more detail, and binding assays are finding greater use in elucidating the structure of the binding sites. This chapter describes several ligand binding techniques: radioligand binding by centrifugation assay, radioligand binding by DE-81 filter binding, and fluorescent ligand binding. In addition, the chapter discusses the way direct binding measurements, indirect binding by competition, and noncompetitive allosteric effects can be analyzed.

Interactions of acetylcholine binding site residues contributing to nicotinic acetylcholine receptor gating: Role of residues Y93, Y190, K145 and D200

The nicotinic acetylcholine receptor exhibits multiple conformational states, resting (channel closed), active (channel open) and desensitized (channel closed). The resting state may be distinguished from the active and desensitized states by the orientation of loop C in the extracellular ligand binding domain (LBD). Homology modeling was used to generate structures of the Torpedo californica α2βδγ nAChR that initially represent the resting state (loop C open) and the desensitized state (loop C closed). Molecular dynamics (MD) simulations were performed on the extracellular LBD on each nAChR conformational state, with and without the agonist anabaseine present in each binding site (the αγ and the αδ sites). Three MD simulations of 10ns each were performed for each of the four conditions. Comparison of dynamics revealed that in the presence of agonist, loop C was drawn inward and attains a more stable conformation. Examination of side-chain interactions revealed that residue αY190 exhibited hydrogen-bonding interactions either with residue αY93 in the ligand binding site or with residue αK145 proximal to the binding site. αK145 also exhibited side chain (salt bridge) interactions with αD200 and main chain interactions with αY93. Residues αW149, αY198, γY116/δT119, γL118/δL121 and γL108/δL111 appear to play the role of stabilizing ligand in the binding site. In MD simulations for the desensitized state, the effect of ligand upon the interactions among αK145, αY190, and αY93 as well as ligand-hydrogen-bonding to αW149 were more pronounced at the αγ interface than at the αδ interface. Differences in affinity for the desensitized state were determined experimentally to be 10-fold. The changes in side chain interactions observed for the two conformations and induced by ligand support a model wherein hydrogen bond interactions between αD200 and αY93 are broken and rearrange to form a salt-bridge between αK145 and αD200 and hydrogen bond interactions between αY93 and αY190 and between αK145 and αY190.