Biology subjects

Gochman, A.

Publications and source records attributed to Gochman, A..

3 recordsLinked to original sources

bioRxiv2023

ent-Verticilide B1 inhibits type 2 ryanodine receptor channels and is antiarrhythmic in Casq2-/- mice

Ca2+ leak from cardiac ryanodine receptor (RyR2) is an established mechanism of sudden cardiac death (SCD), whereby dysregulated Ca2+ handling causes ventricular arrhythmias. We previously discovered the RyR2-selective inhibitor ent-(+)-verticilide (ent-1), a 24-membered cyclooligomeric depsipeptide that is the enantiomeric form of a natural product (nat-(-)-verticilide). Here, we examined its 18-membered ring-size oligomer (ent-verticilide B1; "ent-B1") in single RyR2 channel assays, [3H]ryanodine binding assays, and in Casq2-/- cardiomyocytes and mice, a gene-targeted model of SCD. ent-B1 inhibited RyR2 single-channels and [3H]ryanodine binding with low micromolar potency, and RyR2-mediated spontaneous Ca2+ release in Casq2-/- cardiomyocytes with sub-micromolar potency. ent-B1 was a partial RyR2 inhibitor, with maximal inhibitory efficacy of less than 50%. ent-B1 was stable in plasma, with a peak plasma concentration of 1460 ng/ml at 10 min and half-life of 45 min after intraperitoneal administration of 3 mg/kg in mice. Both 3 mg/kg and 30 mg/kg ent-B1 significantly reduced catecholamine-induced ventricular arrhythmia in Casq2-/- mice. Hence, we have identified a novel chemical entity - ent-B1 - that preserves the mechanism of action of a hit compound and shows therapeutic efficacy. These findings strengthen RyR2 as an antiarrhythmic drug target and highlight the potential of investigating the mirror-image isomers of natural products to discover new therapeutics. Significance statementThe cardiac ryanodine receptor (RyR2) is an untapped target in the stagnant field of antiarrhythmic drug development. We have confirmed RyR2 as an antiarrhythmic target in a mouse model of sudden cardiac death and shown the therapeutic efficacy of a second enantiomeric natural product.

pharmacology and toxicology↗

bioRxiv2023

Cannabidiol sensitizes TRPV2 channels to activation by 2-APB

The cation-permeable TRPV2 channel is essential for cardiac and immune cells. Cannabidiol (CBD), a non-psychoactive cannabinoid of clinical relevance, is one of the few molecules known to activate TRPV2. Using the patch-clamp technique we discover that CBD can sensitize current responses of the rat TRPV2 channel to the synthetic agonist 2-aminoethoxydiphenyl borate (2- APB) by over two orders of magnitude, without sensitizing channels to activation by moderate (40 C) heat. Using cryo-EM we uncover a new small-molecule binding site in the pore domain of rTRPV2 that can be occupied by CBD in addition to a nearby CBD site that had already been reported. The TRPV1 and TRPV3 channels share >40% sequence identity with TRPV2 are also activated by 2-APB and CBD, but we only find a strong sensitizing effect of CBD on the response of mouse TRPV3 to 2-APB. Mutations at non-conserved positions between rTRPV2 and rTRPV1 in either the pore domain or the CBD sites failed to confer strong sensitization by CBD in mutant rTRPV1 channels. Together, our results indicate that CBD-dependent sensitization of TRPV2 channels engages multiple channel regions and possibly involves more than one CBD and 2-APB sites. The remarkably robust effect of CBD on TRPV2 and TRPV3 channels offers a promising new tool to both understand and overcome one of the major roadblocks in the study of these channels - their resilience to activation.

biophysics↗

bioRxiv2020

NMDA receptors require multiple pre-opening gating steps for efficient synaptic activity

NMDA receptors (NMDAR) are glutamate-gated ion channels that mediate the majority of fast excitatory synaptic transmission in the nervous system. A central feature of NMDAR physiology is the opening of the ion channel driven by presynaptically-released glutamate. Using glutamate applications to outside-out patches containing a single NMDAR in the continuous presence of the co-agonist glycine, we find that agonist-bound receptors transition to the open state via two conformations, an unconstrained pre-active state that can rapidly transition to the open state and contributes to synaptic events, and a constrained pre-active state that requires more energy and hence time to open and does not contribute to fast signaling. To define how agonist binding might drive these conformations, we decoupled the ligand-binding domains from specific transmembrane segments for the GluN1 and GluN2A subunits. Displacements of the central pore-forming M3 segments define the energy of fast channel opening. However, to enter the unconstrained conformation and contribute to fast signaling, a peripheral helix, the GluN2 pre-M1, must be displaced before the M3 segments move. This pre-M1 displacement is facilitated by the flexibility of another nearby peripheral element, the GluN1 and GluN2A S2-M4. We conclude that peripheral structural elements - pre-M1 and S2-M4 - work in concert to remove constraints and prime the channel for rapid opening, thus facilitating fast synaptic transmission.

neuroscience↗