Probing the Structural Dynamics of the Activation Gate of KcsA Using Homo-FRET Measurements

Díaz-García, Clara; Renart, M. Lourdes; Poveda, José A.; Giudici, Ana Marcela; González-Ros, José M.; Prieto, Manuel; Coutinho, Ana

doi:10.3390/ijms222111954

Cited by 8 publications

(4 citation statements)

References 79 publications

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“…To label the proteins with dye, a 1:1 mixture of sulfo-Cy3 maleimide (Lumiprobe) and sulfo-Cy5 maleimide (Lumiprobe) was added to the proteins at a protein:fluorophore molar ratio of 1:10. The labeling reaction was performed at 4 °C for 2 h. The labeled proteins were then diluted and added to anti-FLAG resin, and the excess free dyes were effectively removed by extensive washing 29 . The proteins were eluted by elution buffer and concentrated to 0.3–0.4 mg ml −1 (Supplementary Fig.…”

Section: Methodsmentioning

confidence: 99%

FRET analysis of the temperature-induced structural changes in human TRPV3

Kim

Won

Chung

et al. 2023

Sci Rep

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Transient receptor potential vanilloid member 3 (TRPV3) is an ion channel that plays a critical role in temperature sensing in skin. There have been active studies on how TRPV3, which is also known as one of the temperature-sensitive transient receptor potential (thermoTRP) channels, responds to temperature. However, the previous studies were mostly based on TRPV3 originating from mice or rats. Here, we focus on human TRPV3 (hTRPV3) and show that which domain of hTRPV3 undergoes conformational changes as temperature increases by Förster resonance energy transfer (FRET) assay. During the heat-induced activation of hTRPV3, the linker domain close to C-terminus, that is, the C-terminal domain shows a largest structural change whereas there is little change in the ankyrin repeat domain (ARD). Interestingly, the activation of hTRPV3 by an agonist shows structural change patterns that are completely different from those observed during activation by heat; we observe structural changes in ARD and S2–S3 linker after ligand stimulation whereas relatively little change is observed when stimulated by heat. Our results provide insight into the thermal activation of hTRPV3 channel.

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Section: Methodsmentioning

confidence: 99%

FRET analysis of the temperature-induced structural changes in human TRPV3

Kim

Won

Chung

et al. 2023

Sci Rep

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“…Previous studies in the KcsA channel have shown that the fluorescence emitted by W67, located at the pore helix, nicely reports on cation occupancy-induced conformational changes in the nearby SF [ 25 , 26 , 37 ]. Furthermore, the occurrence of a homo-FRET process among the W67 residues present in the four KcsA channel subunits allowed for the calculation of intersubunit distances under different ionic conditions through the analysis of both steady-state and time-resolved fluorescence anisotropy [ 25 ].…”

Section: Resultsmentioning

confidence: 82%

Molecular Events behind the Selectivity and Inactivation Properties of Model NaK-Derived Ion Channels

Giudici

Renart

Coutinho

et al. 2022

IJMS

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Y55W mutants of non-selective NaK and partly K+-selective NaK2K channels have been used to explore the conformational dynamics at the pore region of these channels as they interact with either Na+ or K+. A major conclusion is that these channels exhibit a remarkable pore conformational flexibility. Homo-FRET measurements reveal a large change in W55–W55 intersubunit distances, enabling the selectivity filter (SF) to admit different species, thus, favoring poor or no selectivity. Depending on the cation, these channels exhibit wide-open conformations of the SF in Na+, or tight induced-fit conformations in K+, most favored in the four binding sites containing NaK2K channels. Such conformational flexibility seems to arise from an altered pattern of restricting interactions between the SF and the protein scaffold behind it. Additionally, binding experiments provide clues to explain such poor selectivity. Compared to the K+-selective KcsA channel, these channels lack a high affinity K+ binding component and do not collapse in Na+. Thus, they cannot properly select K+ over competing cations, nor reject Na+ by collapsing, as K+-selective channels do. Finally, these channels do not show C-type inactivation, likely because their submillimolar K+ binding affinities prevent an efficient K+ loss from their SF, thus favoring permanently open channel states.

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“…KcsA is among the best studied channels by X-ray crystallography, yet among more than 100 structures deposited to the Protein Data Bank, there is not a single structure of the open wild-type protein. Other methods, such as EPR or SANS appear to detect structural changes at low pH [63][64][65] . As none of the deposited structures are solved below pH of 4.0, the pH at which the HBC in KcsA was suggested to open, it might be that the open conformation of KcsA displays substantial heterogeneity which precludes crystallization.…”

Section: Discussionmentioning

confidence: 99%

Reassessing the helix bundle crossing model for gating in a non-selective ion channel

Kurauskas

Tonelli

Henzler‐Wildman

2022

Preprint

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A critical part of ion channel function is the ability to open and close in response to stimuli, and thus conduct ions in a regulated fashion. While X-ray diffraction studies of ion channels suggested a general steric gating mechanism located at the helix bundle crossing (HBC), recent functional studies on several channels indicate that the helix bundle crossing is open even in closed, non-conductive channels. Two NaK channel variants were crystallized in very different, open and closed conformations and served as an important model of the HBC gating hypothesis. However, neither of these NaK variants are conductive in liposomes unless phenylalanine 92 is mutated to alanine (F92A). Here we use NMR to probe distances at near-atomic resolution of the two NaK variants in lipid bicelles. We demonstrate that in contrast to the crystal structures, both NaK variants are in a fully open conformation, akin to the well known MthK channel structure were the HBC is widely open. Further inquiry into the gating mechanism suggests that the selectivity filter and pore helix are coupled to the M2 helix below and undergo changes in structure when F92 is mutated. Overall, our data shows that NaK exhibits coupling between the selectivity filter and HBC similar to K+ channels and has a more complex gating mechanism than previously thought.

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Probing the Structural Dynamics of the Activation Gate of KcsA Using Homo-FRET Measurements

Cited by 8 publications

References 79 publications

FRET analysis of the temperature-induced structural changes in human TRPV3

FRET analysis of the temperature-induced structural changes in human TRPV3

Molecular Events behind the Selectivity and Inactivation Properties of Model NaK-Derived Ion Channels

Reassessing the helix bundle crossing model for gating in a non-selective ion channel

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