Richard W. Mercier

Electrophysiological Analysis of Cloned Cyclic Nucleotide-Gated Ion Channels

Electrophysiological studies were conducted on the cloned plant cyclic nucleotide-gated ion channels AtCNGC2 and AtCNGC1 from Arabidopsis, and NtCBP4 from tobacco (Nicotiana tobacum). The nucleotide coding sequences for these proteins were expressed in Xenopus laevis oocytes or HEK 293 cells. Channel characteristics were evaluated using voltage clamp analysis of currents in the presence of cAMP. AtCNGC2 was demonstrated to conduct K+ and other monovalent cations, but exclude Na+; this conductivity profile is unique for any ion channel not possessing the amino acid sequence found in the selectivity filter of K+-selective ion channels. Application of cAMP evoked currents in membrane patches of oocytes injected with AtCNGC2 cRNA. Direct activation of the channel by cyclic nucleotide, demonstrated by application of cyclic nucleotide to patches of membranes expressing such channels, is a hallmark characteristic of this ion channel family. Voltage clamp studies (two-electrode configuration) demonstrated that AtCNGC1 and NtCBP4 are also cyclic nucleotide-gated channels. Addition of a lipophilic analog of cAMP to the perfusion bath of oocytes injected with NtCBP4 and AtCNGC1 cRNAs induced inward rectified, noninactivating K+currents.

Plants Do It Differently. A New Basis for Potassium/Sodium Selectivity in the Pore of an Ion Channel

Understanding of the molecular architecture necessary for selective K+ permeation through the pore of ion channels is based primarily on analysis of the crystal structure of the bacterial K+ channel KcsA, and structure:function studies of cloned animal K+ channels. Little is known about the conduction properties of a large family of plant proteins with structural similarities to cloned animal cyclic nucleotide-gated channels (CNGCs). Animal CNGCs are nonselective cation channels that do not discriminate between Na+ and K+ permeation. These channels all have the same triplet of amino acids in the channel pore ion selectivity filter, and this sequence is different from that of the selectivity filter found in K+-selective channels. Plant CNGCs have unique pore selectivity filters; unlike those found in any other family of channels. At present, the significance of the unique pore selectivity filters of plant CNGCs, with regard to discrimination between Na+ and K+ permeation is unresolved. Here, we present an electrophysiological analysis of several members of this protein family; identifying the first cloned plant channel (AtCNGC1) that conducts Na+. Another member of this ion channel family (AtCNGC2) is shown to have a selectivity filter that provides a heretofore unknown molecular basis for discrimination between K+ and Na+ permeation. Specific amino acids within the AtCNGC2 pore selectivity filter (Asn-416, Asp-417) are demonstrated to facilitate K+ over Na+ conductance. The selectivity filter of AtCNGC2 represents an alternative mechanism to the well-known GYG amino acid triplet of K+ channels that has been identified as the critical basis for K+ over Na+ permeation through the pore of ion channels.

Mass spectrometry-based proteomics of human cannabinoid receptor 2: covalent cysteine 6.47(257)-ligand interaction affording megagonist receptor activation.

The lack of experimental characterization of the structures and ligand-binding motifs of therapeutic G-protein coupled receptors (GPCRs) hampers rational drug discovery. The human cannabinoid receptor 2 (hCB2R) is a class-A GPCR and promising therapeutic target for small-molecule cannabinergic agonists as medicines. Prior mutational and modeling data constitute provisional evidence that AM-841, a high-affinity classical cannabinoid, interacts with cysteine C6.47(257) in hCB2R transmembrane helix 6 (TMH6) to afford improved hCB2R selectivity and unprecedented agonist potency. We now apply bottom-up mass spectrometry (MS)-based proteomics to define directly the hCB2R-AM-841 interaction at the amino-acid level. Recombinant hCB2R, overexpressed as an N-terminal FLAG-tagged/C-terminal 6His-tagged protein (FLAG-hCB2R-6His) with a baculovirus system, was solubilized and purified by immunochromatography as functional receptor. A multiplex multiple reaction monitoring (MRM)-MS method was developed that allowed us to observe unambiguously all seven discrete TMH peptides in the tryptic digest of purified FLAG-hCB2R-6His and demonstrate that AM-841 modifies hCB2R TMH6 exclusively. High-resolution mass spectra of the TMH6 tryptic peptide obtained by Q-TOF MS/MS analysis demonstrated that AM-841 covalently and selectively modifies hCB2R at TMH6 cysteine C6.47(257). These data demonstrate how integration of MS-based proteomics into a ligand-assisted protein structure (LAPS) experimental paradigm can offer guidance to structure-enabled GPCR agonist design.

Interaction of a fragment of the cannabinoid CB1 receptor C-terminus with arrestin-2

Desensitization of the cannabinoid CB1 receptor is mediated by the interaction with arrestin. In this study, we report the structural changes of a synthetic diphosphorylated peptide corresponding to residues 419–439 of the CB1 C‐terminus upon binding to arrestin‐2. This segment is pivotal to the desensitization of CB1. Using high‐resolution proton NMR, we observe two helical segments in the bound peptide that are separated by the presence a glycine residue. The binding we observe is with a diphoshorylated peptide, whereas a previous study reported binding of a highly phosphorylated rhodopsin fragment to visual arrestin. The arrestin bound conformations of the peptides are compared.

Binding between a Distal C-Terminus Fragment of Cannabinoid Receptor 1 and Arrestin-2

Internalization of G-protein-coupled receptors is mediated by phosphorylation of the C-terminus, followed by binding with the cytosolic protein arrestin. To explore structural factors that may play a role in internalization of cannabinoid receptor 1 (CB1), we utilize a phosphorylated peptide derived from the distal C-terminus of CB1 (CB1(5P)(454-473)). Complexes formed between the peptide and human arrestin-2 (wt-arr2(1-418)) were compared to those formed with a truncated arrestin-2 mutant (tr-arr2(1-382)) using isothermal titration calorimetry and nuclear magnetic resonance spectroscopy. The pentaphosphopeptide CB1(5P)(454-473) adopts a helix-loop conformation, whether binding to full-length arrestin-2 or its truncated mutant. This structure is similar to that of a heptaphosphopeptide, mimicking the distal segment of the rhodopsin C-tail (Rh(7P)(330-348)), binding to visual arrestin, suggesting that this adopted structure bears functional significance. Isothermal titration calorimetry (ITC) experiments show that the CB1(5P)(454-473) peptide binds to tr-arr2(1-382) with higher affinity than to the full-length wt-arr2(1-418). As the observed structure of the bound peptides is similar in either case, we attribute the increased affinity to a more exposed binding site on the N-domain of the truncated arrestin construct. The transferred NOE data from the bound phosphopeptides are used to predict a model describing the interaction with arrestin, using the data driven HADDOCK docking program. The truncation of arrestin-2 provides scope for positively charged residues in the polar core of the protein to interact with phosphates present in the loop of the CB1(5P)(454-473) peptide.

Human Cannabinoid Receptor 2 Ligand-Interaction Motif: Transmembrane Helix 2 Cysteine, C2.59(89), as Determinant of Classical Cannabinoid Agonist Activity and Binding Pose

Cannabinoid receptor 2 (CB2R)-dependent signaling is implicated in neuronal physiology and immune surveillance by brain microglia. Selective CB2R agonists hold therapeutic promise for inflammatory and other neurological disorders. Information on human CB2R (hCB2R) ligand-binding and functional domains is needed to inform the rational design and optimization of candidate druglike hCB2R agonists. Prior demonstration that hCB2R transmembrane helix 2 (TMH2) cysteine C2.59(89) reacts with small-molecule methanethiosulfonates showed that this cysteine residue is accessible to sulfhydryl derivatization reagents. We now report the design and application of two novel, pharmacologically active, high-affinity molecular probes, AM4073 and AM4099, as chemical reporters to interrogate directly the interaction of classical cannabinoid agonists with hCB2R cysteine residues. AM4073 has one electrophilic isothiocyanate (NCS) functionality at the C9 position of its cyclohexenyl C-ring, whereas AM4099 has NCS groups at that position and at the terminus of its aromatic A-ring C3 side chain. Pretreatment of wild-type hCB2R with either probe reduced subsequent [3H]CP55,940 specific binding by ∼60%. Conservative serine substitution of any hCB2R TMH cysteine residue except C2.59(89) did not affect the reduction of [3H]CP55,940 specific binding by either probe, suggesting that AM4073 and AM4099 interact irreversibly with this TMH2 cysteine. In contrast, AM841, an exceptionally potent hCB2R megagonist and direct AM4073/4099 congener bearing a single electrophilic NCS group at the terminus of its C3 side chain, had been demonstrated to bind covalently to TMH6 cysteine C6.47(257) and not C2.59(89). Molecular modeling indicates that the AM4073-hCB2R* interaction at C2.59(89) orients this classical cannabinoid away from TMH6 and toward the TMH2-TMH3 interface in the receptors hydrophobic binding pocket, whereas the AM841-hCB2R* interaction at C6.47(257) favors agonist orientation toward TMH6/7. These data constitute initial evidence that TMH2 cysteine C2.59(89) is a component of the hCB2R binding pocket for classical cannabinoids. The results further demonstrate how interactions between classical cannabinoids and specific amino acids within the hCB2R* ligand-binding domain act as determinants of agonist pharmacological properties and the architecture of the agonist-hCB2R* conformational ensemble, allowing the receptor to adopt distinct activity states, such that interaction of classical cannabinoids with TMH6 cysteine C6.47(257) favors a binding pose more advantageous for agonist potency than does their interaction with TMH2 cysteine C2.59(89).