This work provides a potential therapeutic mechanism for the development of antiarrhythmic compounds that inhibit leaky RyR2 resulting from CaM dissociation, which is often associated with failing hearts. Our data also suggest that CaM dissociation may contribute to the pathogenesis of arrhythmias with the CPVT-linked R176Q mutation.

Acid-sensing ion channels (ASICs) are sensitized to activation by inflammatory mediators such as the polyunsaturated fatty acid (PUFA) arachidonic acid (AA). Previous work has shown that AA can potentiate ASIC currents at subsaturating proton concentrations, but the structural mechanisms of this change in gating are not understood. Here we show that PUFAs cause multiple gating changes in ASIC3, including shifting the pH dependence of activation, slowing the rate of desensitization, and increasing the current even at a saturating pH. The impact on gating depends on the nature of both the head and tail of the lipid, with the head group structure primarily determining the magnitude of the effect on the channel. An N-acyl amino acid (NAAA), arachidonyl glycine (AG), is such a strong regulator that it can act as a ligand at neutral pH. Mutation of an arginine in the outer segment of TM1 (R64) eliminated the effect of docosahexaenoic acid (DHA) even at high concentrations, suggesting a potential interaction site for the lipid on the channel. Our results suggest a model in which PUFAs bind to ASICs via both their tail group and an electrostatic interaction between the negatively charged PUFA head group and the positively charged arginine side chain. These data provide the first look at the structural features of lipids that are important for modulating ASICs and suggest a potential binding site for PUFAs on the channel.

Rationale Catecholaminergic polymorphic ventricular tachycardia (CPVT) is a potentially lethal arrhythmic disorder caused by mutations in the type-2 ryanodine receptor (RyR2). Mutant RyR2 cause abnormal Ca2+ leak from the sarcoplasmic reticulum (SR), which is associated with the development of arrhythmias. Objective To determine whether derivatives of tetracaine, a local anesthetic drug with known RyR2 inhibiting action, could prevent CPVT induction by suppression of RyR2-mediated SR Ca2+ leak. Methods and results Confocal microscopy was used to assess the effects of tetracaine and 9 derivatives (EL1–EL9) on spontaneous Ca2+ sparks in ventricular myocytes isolated from RyR2-R176Q/+ mice with CPVT. Whereas each derivative suppressed the Ca2+ spark frequency, derivative EL9 was most effective at the screening dose of 500 nmol/L. At this high dose, the Ca2+ transient amplitude was not affected in myocytes from WT or R176Q/+ mice. The IC50 of EL9 was determined to be 13 nmol/L, which is about 400x time lower than known RyR2 stabilizer K201. EL9 prevented the induction of ventricular tachycardia observed in placebo-treated R176Q/+ mice, without affecting heart rate or cardiac contractility. Conclusions Tetracaine derivatives represent a novel class of RyR2 stabilizing drugs that could be used for the treatment of the potentially fatal disorder catecholaminergic polymorphic ventricular tachycardia.

Stomatin (STOM) is a monotopic integral membrane protein found in all classes of life that has been shown to regulate members of the acid-sensing ion channel (ASIC) family. However, the mechanism by which STOM alters ASIC function is not known. Using chimeric channels, we combined patch-clamp electrophysiology and FRET to search for regions of ASIC3 critical for binding to and regulation by STOM. With this approach, we found that regulation requires two distinct sites on ASIC3: the distal C-terminus and the first transmembrane domain (TM1). The C-terminal site is critical for formation of the STOM–ASIC3 complex, while TM1 is required only for the regulatory effect. We then looked at the mechanism of STOM-dependent regulation of ASIC3 and found that STOM does not alter surface expression of ASIC3 or shift the pH dependence of channel activation. However, a point mutation (Q269G) that prevents channel desensitization also prevents STOM regulation, suggesting that STOM may alter ASIC3 currents by stabilizing the desensitized state of the channel. Based on these findings, we propose a model whereby STOM is anchored to the channel via a site on the distal C-terminus and stabilizes the desensitized state of the channel via an interaction with TM1.

Carborane-capped gold nanoparticles (Au/carborane NPs, 2–3 nm) can act as artificial ion transporters across biological membranes. The particles themselves are large hydrophobic anions that have the ability to disperse in aqueous media and to partition over both sides of a phospholipid bilayer membrane. Their presence therefore causes a membrane potential that is determined by the relative concentrations of particles on each side of the membrane according to the Nernst equation. The particles tend to adsorb to both sides of the membrane and can flip across if changes in membrane potential require their repartitioning. Such changes can be made either with a potentiostat in an electrochemical cell or by competition with another partitioning ion, for example, potassium in the presence of its specific transporter valinomycin. Carborane-capped gold nanoparticles have a ligand shell full of voids, which stem from the packing of near spherical ligands on a near spherical metal core. These voids are normally filled with sodium or potassium ions, and the charge is overcompensated by excess electrons in the metal core. The anionic particles are therefore able to take up and release a certain payload of cations and to adjust their net charge accordingly. It is demonstrated by potential-dependent fluorescence spectroscopy that polarized phospholipid membranes of vesicles can be depolarized by ion transport mediated by the particles. It is also shown that the particles act as alkali-ion-specific transporters across free-standing membranes under potentiostatic control. Magnesium ions are not transported.