Gisela E. Rangel-Yescas

Coarse architecture of the transient receptor potential vanilloid 1 (TRPV1) ion channel determined by fluorescence resonance energy transfer.

Background: Little is known about the structural characteristics of the multimodal TRPV1 ion channel. Results: FRET measurements show the C terminus surrounded by the N terminus arranged with 4-fold symmetry. The N terminus is further away from the plasma membrane than the C terminus. Conclusion: Domain organization is consistent with a compact structure of the channel. Significance: This work presents novel insights regarding the structure of TRPV1. The transient receptor potential vanilloid 1 ion channel is responsible for the perception of high temperatures and low extracellular pH, and it is also involved in the response to some pungent compounds. Importantly, it is also associated with the perception of pain and noxious stimuli. Here, we attempt to discern the molecular organization and location of the N and C termini of the transient receptor potential vanilloid 1 ion channel by measuring FRET between genetically attached enhanced yellow and cyan fluorescent protein to the N or C terminus of the channel protein, expressed in transfected HEK 293 cells or Xenopus laevis oocytes. The static measurements of the domain organization were mapped into an available cryo-electron microscopy density of the channel with good agreement. These measurements also provide novel insights into the organization of terminal domains and their proximity to the plasma membrane.

Uncoupling Charge Movement from Channel Opening in Voltage-gated Potassium Channels by Ruthenium Complexes

The Kv2.1 channel generates a delayed-rectifier current in neurons and is responsible for modulation of neuronal spike frequency and membrane repolarization in pancreatic β-cells and cardiomyocytes. As with other tetrameric voltage-activated K+-channels, it has been proposed that each of the four Kv2.1 voltage-sensing domains activates independently upon depolarization, leading to a final concerted transition that causes channel opening. The mechanism by which voltage-sensor activation is coupled to the gating of the pore is still not understood. Here we show that the carbon-monoxide releasing molecule 2 (CORM-2) is an allosteric inhibitor of the Kv2.1 channel and that its inhibitory properties derive from the CORM-2 ability to largely reduce the voltage dependence of the opening transition, uncoupling voltage-sensor activation from the concerted opening transition. We additionally demonstrate that CORM-2 modulates Shaker K+-channels in a similar manner. Our data suggest that the mechanism of inhibition by CORM-2 may be common to voltage-activated channels and that this compound should be a useful tool for understanding the mechanisms of electromechanical coupling.

Voltage-dependent gating and gating charge measurements in the Kv1.2 potassium channel

Kv1.2’s gating charge is less than Shaker’s, and the specific contributions of charged S4 residues differ, suggesting that the electric field distribution in the Kv1.2 voltage-sensing domain is different than Shaker’s.

Currents through Hv1 channels deplete protons in their vicinity

The pH-sensitive fluorescent protein Venus can be used as an optical reporter for proton flux when fused to an intracellular domain of Hv1 channels.

The helical character of the S6 segment of TRPV1 channels.

The era of the molecular structure of ion channels has revealed that their transmembrane segments are alpha helices, as was suspected from hydropathy analysis and experimental data. TRP channels are recent additions to the known families of ion channels and little structural data is available. In a recent work, we explored the conformational changes occurring at the putative S6 segment of TRPV1 channels and observed a periodicity of chemical modification of residues suggestive of an alpha helical structure. Further analysis of the periodicity of the disposition of hydrophobic residues in the S6 segment, suggests that the general architecture of the TRPV1 S6 segment, is very similar to that of voltage-dependent channels of known structure—an aqueous cavity lined by an amphipathic alpha helix, with most of the hydrophobic residues pointing into it.

A simple method for fast temperature changes and its application to thermal activation of TRPV1 ion channels

BACKGROUND Thermally activated ion channels function as molecular thermometers and participate in other physiological important functions. The mechanism by which they acquire their exquisite temperature sensitivity is unknown and is currently an area of intense research. For this reason, there is a need for diverse methods to deliver controlled temperature stimuli. NEW METHOD We have developed a simple, inexpensive and reliable method to deliver temperature pulses to small volumes surrounding the recording area, which can be either a patch-clamp pipette containing a cell-free membrane with thermally activated channels or a whole cell attached to a pipette. RESULTS Here we developed a micro-heater based on resistive heating of a copper filament enclosed in a glass capillary that is capable of delivering fast and localized temperature changes. We validated the performance of the micro-heaters by analyzing the heat-induced activation of TRPV1 thermoTRP channels recorded in inside-out patches and demonstrate the use of the micro-heaters. COMPARISON WITH EXISTING METHOD(S) The micro-heaters we introduce here are compact, easy to fabricate and to operate. In contrast with bulk solution heaters commercially available, our method is extremely affordable and simple to operate. To the best of our knowledge there are no other similar, commercially available heating methods. CONCLUSIONS The micro-heater method is simple and should provide a straightforward and rapid experimental tool to study mechanisms in thermally activated ion channels.

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 functional and structural information, 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. We propose that this form of gating in TRPV1 channels is a consequence of the heat absorption process that leads to channel opening.