Single-molecule FRET studies of ion channels

Accepted Manuscript Single-molecule FRET studies of ion channels Boris Martinac PII:

S0079-6107(17)30062-7

DOI:

10.1016/j.pbiomolbio.2017.06.014

Reference:

JPBM 1225

To appear in:

Progress in Biophysics and Molecular Biology

Received Date: 14 March 2017 Revised Date:

19 June 2017

Accepted Date: 21 June 2017

Please cite this article as: Martinac, B., Single-molecule FRET studies of ion channels, Progress in Biophysics and Molecular Biology (2017), doi: 10.1016/j.pbiomolbio.2017.06.014. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

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Boris Martinac*

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Single-molecule FRET studies of ion channels

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Victor Chang Cardiac Research Institute, Lowy Packer Building, Darlinghurst, NSW 2010, Australia and St Vincent’s Clinical School, University of New South Wales, Darlinghurst, NSW 2010, Australia

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E-mail: [email protected]

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Running title: smFRET and ion channels

Key words:

Patch clamp, FRAP, Gramicidin, MscL, KirBac, NMDA

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Abstract Different types of fluorescence spectroscopy approaches have over the last two decades become important techniques in studies of ion channel structure and dynamics. Many fluorescence

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methods have been used to examine a huge variety of ion channels. Any fluorescence study of ion channels requires the presence of fluorophores, which may be intrinsic to the channel protein, attached either extrinsically to the protein, or be simply located nearby the channel to monitor

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local conditions such as for many of the ion-sensitive dyes. Many ion channel studies utilize protein-bound or intrinsic protein fluorophores. Single-molecule Förster resonance energy

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transfer (smFRET) spectroscopy has been particularly useful in gaining detailed structural information for multimeric membrane proteins including ion channels. This technique presents a major advancement in studies of structural dynamics of these membrane proteins. Although it has required different approaches to protein labelling, control of the protein state, as well as

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careful analysis of the orientations, geometries, and number of fluorescent probes, the smFRET

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methodology provides an excellent tool for studying the structure of ion channels.

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Introduction Förster resonance energy transfer (FRET) spectroscopy is a powerful tool for structural analysis of proteins (Geddes 2006). Over the last two decades it has become one of the frequently used

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techniques for studying the conformational changes of membrane proteins, including ion channels. Although structures of ion channels obtained by X-ray crystallography provide invaluable information of their 3D architecture they only present snapshots of distinct structural

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states of detergent solubilized channel proteins without providing information about time trajectories between these states. Even the very recent developments in the field of cryo-

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electronmicroscopy (cryoEM), which have resulted in cryoEM superseding crystallography as the technique of choice for determining specific states for ion channels near atomic resolution (Hite and MacKinnon 2017), cannot provide dynamic information on changes in protein conformations. In contrast, spectroscopy techniques, such as FRET, provide information about

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structural dynamics of intramolecular and intermolecular motions underlying ion channel function within the lipid bilayer, the natural environment of membrane proteins. Single molecule FRET (smFRET) is a variant of FRET enabling measurements of time-dependent processes

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associated with protein conformational changes on a single pair of fluorophores attached to the specific sites within the protein structure. Since the first report on smFRET measurements of

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emission spectra from donor and acceptor fluorophores linked by a short DNA molecule (Ha, Enderle et al. 1996) this new approach has rapidly gained in popularity in studies of a large array of biological phenomena ranging from DNA and RNA molecular dynamics to protein folding and conformational changes (Roy, Hohng et al. 2008).

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In this review, I will briefly revisit the principles of smFRET as well as its application for characterization of several types of ion channels including potassium, mechanosensitive and ligand-gated channels. Interested readers can find detailed information on FRET microscopy and

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spectroscopy in the references listed below (Lakowicz 2006 ; Kramer 2011; Geddes 2006).

Basic principle of FRET spectroscopy

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Fluorescence resonance energy transfer (FRET) has been widely used for biological studies of

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molecular interactions at the level of single cells, cell organelles, and single molecules. It is

Figure 1. Basic principle of FRET spectroscopy. A donor fluorophore (D) is excited and transfers this energy in a distance- and orientation-dependent manner (r/R0) to an acceptor molecule (A). The energy transfer between an excited donor to an acceptor occurs through nonradiative dipole-dipole coupling (E). This mechanism is called "Förster resonance energy transfer". If the acceptor is itself a fluorescent molecule it will become excited, which can emit its own photon, albeit of a lesser energy corresponding to a longer wavelength. The spectral overlap integral J( λ) can be calculated using the expression: J(λ) = ∫fD(λ) εA(λ) λ4 dλ, where fD(λ) is the normalized donor emission spectrum, and εA(λ) is the acceptor molar extinction coefficient, and λ is the wavelength (Martinac 2012 ). 4

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today a standard tool for investigating inter- and intramolecular distances in the 1- to 8-nm (10to 80-Å) range (Selvin 1995; Förster 1965). FRET involves the transfer of energy from a

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fluorescent donor molecule to an acceptor molecule quenching the fluorescence intensity of the donor. As chromophores, donor molecules can be excited by light or chemical stimulation by transiting into an excited state, whose energy can be transferred to a neighboring acceptor

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provided that conditions favour resonance transfer. Worth mentioning is another closely related spectroscopic technique known as BRET (Bioluminescence Resonance Energy Transfer). This

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variant of FRET is predominantly used for studies of protein-protein interactions (Pfleger 2006; Martinac 2012 ). The transfer of energy through FRET is a radiation-less process because the energy between a donor and an acceptor is transferred through dipole-dipole coupling (Fig. 1). It is not transferred by an emitted photon. The fraction of energy transferred per donor excitation event is termed FRET efficiency of energy transfer (E), which depends on the donor-acceptor

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separation distance r (typically ≤10 nm) with an inverse 6th power relation: E = 1/[1+ (r/R0)6]

(1)

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where R0 is the Förster distance of a donor/acceptor pair corresponding to the distance at which FRET efficiency is 50%. R0 depends on the overlap integral J(λ) (Fig. 1) of the donor emission

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spectrum with the acceptor absorption spectrum and their mutual molecular orientation known as the dipole orientation factor κ2: R06 =

9Q0 ln(10) κ2 J(λ) (2) 5

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128 π n NA

where Q0 is the fluorescence quantum yield in the absence of the acceptor, n is the refractive index of the medium in which fluorescence is measured and NA is Avogadro’s number. The

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dipole orientation factor κ2 is usually assumed to be 2/3 because this value applies to a freely rotating pair of dyes isotropically oriented during the lifetime of the excited state. Once a value of the FRET efficiency E is obtained, the distance between the donor and acceptor, can be

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calculated from Eq (1) as:

Figure 2. Change in channel radius determined from FRET. FRET efficiency for an ensemble of pentameric MscL channels determined in closed and open channel conformations (top) modified after (Corry, Rigby et al. 2005). The FRET efficiency curve, which relates a pentamer size to transfer efficiency in two conformations (bottom), can be determined using a Monte Carlo scheme.

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r = [(1/E) -1]1/6 R0

(3)

FRET is most often used as a tool for measuring changes in intra- and inter-molecular distances such as during opening of an ion channel (Corry, Rigby et al. 2005; Corry, Hurst et al. 2010),

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which is of particular interest here. Because of the difficulties in establishing the Förster distance R0 (Förster distance of the donor-acceptor pair corresponds to the distance at which the energy transfer efficiency is 50%), and accurately measuring the FRET is often measured on an

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ensemble of multimeric protein molecules, such as ion channels of oligomeric structure. In multimeric proteins FRET measurement is more complicated because each protein contains a

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random mix of combinations of donor and acceptor fluorophores depending on the multimeric structure of the particular protein. In such measurements one cannot directly determine FRET efficiency but has to determine it using probability distributions based on random sampling, such

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as Monte Carlo methods (Fig. 2).

Single molecule FRET (smFRET)

Extending the usage of FRET to the single molecule level (Weiss 2000; Roy, Hohng et al. 2008)

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presented an important advancement for studies of membrane proteins, including ion channels, because for single donor–acceptor pairs the fluorophore separation can easily be determined

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from transfer efficiency. Another advantage of smFRET for studies of ion channels compared to the ensemble FRET consists in detecting structural dynamics within a single channel molecule rather than detecting interactions between the ion channels in an ensemble. This is important because smFRET provides information on unsynchronized structural changes in a protein, which can be concealed by ensemble averaging of structurally heterogeneous subpopulations (Ha, Enderle et al. 1996; Roy, Hohng et al. 2008). However, only a small number of membrane

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proteins have thus far been studied by smFRET (Zhao, Terry et al. 2010; Akyuz, Altman et al. 2013; Erkens, Hanelt et al. 2013; Wang, Liu et al. 2014; Vafabakhsh, Levitz et al. 2015) given the technical difficulties associated with fluorophore labeling in multimeric proteins.

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Nevertheless, smFRET has been used to detect conformational changes associated with gating in several ion channel types. The examples discussed here should illustrate the power of smFRET spectroscopy for studies of ion channel structural dynamics that would be difficult to study in

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Gramicidin

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such a detail using other methods.

Gramicidin A (gA) is one of the three antibiotic compounds isolated from the soil bacterium Bacillus brevis. It is a simple hydrophobic peptide that forms cation-selective channels by

forming dimers in lipid bilayers through association of one monomer from each monolayer

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(Urry, Goodall et al. 1971; Koeppe R.E. 1996). gA, which has for many years served as a model channel in studies of the effects of protein inclusions on acyl chain order and dynamics in lipid bilayers (Killian 1992; Herold, Sanford et al. 2017), was the first ion channel examined In this study, fluorescence

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structurally using smFRET (Borisenko, Lougheed et al. 2003).

imaging and electrical recording of single ion channels in planar bilayers were simultaneously

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performed on two different fluorescently labeled derivatives of the gA peptide. One peptide that forms channels of low conductance was labeled with a donor Cy3 dye, whereas the other that forms channels of high conductance was labelled with an acceptor Cy5 dye. The currents of the two types of the gA homodimer, i.e. Cy3/Cy3 and Cy5/Cy5, and a heterodimer Cy3/Cy5 could be electrically recorded. The current recordings revealed that in addition to channels of low and high conductance corresponding to Cy3/Cy3 and Cy5/Cy5 homodimers, respectively, a channel

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of an intermediate conductance corresponding to the Cy3/Cy5 heterodimer, was also present in the planar bilayer. The gA example clearly shows that the combined smFRET and electrophysiological recording present a powerful approach for studies of gating mechanisms in a

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wide variety of ion channels, as demonstrated by the examples of the bacterial mechanosensitive channel MscL and potassium KirBac channels as well as the mammalian ligand-gated NMDA

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receptor channel briefly discussed in the next sections.

MscL

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MscL, the bacterial mechanosensitive (MS) channel of large conductance, was the first MS channel identified at the molecular level by following its channel activity in liposomereconstituted chromatographically separated protein fractions by the patch clamp technique (Sukharev, Blount et al. 1994; Sukharev 1997 ). Determination of the 3D crystal structure from

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MscL of Mycobacterium tuberculosis (Chang, Spencer et al. 1998; Steinbacher 2007) presented a major advancement for the research on MS channels by facilitating studies of the structural dynamics of this channel protein by spectroscopic techniques combined with computational

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modelling (Perozo, Cortes et al. 2002; Corry, Hurst et al. 2010; Wang, Liu et al. 2014). Since then MscL has served as a standard molecule for studies of the basic biophysical principles of

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cellular mechanotransduction processes mediated by MS channels (Martinac 2011; Cox, Bae et al. 2016).

Prior to applying smFRET to studies of MscL ensemble FRET spectroscopy was used to examine MscL transition from the closed to the open conformation by analyzing the intensity of light emitted by Alexa-Fluor-labeled cysteine mutants of MscL reconstituted into liposomes

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(Corry, Rigby et al. 2005; Corry, Hurst et al. 2010). Using fluorescence recovery after photobleaching (FRAP), the FRET acceptor photobleaching method, allowed for determination of the change in diameter of the MscL protein of ∼16 Å (Fig. 2) (Corry, Rigby et al. 2005) and

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diameter >25Å of the channel pore upon channel opening (Corry, Hurst et al. 2010). Both results

Figure 3. Scheme of single molecule FRET setup. (Top) Labeled MscL proteins were reconstituted into liposomes, which were then immobilized on a coverslip and used for smFRET experiments. The addition of LPC traps the protein in the open conformation (Perozo et al., 2002). (Bottom) Examples of fluorescent intensity traces showing (a) a single photobleaching step, (b) multiple photobleaching steps, and (c) a single photobleaching step but the acceptor photobleached first. Only the traces showing a single photobleaching step in both the donor and acceptor channels were used to ensure that only a single donor and/or acceptor fluorophore were included in the analysis of the smFRET results. (Reproduced and adapted from (Wang, Liu et al. 2014) and (Bavi, Cortes et al. 2016)).

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are in a close agreement with the open channel structure previously determined by EPR spectroscopy (Perozo, Cortes et al. 2002), which could not be achieved by X-ray crystallography. Nevertheless, due to multiple labeling in the pentameric structure of the channel, potential

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problems with MscL protein clustering (Grage, Keleshian et al. 2011) and the need for MonteCarlo simulations to determine the distance changes during MscL opening (Corry, Rigby et al. 2005; Corry, Hurst et al. 2010), ensemble FRET left the question about variability and

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uncertainty in those results unanswered. smFRET was consequently used to explore the structure of the open MscL channel from E. coli in order to determine its open structure without

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ambiguity.

In the smFRET technique an individual channel molecule is labeled with only one donor and one acceptor fluorescent molecule (Fig. 3). By illuminating the channel with light of a wavelength

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that excites the donor molecule, and measuring fluorescence efficiency from the acceptor molecule (Fig. 1), one can simply calculate the distance between the two molecules using Eq. (3). From this, by labeling different residues within the channel molecule the structural dynamics

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of the channel when it opens and closes can be explored. The analysis of the data obtained from a series of individual MscL proteins using smFRET the diameter of the fully open channel pore

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of 28 Å was determined (Wang, Liu et al. 2014). In addition, smFRET results provided a further strong support for the helix-tilt model of the MscL opening that was previously proposed in several studies (Betanzos, Chiang et al. 2002; Perozo, Cortes et al. 2002; Corry, Hurst et al. 2010).

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KirBac

Among potassium ion channels the voltage-gated Shaker K+ channel was among the first ion channels examined by FRET (Cha, Snyder et al. 1999; Glauner, Mannuzzu et al. 1999). FRAP

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and fluorescence lifetime measurements were used in these studies. Only very recently two smFRET studies of KirBac, the bacterial homologue of mammalian inwardly rectifying potassium (Kir) channels, were reported (Sadler, Kapanidis et al. 2016; Wang 2016). In their

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study Wang et al. (Wang 2016) engineered concatemeric KirBac1.1 channel proteins containing

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Figure 4. Fluorophore labelling of KirBac for smFRET. (A) The relative locations of the two separate cysteine mutation reporter sites used in the study by Sadler et al. (2016) are shown; R151C is located at the base of the second transmembrane helix TM2 (green) and is below the helix bundle crossing (HBC) gate. G249C is in the C-terminal domain (CTD). K+ ions within the filter are shown as pink spheres. For clarity, only two of the four identical KirBac1.1 subunits are shown. (B) Due to the multimeric nature of the channel, even when the channel is labeled by only single donor and acceptor fluorophores, there are still two possible labeling schemes in which the inter-dye distances are different. The relative proximal (dP) and distal (dD) distances are shown. Note that dP
only two cysteines within the channel tetramers, which upon expression and fluorophore labeling were reconstituted into liposomes and examined by smFRET. Sadler et al. (2016) on the other hand, used nanodiscs to solubilize the channels, which allowed them to examine transition between the closed and open channel states in solution and nevertheless study the gating KirBac

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in a bilayer-like environment. For their measurements they combined alternating-laser excitation (ALEX) confocal-in-solution microscopy with smFRET. A significant advantage of the solutionbased smFRET technique is that it does not require tracking of single molecules or wide-field

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imaging of surface-tethered molecules.

Common to both studies was the use of phosphatidylinositol 4,5-bisphosphate (PIP2) to induce

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closed to open transitions in KirBac, which were reflected in the distance changes observed between the donor and acceptor fluorofores. (Note: eukaryotic Kir channels exhibit PIP2-

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dependent activation, whereas increase in PIP2 concentration stabilizes the closed state in KirBac.) Interestingly, the distance measurements during the channel closing in the study by Sadler et al (2016) strongly support the “twist-to-shrink mechanism” for the channel closing rather than a simple pore-dilation model in which the R151 sites (Fig. 4) would move closer

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together upon the channel closure suggesting that structural dynamics in KirBac are more complex than previously anticipated. In addition, the studies indicate that the extracellular region in KirBac is structurally rigid in both closed and open states, whereas the N- and C-terminal

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domains undergo large conformational fluctuations. Consequently, the existence of mobile and rigid structural motifs relevant for KirBac function may also be expected to be important in

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mammalian Kir channels.

NMDA

N‑methyl‑D‑aspartate (NMDA) receptors are ligand‑gated ion channels requiring glutamate and glycine as well as membrane depolarization removing Mg2+ block for their activation (Kloda, Martinac et al. 2007). As Ca2+ permeable ion channels they play an important role in

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synaptic transmission, long - term potentiation, synaptic plasticity and neurodegeneration. The NMDA receptor forms functional heterotetramers (Laube, Kuhse et al. 1998) consisting of two GluN1 (NR1) and two GluN2 (NR2) subunits with glycine binding to GluN1 and glutamate

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binding to GluN2 subunit. (Lu, Du et al. 2017).

At present, the NMDA receptor is the only ligand-gated channel, whose structural dynamics

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were examined by smFRET technique (Sasmal and Lu 2014). smFRET imaging and patch clamp recording were applied simultaneously to record single-channel currents of NMDA receptors

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expressed in HEK-293 cells and correlate them with FRET efficiency time trajectories resulting from the conformational changes occurring during the channel gating. For FRET measurements GluN2b subunit of the NMDA receptor was labeled by antibody covalently labeled with ATTO594 as the FRET acceptor and the ligand glycine was covalently labeled with Alexa-532 as the

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FRET donor. The results of this study suggest that NMDA was undergoing a wide distribution of multiple closed conformational states during the channel gating corresponding to apparently similar electrically silent states, whereas the electrically active state corresponded to a narrowly

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distributed subset of open states revealing complex structure and function fluctuations as indicated by a recent cryoEM study of the triheteromeric NMDA receptor (Lu, Du et al. 2017).

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This novel experimental approach combining smFRET with the patch clamp technique for simultaneous recording of the NMDA single-channel currents and FRET efficiency time trajectories demonstrate a great potential of this approach for structure - function analysis of not only glutamate receptor ion channels but also of other types of ion channels.

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Limitations of FRET FRET imaging, including smFRET, suffers from several limitations that have to be considered before employing it to study molecular dynamics of multimeric proteins such as ion channels.

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Some of the limitations are associated with the physics of FRET itself resulting in the low signalto-noise ratio due to the loss of energy associated with the FRET process between two fluorescent molecules often requiring long exposure times (>1–2 sec). This may limit the

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interpretation of results based on small changes in FRET efficiency (Leavesley 2016). Other limitations result from the fluorescence properties of fluorescent probes, which may be sensitive

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to changes in pH, ion concentrations or temperature. Furthermore, the finite size and length of fluorophore probes such as Alexa Fluor® dyes (∼ 17 Å), brings additional difficulty to converting FRET measurements to the estimation of distances (Wang, Liu et al. 2014). Also, due to their finite size fluorescent probes, especially fluorescent proteins may not be readily attached

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to any residue within an ion channel protein because of the very close proximity of the residue to other residues of the channel multimer. In such a case, the size of a fluorophore label could either prevent the residue labeling itself or affect the channel gating if attached to a site involved in the

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gating process. Despite these limitations, smFRET provides an excellent tool for studies of ion channel structures and dynamics, if combined with analysis of the orientations and geometries of

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fluorescent probes and applied together with another experimental technique, such as the patch clamp recording.

Concluding remarks The smFRET imaging has become a powerful technique for studies of structural dynamics of membrane proteins, in particular of multimeric membrane proteins such as ion channels. Though

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only a few ion channels have thus far been examined by smFRET its use led to a major advancement in our understanding of the conformational changes occurring during the closed to open transitions in ion channels. As briefly illustrated in this review on the examples of the

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gramicidin peptide, MscL mechanosensitive channel, KirBac potassium channel and NMDA ligand-gated receptor channel, smFRET not only enabled exact measurements of distance changes between the intramolecular domains during opening of the MscL channels, but also gave

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an insight into multiple intermediate conformational states occurring in both closed and open states of the NMDA receptor. Given the large variety of ion channels known or believed to be

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involved in cardiac mechano-electric coupling, such as TRPC6, TRPC3, TRPM4 or Piezo channels, smFRET holds a great potential to provide access to real-time dynamics and interactions of these channels at the single molecule level towards understanding their function, activity and gating mechanism that working in concert empower the billions of heart beats during

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a human life.

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Acknowledgments

I wish to thank Dr Navid Bavi for his assistance with preparation of figures. I also wish to thank him and Dr Charles Cox for their suggestions and critical reading of the manuscript. I also like to acknowledge support from the National Health and Medical Research Council of Australia.

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References

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Akyuz, N., R. B. Altman, et al. (2013). "Transport dynamics in a glutamate transporter homologue." Nature 502(7469): 114-118. Bavi, N., D. M. Cortes, et al. (2016). "The role of MscL amphipathic N terminus indicates a blueprint for bilayer-mediated gating of mechanosensitive channels." Nat Commun 7: 11984. Betanzos, M., C. S. Chiang, et al. (2002). "A large iris-like expansion of a mechanosensitive channel protein induced by membrane tension." Nat Struct Biol 9(9): 704-710. Borisenko, V., T. Lougheed, et al. (2003). "Simultaneous optical and electrical recording of single gramicidin channels." Biophys J 84(1): 612-622. Cha, A., G. E. Snyder, et al. (1999). "Atomic scale movement of the voltage-sensing region in a potassium channel measured via spectroscopy." Nature 402(6763): 809-813. Chang, G., R. H. Spencer, et al. (1998). "Structure of the MscL homolog from Mycobacterium tuberculosis: a gated mechanosensitive ion channel." Science 282(5397): 2220-2226. Corry, B., A. C. Hurst, et al. (2010). "An improved open-channel structure of MscL determined from FRET confocal microscopy and simulation." J Gen Physiol 136(4): 483-494. Corry, B., P. Rigby, et al. (2005). "Conformational changes involved in MscL channel gating measured using FRET spectroscopy." Biophys J 89(6): L49-51. Cox, C. D., C. Bae, et al. (2016). "Removal of the mechanoprotective influence of the cytoskeleton reveals PIEZO1 is gated by bilayer tension." Nat Commun 7: 10366. Erkens, G. B., I. Hanelt, et al. (2013). "Unsynchronised subunit motion in single trimeric sodium-coupled aspartate transporters." Nature 502(7469): 119-123. Geddes, C. D., J.R. Lakowicz, and R. Clegg ( 2006). The History of Fret. Reviews in Fluorescence. C. G. a. J. Lakowicz, Springer US: 1-45. Glauner, K. S., L. M. Mannuzzu, et al. (1999). "Spectroscopic mapping of voltage sensor movement in the Shaker potassium channel." Nature 402(6763): 813-817. Ha, T., T. Enderle, et al. (1996). "Probing the interaction between two single molecules: fluorescence resonance energy transfer between a single donor and a single acceptor." Proc Natl Acad Sci U S A 93(13): 6264-6268. Herold, K. F., R. L. Sanford, et al. (2017). "Clinical concentrations of chemically diverse general anesthetics minimally affect lipid bilayer properties." Proc Natl Acad Sci U S A. Hite, R. K. and R. MacKinnon (2017). "Structural Titration of Slo2.2, a Na+-Dependent K+ Channel." Cell 168(3): 390-399 e311. Killian, J. A. (1992). "Gramicidin and gramicidin-lipid interactions." Biochim. Biophys. Acta 1113: 391–425. Kloda, A., B. Martinac, et al. (2007). "Polymodal regulation of NMDA receptor channels." Channels (Austin) 1(5): 334-343. Koeppe R.E., n., and Anderson O.S.. (1996). "Engineering the gramicidin channel." Annu Rev Biophys Biomol Struct 25: 231–258. Kramer, H. E. A. a. F., p. (2011). "The Scientific Work of Theodor Förster: A Brief Sketch of his Life and Personality." Chem. Phys. Chem. 12(3): 555-558. Lakowicz, J. R. (2006 ). Principles of fluorescence spectroscopy. New York, NY, Springer. Laube, B., J. Kuhse, et al. (1998). "Evidence for a tetrameric structure of recombinant NMDA receptors." J Neurosci 18(8): 2954-2961. 17

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EP

TE D

M AN U

SC

RI PT

Leavesley, S. J. a. R., T.C. (2016). "Overcoming Limitations of FRET Measurements." Cytometry 89A: 325-327. Lu, W., J. Du, et al. (2017). "Cryo-EM structures of the triheteromeric NMDA receptor and its allosteric modulation." Science. Martinac, B. (2011). "Bacterial mechanosensitive channels as a paradigm for mechanosensory transduction." Cell Physiol Biochem 28(6): 1051-1060. Martinac, B., and Cranfield, C.G. (2012 ). "Shining a light on the structural dynamics of ion channels using Förster resonance energy transfer (FRET)." IPSI Transactions 9(1): 19-24. Perozo, E., D. M. Cortes, et al. (2002). "Open channel structure of MscL and the gating mechanism of mechanosensitive channels." Nature 418(6901): 942-948. Pfleger, K. D. G., Seeber, R.M., and Eidne, K.A. (2006). "Bioluminescence resonance energy transfer (BRET) for the real-time detection of protein-protein interactions." Nat. Protocols 1(1): 337-345. Roy, R., S. Hohng, et al. (2008). "A practical guide to single-molecule FRET." Nat Methods 5(6): 507-516. Sadler, E. E., A. N. Kapanidis, et al. (2016). "Solution-Based Single-Molecule FRET Studies of K(+) Channel Gating in a Lipid Bilayer." Biophys J 110(12): 2663-2670. Sasmal, D. K. and H. P. Lu (2014). "Single-molecule patch-clamp FRET microscopy studies of NMDA receptor ion channel dynamics in living cells: revealing the multiple conformational states associated with a channel at its electrical off state." J Am Chem Soc 136(37): 12998-13005. Steinbacher, S., Bass, R., Strop, P., Rees, D.C. (2007). "Mechanosensitive Channel of Large Conductance (MscL)." from http://www.rcsb.org/pdb/results/results.do. Sukharev, S. I., P. Blount, et al. (1994). "A large-conductance mechanosensitive channel in E. coli encoded by mscL alone." Nature 368(6468): 265-268. Sukharev, S. I., Blount, P., Martinac, B. and Kung, C. (1997 ). "Mechanosensitive channels of Escherichia coli: the MscL gene, protein, and activities." Ann. Rev. Physiol. 59: 633-657 Urry, D. W., M. C. Goodall, et al. (1971). "The gramicidin A transmembrane channel: characteristics of head-to-head dimerized (L,D) helices." Proc Natl Acad Sci U S A 68(8): 1907-1911. Vafabakhsh, R., J. Levitz, et al. (2015). "Conformational dynamics of a class C G-proteincoupled receptor." Nature 524(7566): 497-501. Wang, S., Vafabakhsh, R., Borschel, W.F., Ha, T., and Nichols, C.G. (2016). "Structural dynamics of potassium-channel gating revealed by single-molecule FRET." Nature Struct Mol Biol 23(1): 31-36. Wang, Y., Y. Liu, et al. (2014). "Single molecule FRET reveals pore size and opening mechanism of a mechano-sensitive ion channel." Elife 3: e01834. Weiss, S. (2000). "Measuring conformational dynamics of biomolecules by single molecule fluorescence spectroscopy." Nature Struct. Biol. 7(9): 724-729. Zhao, Y., D. Terry, et al. (2010). "Single-molecule dynamics of gating in a neurotransmitter transporter homologue." Nature 465(7295): 188-193.

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