Biology subjects

Islas, L. D.

Publications and source records attributed to Islas, L. D..

7 recordsLinked to original sources

bioRxiv2023

Cation permeability and pore dynamics in TRPV1 ion channels

The Transient Receptor Vanilloid 1 (TRPV1) is a non-selective ion channel, which is activated by several chemical ligands and heat. We have previously shown that activation of TRPV1 by different ligands result in single-channel openings with different conductance, suggesting that the selectivity filter is highly dynamic. TRPV1 is weakly voltage-dependent, here we sought to explore whether the permeation of different monovalent ions could influence the voltage-dependence of this ion channel. By using single-channel recordings, we show that TRPV1 channels undergo rapid transitions to closed states that are directly connected to the open state, which may result from structural fluctuations of their selectivity filters. Moreover, we demonstrate that the rates of these transitions are strongly influenced by the permeant ion, suggesting that ion permeation regulates the voltage dependence of these channels.

biophysics↗

bioRxiv2022

Activation-pathway transitions in human voltage-gated proton channels revealed by a non-canonical fluorescent amino acid.

Voltage-dependent gating of the voltage-gated proton channels (HV1) remains poorly understood, partly because of the difficulty of obtaining direct measurements of voltage sensor movement in the form of gating currents. To circumvent this problem, we have implemented patch-clamp fluorometry in combination with the incorporation of the fluorescent non-canonical amino acid Anap to monitor channel opening and movement of the S4 segment. Simultaneous recording of currents and fluorescence signals allows for direct correlation of these parameters and investigation of their dependence on voltage and the pH gradient ({Delta}pH). We present data that indicate that Anap incorporated in the S4 helix is quenched by an aromatic residue located in the S2 helix, and that motion of the S4 relative to this quencher is responsible for fluorescence increases upon depolarization. The kinetics of the fluorescence signal reveals the existence of a very slow transition in the deactivation pathway, which seems to be singularly regulated by {Delta}pH. Our experiments also suggest that the voltage sensor can move after channel opening and that the absolute value of the pH can influence the channel opening step. These results shed light on the complexities of voltage-dependent opening of human HV1 channels. Significance statementThe activation mechanisms of voltage-gated proton channels (HV1) are not well understood. Here we have combined patch-clamp fluorometry and a fluorescent non-canonical amino acid to uncover transitions in the activation pathway of human HV1 that are modulated by voltage and the pH gradient.

biophysics↗

bioRxiv2021

Discovery and characterization of Hv1-type proton channels in reef-building corals.

Voltage-dependent proton-permeable channels are membrane proteins mediating a number of important physiological functions. Here we report the presence of a gene encoding for Hv1 voltage-dependent, proton-permeable channels in two species of reef-building corals. We performed a characterization of their biophysical properties and found that these channels are fast-activating and modulated by the pH gradient in a distinct manner. The biophysical properties of these novel channels make them interesting model systems. We have also developed an allosteric gating model that provides mechanistic insight into the modulation of voltage-dependence by protons. This work also represents the first functional characterization of any ion channel in scleractinian corals. We discuss the implications of the presence of these channels in the membranes of coral cells in the calcification and pH regulation processes and possible consequences of ocean acidification related to the function of these channels.

biophysics↗

bioRxiv2019

KV1.2 channels inactivate through a mechanism similar to C-type inactivation

C-type inactivation has been described in multiple voltage-gated K+ channels and in great detail in the Drosophila Shaker channel. As channels have moved into the structural era, atomic details of this and other gating mechanisms have started to be better understood. To date, the only voltage-gated channels whose structure has been solved are KvAP (X-ray diffraction), the KV1.2- KV2.1 \"paddle\" chimera (X-ray diffraction), KV1.2 (Cryo-EM); and ether-a-go-go (Cryo-EM) (Wang and MacKinnon, 2017), however, the characteristics and mechanisms of slow inactivation in these channels are unknown or poorly characterized. Here we present a detailed study of slow inactivation in the rat KV1.2 and show that it has some properties consistent with the C-type inactivation described in Shaker. We also study the effects of some mutations that are known to modulate C-type inactivation in Shaker and show that qualitative and quantitative differences exist in their functional effects, possibly underscoring subtle but important structural differences between the C-inactivated states in Shaker and KV1.2.

biophysics↗

bioRxiv2019

The contribution of the ankyrin repeat domain of TRPV1 as a thermal module

The TRPV1 cation non-selective ion channel plays an essential role in thermosensation and perception of other noxious stimuli. TRPV1 can be activated by low pH, high temperature or naturally occurring pungent molecules such as allicin, capsaicin or resiniferatoxin. Its noxious thermal sensitivity makes it an important participant as a thermal sensor in mammals. However, details of the mechanism of channel activation by increases in temperature remain unclear. Here we used a combination of approaches to try to understand the role of the ankyrin repeat domain (ARD) in channel behavior. First, a computational modeling approach by coarse-grained molecular dynamics simulation of the whole TRPV1 embedded in a phosphatidylcholine (POPC) and phosphatidylethanolamine (POPE) membrane provides insight into the dynamics of this channel domain. Global analysis of the structural ensemble shows that the ankyrin repeat domain is a region that sustains high fluctuations during dynamics at different temperatures. We then performed biochemical and thermal stability studies of the purified ARD by means of circular dichroism and tryptophan fluorescence and demonstrate that this region undergoes structural changes at similar temperatures that lead to TRPV1 activation. Our data suggest that the ARD is a dynamic module and that it may participate in controlling the temperature sensitivity of TRPV1.\n\nStatement of SignificanceThis work demonstrates that the temperature-dependent dynamics of the ankyrin repeat domain (ARD) of TRPV1 channels, as probed by coarse-grained molecular dynamics, corresponds to the experimentally determined dynamics of an isolated ARD domain. These results show that this region of TRPV1 channels undergoes significant conformational change as a function of increased temperature and suggest that it participates in the temperature-dependent structural changes that lead to channel opening.

biophysics↗

bioRxiv2018

Irreversible temperature gating in trpv1 sheds light on channel activation

Temperature activated TRP channels or thermoTRPs are among the only proteins that can directly convert temperature changes into changes in channel open probability. In spite of a wealth of information, including several experimentally determined structural models of TRP channels, the mechanism of temperature activation remains unknown. We have carefully characterized the repeated activation of TRPV1 by thermal stimuli and discovered a previously unknown inactivation process, which is irreversible. This inactivation is associated with specific conformational changes in the membrane proximal domain. We propose that this form of gating in TRPV1 channels is a consequence of the heat absorption process that leads to channel opening.

biophysics↗

bioRxiv2017

Conformational Dynamics In TRPV1 Channels Reported By An Encoded Coumarin Amino Acid

Transient Receptor Potential Vanilloid (TRPV1) channels support the detection and integration of nociceptive input. Currently available functional and structural data suggest that that TRPV1 channels have two potential gates within their cation selective permeation pathway: a barrier formed by a bundle crossing at the intracellular entrance and a second constriction created by the ion selectivity filter. To describe conformational changes associated with channel gating within the pore, the fluorescent non-canonical amino acid (f- ncAA) coumarin-tyrosine was genetically encoded at Y671, a residue proximal to the selectivity filter. TRPV1 channels expressing coumarin at either site displayed normal voltage- and agonist-dependent gating. Next, total internal reflection microscopy (TIRF) was performed to enable ultra-rapid, millisecond imaging of the conformational dynamics in single TRPV1 channels in live cells. Here, the data obtained from channels expressed in human derived cells show that optical fluctuations, photon counts, and variance of noise analysis from Y671 coumarin encoded in TRPV1 tetramers correlates closely with channel activation by capsaicin, thus providing an direct optical marker of channel activation at the selectivity filter. In companion molecular dynamics simulations, Y671 displays alternating solvent exposure between the closed and open states, giving support to the optical data. These calculations further suggest a direct involvement of Y671 in controlling the relative position of the pore helix and its role in supporting ionic conductance at the TRPV1 selectivity filter.

biophysics↗