Biology subjects

Rangel-Yescas, G. E.

Publications and source records attributed to Rangel-Yescas, G. E..

3 recordsLinked to original sources

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↗

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↗