Aysenur Yazici

Inhibition of Transient Receptor Potential Melastatin 3 ion channels by G-protein βγ subunits

Transient receptor potential melastatin 3 (TRPM3) channels are activated by heat, and chemical ligands such as pregnenolone sulphate (PregS) and CIM0216. Here, we show that activation of receptors coupled to heterotrimeric Gi/o proteins inhibits TRPM3 channels. This inhibition was alleviated by co-expression of proteins that bind the βγ subunits of heterotrimeric G-proteins (Gβγ). Co-expression of Gβγ, but not constitutively active Gαi or Gαo, inhibited TRPM3 currents. TRPM3 co-immunoprecipitated with Gβ, and purified Gβγ proteins applied to excised inside-out patches inhibited TRPM3 currents, indicating a direct effect. Baclofen and somatostatin, agonists of Gi-coupled receptors, inhibited Ca2+ signals induced by PregS and CIM0216 in mouse dorsal root ganglion (DRG) neurons. The GABAB receptor agonist baclofen also inhibited inward currents induced by CIM0216 in DRG neurons, and nocifensive responses elicited by this TRPM3 agonist in mice. Our data uncover a novel signaling mechanism regulating TRPM3 channels. DOI: http://dx.doi.org/10.7554/eLife.26147.001

Ion Channel Sensing: Are Fluctuations the Crux of the Matter?

The nonselective cation channel TRPV1 is responsible for transducing noxious stimuli into action potentials propagating through peripheral nerves. It is activated by temperatures greater than 43 °C, while remaining completely nonconductive at temperatures lower than this threshold. The origin of this sharp response, which makes TRPV1 a biological temperature sensor, is not understood. Here we used molecular dynamics simulations and free energy calculations to characterize the molecular determinants of the transition between nonconductive and conductive states. We found that hydration of the pore and thus ion permeation depends critically on the polar character of its molecular surface: in this narrow hydrophobic enclosure, the motion of a polar side-chain is sufficient to stabilize either the dry or wet state. The conformation of this side-chain is in turn coupled to the hydration state of four peripheral cavities, which undergo a dewetting transition at the activation temperature.

TRPV1 activation relies on hydration/dehydration of nonpolar cavities

TRPV1 promotes cationic currents across cellular membranes in response to multiple stimuli such as increased temperature or pressure, binding of chemicals, low pH and voltage. The molecular underpinnings of TRPV1 gating, in particular the mechanism of temperature sensitivity, are still largely unknown. Here, we used molecular simulations and electrophysiology to shed light on the closed to open transition. Specifically, we found that gating of TRPV1 relies on the motion of an evolutionarily conserved amino acid (N676) in the middle of the S6 helix. On rotation, the side chain of this asparagine faces either the central pore or the S4-S5 linker. Only in the former case is the central pore hydrated and thus conductive. Interestingly, when N676 rotates toward the linker, we observe hydration of four so far unreported small nonpolar cavities. Based on these findings, we propose a model for TRPV1 gating involving the dynamic hydration of these four cavities. Free energy calculations indicate that this gating mechanisms is markedly temperature dependent favoring the open state at high temperature. On the basis of this model, which is able to rationalize a wealth of seemingly conflicting and/or unrelated experimental observations, we predicted the behavior of a single residue mutant, F580Y, the consequences of which are confirmed experimentally and give support to the model.

A consistent picture of TRPV1 activation emerges from molecular simulations and experiments

Although experimental structures of both the TRPV1 closed and open states are available, the conformational changes occurring in the pore domain and resulting in ionic conduction remain elusive. Here, we use bioinformatics analyses, molecular dynamics simulations and site-directed mutagenesis to shed light on this issue and suggest a possible molecular mechanism for TRPV1 activation. In light of our hypothesis, we re-examine the results of the previously published water accessibility and mutagenesis experiments, and analyze the newly available structures of TRP and other evolutionary related ion channels. Overall, we show that several independent lines of evidence corroborate our hypothesis, which highlights the rotation of a conserved asparagine toward the pore and the dehydration of hydrophobic cavities as key components of TRPV1 activation. Importantly, this molecular mechanism provides also a rationale for the coupling between the TRPV1 upper and lower gates.

A hypothetical molecular mechanism for TRPV1 activation that invokes rotation of an S6 asparagine

The transient receptor potential channel vanilloid type 1 (TRPV1) is activated by a variety of endogenous and exogenous stimuli and is involved in nociception and body temperature regulation. Although the structure of TRPV1 has been experimentally determined in both the closed and open states, very little is known about its activation mechanism. In particular, the conformational changes that occur in the pore domain and result in ionic conduction have not yet been identified. Here we suggest a hypothetical molecular mechanism for TRPV1 activation, which involves rotation of a conserved asparagine in S6 from a position facing the S4–S5 linker toward the pore. This rotation is associated with hydration of the pore and dehydration of the four peripheral cavities located between each S6 and S4–S5 linker. In light of our hypothesis, we perform bioinformatics analyses of TRP and other evolutionary related ion channels, evaluate newly available structures, and reexamine previously reported water accessibility and mutagenesis experiments. These analyses provide several independent lines of evidence to support our hypothesis. Finally, we show that our proposed molecular mechanism is compatible with the prevailing theory that the selectivity filter acts as a secondary gate in TRPV1.