Structural basis of AMPA receptor inhibition by trans-4-butylcyclohexane carboxylic acid.

BACKGROUND AND PURPOSE: AMPA receptors, which shape excitatory postsynaptic currents and are directly involved in overactivation of synaptic function during seizures, represent a well-accepted target for anti-epileptic drugs. Trans-4-butylcyclohexane carboxylic acid (4-BCCA) has emerged as a new promising anti-epileptic drug in several in vitro and in vivo seizure models, but the mechanism of its action remained unknown. The purpose of this study is to characterize structure and dynamics of 4-BCCA interaction with AMPA receptors. EXPERIMENTAL APPROACH: We studied the molecular mechanism of AMPA receptor inhibition by 4-BCCA using a combination of X-ray crystallography, mutagenesis, electrophysiological assays, and molecular dynamics simulations. KEY RESULTS: We identified 4-BCCA binding sites in the transmembrane domain (TMD) of AMPA receptor, at the lateral portals formed by transmembrane segments M1-M4. At this binding site, 4-BCCA is very dynamic, assumes multiple poses, and can enter the ion channel pore. CONCLUSION AND IMPLICATIONS: 4-BCCA represents a low-affinity inhibitor of AMPA receptors that acts at the TMD sites distinct from non-competitive inhibitors, such as the anti-epileptic drug perampanel and the ion channel blockers. Further studies might examine the possibsility of synergistic use of these inhibitors in treatment of epilepsy and a wide range of neurological disorders and gliomas. LINKED ARTICLES: This article is part of a themed issue on Structure Guided Pharmacology of Membrane Proteins (BJP 75th Anniversary). To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v179.14/issuetoc.

Cryo-EM structure of human GPR158 receptor coupled to the RGS7-Gß5 signaling complex.

GPR158 is an orphan G protein­coupled receptor (GPCR) highly expressed in the brain, where it controls synapse formation and function. GPR158 has also been implicated in depression, carcinogenesis, and cognition. However, the structural organization and signaling mechanisms of GPR158 are largely unknown. We used single-particle cryo­electron microscopy (cryo-EM) to determine the structures of human GPR158 alone and bound to an RGS signaling complex. The structures reveal a homodimeric organization stabilized by a pair of phospholipids and the presence of an extracellular Cache domain, an unusual ligand-binding domain in GPCRs. We further demonstrate the structural basis of GPR158 coupling to RGS7-Gß5. Together, these results provide insights into the unusual biology of orphan receptors and the formation of GPCR-RGS complexes.

Inactivation-mimicking block of the epithelial calcium channel TRPV6.

Epithelial calcium channel TRPV6 plays vital roles in calcium homeostasis, and its dysregulation is implicated in multifactorial diseases, including cancers. Here, we study the molecular mechanism of selective nanomolar-affinity TRPV6 inhibition by (4-phenylcyclohexyl)piperazine derivatives (PCHPDs). We use x-ray crystallography and cryo-electron microscopy to solve the inhibitor-bound structures of TRPV6 and identify two types of inhibitor binding sites in the transmembrane region: (i) modulatory sites between the S1-S4 and pore domains normally occupied by lipids and (ii) the main site in the ion channel pore. Our structural data combined with mutagenesis, functional and computational approaches suggest that PCHPDs plug the open pore of TRPV6 and convert the channel into a nonconducting state, mimicking the action of calmodulin, which causes inactivation of TRPV6 channels under physiological conditions. This mechanism of inhibition explains the high selectivity and potency of PCHPDs and opens up unexplored avenues for the design of future-generation biomimetic drugs.

Expression, Purification, and Crystallization of the Transient Receptor Potential Channel TRPV6.

Transient receptor potential (TRP) channels are polymodal sensory transducers that respond to chemicals, temperature, mechanical stress, and membrane voltage and are involved in vision, taste, olfaction, hearing, touch, thermal perception, and nociception. TRP channels are implicated in numerous devastating diseases, including various forms of cancer, and represent important drug targets. The large sizes, low expression levels, and conformational dynamics of TRP channels make them challenging targets for structural biology. Here, we present the methodology used in structural studies of TRPV6, a TRP channel that is highly selective for calcium and mediates Ca2+ uptake in epithelial tissues. We provide a protocol for the expression, purification, and crystallization of TRPV6. Similar approaches can be used to determine crystal structures of other membrane proteins, including different members of the TRP channel family.

Mechanism of calmodulin inactivation of the calcium-selective TRP channel TRPV6.

Calcium (Ca2+) plays a major role in numerous physiological processes. Ca2+ homeostasis is tightly controlled by ion channels, the aberrant regulation of which results in various diseases including cancers. Calmodulin (CaM)-mediated Ca2+-induced inactivation is an ion channel regulatory mechanism that protects cells against the toxic effects of Ca2+ overload. We used cryo-electron microscopy to capture the epithelial calcium channel TRPV6 (transient receptor potential vanilloid subfamily member 6) inactivated by CaM. The TRPV6-CaM complex exhibits 1:1 stoichiometry; one TRPV6 tetramer binds both CaM lobes, which adopt a distinct head-to-tail arrangement. The CaM carboxyl-terminal lobe plugs the channel through a unique cation-π interaction by inserting the side chain of lysine K115 into a tetra-tryptophan cage at the pore's intracellular entrance. We propose a mechanism of CaM-mediated Ca2+-induced inactivation that can be explored for therapeutic design.

Structural bases of TRP channel TRPV6 allosteric modulation by 2-APB.

Transient receptor potential (TRP) channels are involved in various physiological processes, including sensory transduction. The TRP channel TRPV6 mediates calcium uptake in epithelia and its expression is dramatically increased in numerous types of cancer. TRPV6 inhibitors suppress tumor growth, but the molecular mechanism of inhibition remains unknown. Here, we present crystal and cryo-EM structures of human and rat TRPV6 bound to 2-aminoethoxydiphenyl borate (2-APB), a TRPV6 inhibitor and modulator of numerous TRP channels. 2-APB binds to TRPV6 in a pocket formed by the cytoplasmic half of the S1-S4 transmembrane helix bundle. Comparing human wild-type and high-affinity mutant Y467A structures, we show that 2-APB induces TRPV6 channel closure by modulating protein-lipid interactions. Mutagenesis and functional analyses suggest that the identified 2-APB binding site might be present in other members of vanilloid subfamily TRP channels. Our findings reveal a mechanism of ion channel allosteric modulation that can be exploited for therapeutic design.

Opening of the human epithelial calcium channel TRPV6.

Calcium-selective transient receptor potential vanilloid subfamily member 6 (TRPV6) channels play a critical role in calcium uptake in epithelial tissues. Altered TRPV6 expression is associated with a variety of human diseases, including cancers. TRPV6 channels are constitutively active and their open probability depends on the lipidic composition of the membrane in which they reside; it increases substantially in the presence of phosphatidylinositol 4,5-bisphosphate. Crystal structures of detergent-solubilized rat TRPV6 in the closed state have previously been solved. Corroborating electrophysiological results, these structures demonstrated that the Ca2+ selectivity of TRPV6 arises from a ring of aspartate side chains in the selectivity filter that binds Ca2+ tightly. However, how TRPV6 channels open and close their pores for ion permeation has remained unclear. Here we present cryo-electron microscopy structures of human TRPV6 in the open and closed states. The channel selectivity filter adopts similar conformations in both states, consistent with its explicit role in ion permeation. The iris-like channel opening is accompanied by an α-to-π-helical transition in the pore-lining transmembrane helix S6 at an alanine hinge just below the selectivity filter. As a result of this transition, the S6 helices bend and rotate, exposing different residues to the ion channel pore in the open and closed states. This gating mechanism, which defines the constitutive activity of TRPV6, is, to our knowledge, unique among tetrameric ion channels and provides structural insights for understanding their diverse roles in physiology and disease.

Probing Intersubunit Interfaces in AMPA-subtype Ionotropic Glutamate Receptors.

AMPA subtype ionotropic glutamate receptors (iGluRs) mediate the majority of fast neurotransmission across excitatory synapses in the central nervous system. Each AMPA receptor is composed of four multi-domain subunits that are organized into layers of two amino-terminal domain (ATD) dimers, two ligand-binding domain (LBD) dimers, transmembrane domains and carboxy-terminal domains. We introduced cysteine substitutions at the intersubunit interfaces of AMPA receptor subunit GluA2 and confirmed substituted cysteine crosslink formation by SDS-PAGE. The functional consequence of intersubunit crosslinks was assessed by recording GluA2-mediated currents in reducing and non-reducing conditions. Strong redox-dependent changes in GluA2-mediated currents were observed for cysteine substitutions at the LBD dimer-dimer interface but not at the ATD dimer-dimer interface. We conclude that during gating, LBD dimers undergo significant relative displacement, while ATD dimers either maintain their relative positioning, or their relative displacement has no appreciable effect on AMPA receptor function.