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 (ΔpH). 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 reveal the existence of a very slow transition in the deactivation pathway, which seems to be singularly regulated by ΔpH. 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.

Unconventional interactions of the TRPV4 ion channel with beta-adrenergic receptor ligands.

The transient receptor potential vanilloid 4 (TRPV4) ion channel is present in different tissues including those of the airways. This channel is activated in response to stimuli such as changes in temperature, hypoosmotic conditions, mechanical stress, and chemicals from plants, lipids, and others. TRPV4's overactivity and/or dysfunction has been associated with several diseases, such as skeletal dysplasias, neuromuscular disorders, and lung pathologies such as asthma and cardiogenic lung edema and COVID-19-related respiratory malfunction. TRPV4 antagonists and blockers have been described; nonetheless, the mechanisms involved in achieving inhibition of the channel remain scarce, and the search for safe use of these molecules in humans continues. Here, we show that the widely used bronchodilator salbutamol and other ligands of ß-adrenergic receptors inhibit TRPV4's activation. We also demonstrate that inhibition of TRPV4 by salbutamol is achieved through interaction with two residues located in the outer region of the pore and that salbutamol leads to channel closing, consistent with an allosteric mechanism. Our study provides molecular insights into the mechanisms that regulate the activity of this physiopathologically important ion channel.

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 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.

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

Slow inactivation has been described in multiple voltage-gated K+ channels and in great detail in the Drosophila Shaker channel. Structural studies have begun to facilitate a better understanding of the atomic details of this and other gating mechanisms. To date, the only voltage-gated potassium channels whose structure has been solved are KvAP (x-ray diffraction), the KV1.2-KV2.1 "paddle" chimera (x-ray diffraction and cryo-EM), KV1.2 (x-ray diffraction), and ether-à-go-go (cryo-EM); however, the structural details 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 channel 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.

The Contribution of the Ankyrin Repeat Domain of TRPV1 as a Thermal Module.

The TRPV1 cation nonselective ion channel plays an essential role in thermosensation and perception of other noxious stimuli. TRPV1 can be activated by low extracellular 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 and phosphatidylethanolamine membrane provides insight into the dynamics of this channel domain. Global analysis of the structural ensemble shows that the ARD 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 the 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.

FRET-based analysis and molecular modeling of the human GPN-loop GTPases 1 and 3 heterodimer unveils a dominant-negative protein complex.

GPN-loop GTPases 1 and 3 are required for RNA polymerase II (RNAPII) nuclear import. Gpn1 and Gpn3 display some sequence similarity, physically associate, and their protein expression levels are mutually dependent in human cells. We performed here Fluorescence Resonance Energy Transfer (FRET), molecular modeling, and cell biology experiments to understand, and eventually disrupt, human Gpn1-Gpn3 interaction in live HEK293-AD cells. Transiently expressed EYFP-Gpn1 and Gpn3-CFP generated a strong FRET signal, indicative of a very close proximity, in the cytoplasm of HEK293-AD cells. Molecular modeling of the human Gpn1-Gpn3 heterodimer based on the crystallographic structure of Npa3, the Saccharomyces cerevisiae Gpn1 ortholog, revealed that human Gpn1 and Gpn3 associate through a large interaction surface formed by internal α-helix 7, insertion 2, and the GPN-loop from each protein. In site-directed mutagenesis experiments of interface residues, we identified the W132D and M227D EYFP-Gpn1 mutants as defective to produce a FRET signal when coexpressed with Gpn3-CFP. Simultaneous but not individual expression of Gpn1 and Gpn3, with either or both proteins fused to EYFP, retained RNAPII in the cytoplasm and markedly inhibited global transcription in HEK293-AD cells. Interestingly, the W132D and M227D Gpn1 mutants that showed an impaired ability to interact with Gpn3 by FRET were also unable to delocalize RNAPII in this assay, indicating that an intact Gpn1-Gpn3 interaction is required to display the dominant-negative effect on endogenous Gpn1/Gpn3 function we described here. Altogether, our results suggest that a Gpn1-Gpn3 strong interaction is critical for their cellular function.

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.

Currents through Hv1 channels deplete protons in their vicinity.

Proton channels have evolved to provide a pH regulatory mechanism, affording the extrusion of protons from the cytoplasm at all membrane potentials. Previous evidence has suggested that channel-mediated acid extrusion could significantly change the local concentration of protons in the vicinity of the channel. In this work, we directly measure the proton depletion caused by activation of Hv1 proton channels using patch-clamp fluorometry recordings from channels labeled with the Venus fluorescent protein at intracellular domains. The fluorescence of the Venus protein is very sensitive to pH, thus behaving as a genetically encoded sensor of local pH. Eliciting outward proton currents increases the fluorescence intensity of Venus. This dequenching is related to the magnitude of the current and not to channel gating and is dependent on the pH gradient. Our results provide direct evidence of local proton depletion caused by flux through the proton-selective channel.

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

Much has been learned about the voltage sensors of ion channels since the x-ray structure of the mammalian voltage-gated potassium channel Kv1.2 was published in 2005. High resolution structural data of a Kv channel enabled the structural interpretation of numerous electrophysiological findings collected in various ion channels, most notably Shaker, and permitted the development of meticulous computational simulations of the activation mechanism. The fundamental premise for the structural interpretation of functional measurements from Shaker is that this channel and Kv1.2 have the same characteristics, such that correlation of data from both channels would be a trivial task. We tested these assumptions by measuring Kv1.2 voltage-dependent gating and charge per channel. We found that the Kv1.2 gating charge is near 10 elementary charges (eo), ∼25% less than the well-established 13-14 eo in Shaker. Next, we neutralized positive residues in the Kv1.2 S4 transmembrane segment to investigate the cause of the reduction of the gating charge and found that, whereas replacing R1 with glutamine decreased voltage sensitivity to ∼50% of the wild-type channel value, mutation of the subsequent arginines had a much smaller effect. These data are in marked contrast to the effects of charge neutralization in Shaker, where removal of the first four basic residues reduces the gating charge by roughly the same amount. In light of these differences, we propose that the voltage-sensing domains (VSDs) of Kv1.2 and Shaker might undergo the same physical movement, but the septum that separates the aqueous crevices in the VSD of Kv1.2 might be thicker than Shaker's, accounting for the smaller Kv1.2 gating charge.

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.

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

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.

Cloning and functional analysis of P2X1b, a new variant in rat optic nerve that regulates the P2X1 receptor in a use-dependent manner.

P2X receptors are trimeric, ATP-gated cation channels. In mammals seven P2X subtypes have been reported (P2X1-P2X7), as well as several variants generated by alternative splicing. Variants confer to the homomeric or heteromeric channels distinct functional and/or pharmacological properties. Molecular biology, biochemical, and functional analysis by electrophysiological methods were used to identify and study a new variant of the P2X1 receptor named P2X1b. This new variant, identified in rat optic nerve, was also expressed in other tissues. P2X1b receptors lack amino acids 182 to 208 of native P2X1, a region that includes residues that are highly conserved among distinct P2X receptors. When expressed in Xenopus oocytes, P2X1b was not functional as a homomer; however, when co-expressed with P2X1, it downregulated the electrical response generated by ATP compared with that of oocytes expressing P2X1 alone, and it seemed to form heteromeric channels with a modestly enhanced ATP potency. A decrease in responses to ATP in oocytes co-expressing different ratios of P2X1b to P2X1 was completely eliminated by overnight pretreatment with apyrase. Thus, it is suggested that P2X1b regulates, through a use-dependent mechanism, the availability, in the plasma membrane, of receptor channels that can be operated by ATP.

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 ß-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.

The helical character of the S6 segment of TRPV1 channels.

The era of 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 we 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.