Single-Molecule Patch-Clamp FRET Anisotropy Microscopy Studies of NMDA Receptor Ion Channel Activation and Deactivation under Agonist Ligand Binding in Living Cells.

N-methyl-d-aspartate (NMDA) receptor ion channel is activated by the binding of two pairs of glycine and glutamate along with the application of action potential. Binding and unbinding of ligands changes its conformation that plays a critical role in the open-close activities of NMDA receptor. Conformation states and their dynamics due to ligand binding are extremely difficult to characterize either by conventional ensemble experiments or single-channel electrophysiology method. Here we report the development of a new correlated technical approach, single-molecule patch-clamp FRET anisotropy imaging and demonstrate by probing the dynamics of NMDA receptor ion channel and kinetics of glycine binding with its ligand binding domain. Experimentally determined kinetics of ligand binding with receptor is further verified by computational modeling. Single-channel patch-clamp and four-channel fluorescence measurement are recorded simultaneously to get correlation among electrical on and off states, optically determined conformational open and closed states by FRET, and binding-unbinding states of the glycine ligand by anisotropy measurement at the ligand binding domain of GluN1 subunit. This method has the ability to detect the intermediate states in addition to electrical on and off states. Based on our experimental results, we have proposed that NMDA receptor gating goes through at least one electrically intermediate off state, a desensitized state, when ligands remain bound at the ligand binding domain with the conformation similar to the fully open state.

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.

Conformational dynamics plays a critical role in the activation, deactivation, and open-close activities of ion channels in living cells. Such conformational dynamics is often inhomogeneous and extremely difficult to be directly characterized by ensemble-averaged spectroscopic imaging or only by single channel patch-clamp electric recording methods. We have developed a new and combined technical approach, single-molecule patch-clamp FRET microscopy, to probe ion channel conformational dynamics in living cell by simultaneous and correlated measurements of real-time single-molecule FRET spectroscopic imaging with single-channel electric current recording. Our approach is particularly capable of resolving ion channel conformational change rate process when the channel is at its electrically off states and before the ion channel is activated, the so-called "silent time" when the electric current signals are at zero or background. We have probed NMDA (N-methyl-D-aspartate) receptor ion channel in live HEK-293 cell, especially, the single ion channel open-close activity and its associated protein conformational changes simultaneously. Furthermore, we have revealed that the seemingly identical electrically off states are associated with multiple conformational states. On the basis of our experimental results, we have proposed a multistate clamshell model to interpret the NMDA receptor open-close dynamics.

Solvation dynamics of biological water in a single live cell under a confocal microscope.

Time-resolved confocal microscopy has been applied to study the cytoplasm and nucleus region of a single live Chinese hamster ovary (CHO) cell. To select the cytoplasm and the nucleus region, two different fluorescent probes are used. A hydrophobic fluorescent dye, DCM, localizes preferentially in the cytoplasm region of a CHO cell. A DNA binding dye, DAPI, is found to be inside the nucleus of the cell. The locations of the probes are clearly seen in the image. Emission maxima of the dyes (DCM in cytoplasm and DAPI in the nucleus) are compared to those of the same dyes in different solvents. From this, it is concluded that the polarity (dielectric constant, ε) of the microenvironment of DCM in the cytoplasm is ~15. The nucleus is found to be much more polar with ε ≈ 60 (as reported by DAPI). The diffusion coefficient (and hence viscosity) in the cytoplasm and the nucleus was determined using fluorescence correlation spectroscopy (FCS). The diffusion coefficient (D(t)) of the dye (DCM) in the cytoplasm is 2 µm(2) s(-1) and is ~150 times slower than that in bulk water (buffer). D(t) of DAPI in the nucleus (15 µm(2) s(-1)) is 30 times slower than that in bulk water (buffer). The extremely slow diffusion inside the cell has been ascribed to higher viscosity and also to the binding of the probes (DCM and DAPI) to large biological macromolecules. The solvation dynamics of water in the cytoplasm (monitored by DCM) exhibits an average relaxation time [τ(sol)] of 1250 ± 50 ps, which is about 1000 times slower than in bulk water (1 ps). The solvation dynamics inside the nucleus (studied using DAPI) is about 2-fold faster, [τ(sol)] ≈ 775 ps. The higher polarity, faster diffusion, and faster solvation dynamics in the nucleus indicates that it is less crowded and less restricted than the cytoplasm.

Deoxycholate induced tetramer of αA-crystallin and sites of phosphorylation: fluorescence correlation spectroscopy and femtosecond solvation dynamics.

Structure and dynamics of acrylodan labeled αA-crystallin tetramer formed in the presence of a bile salt (sodium deoxycholate, NaDC) has been studied using fluorescence correlation spectroscopy (FCS) and femtosecond up-conversion techniques. Using FCS it is shown that, the diffusion constant (D(t)) of the αA-crystallin oligomer (mass ~800 kDa) increases from ~35 µm(2) s(-1) to ~68 µm(2) s(-1). This corresponds to a decrease in hydrodynamic radius (r(h)) from ~6.9 nm to ~3.3 nm. This corresponds to about 10-fold decrease in molecular mass to ~80 kDa and suggests formation of a tetramer (since mass of αA-crystallin monomer is ~20 kDa). The steady state emission maximum and average solvation time (<τ(s)>) of acrylodan labeled at cysteine 131 position of αA-crystallin is markedly affected on addition of NaDC, while the tryptophan (trp-9) becomes more exposed. This suggests that NaDC binds near the cys-131 and makes the terminal region of αA-crystallin exposed. This may explain the enhanced auto-phosphorylation activity of αA-crystallin near the terminus of the 173 amino acid protein (e.g., at the threonine 13, serine 45, or serine 169 and 172) and suggests that phosphorylation at ser-122 (close to cys-131) is relatively less important.

An FCS study of unfolding and refolding of CPM-labeled human serum albumin: role of ionic liquid.

The effect of a room temperature ionic liquid (RTIL) on the conformational dynamics of a protein, human serum albumin (HSA), is studied by fluorescence correlation spectroscopy (FCS). For this, the protein was covalently labeled by a fluorophore, 7-dimethylamino-3-(4-maleimidophenyl)-4-methylcoumarin (CPM). On addition of a RTIL ([pmim][Br]) to the native protein, the diffusion coefficient (D(t)) decreases and the hydrodynamic radius (R(h)) increases. This suggests that the RTIL ([pmim][Br]) acts as a denaturant when the protein is in the native state. However, addition of [pmim][Br] to a protein denatured by GdnHCl causes an increases in D(t) and decrease in R(h). This suggests that in the presence of GdnHCl addition of RTIL helps the protein to refold. In the native state, the conformational dynamics of protein is described by three distinct time constants: ~3.6 ± 0.7, ~29 ± 4.5, and 133 ± 23 µs. The faster components (~3.6 ± 0.7 and ~29 ± 4.5 µs) are ascribed to chain dynamics of the protein, while the slowest component (133 µs) is responsible for interchain interaction or concerted motion. On addition of [pmim][Br], the conformational dynamics of HSA becomes slower (~5.1 ± 1, ~43.5 ± 2.8, and ~311 ± 2.3 µs in the presence of 1.5 M [pmim][Br]). The time constants for the protein denatured by 6 M GdnHCl are 3.2 ± 0.4, 34 ± 6, and 207 ± 38 µs. When 1.5 M [pmim][Br] is added to the denatured protein (in 6 M GdnHCl), the time constants become ~5 ± 1, ~41 ± 10, and ~230 ± 45 µs. The lifetime histogram shows that, on addition of GdnHCl to HSA, the contribution of the shorter lifetime component decreases and vanishes at 6 M GdnHCl. The shorter lifetime component immediately reappears after addition of RTIL to unfolded HSA. This suggests recoiling of the unfolded protein by RTIL.

Femtosecond study of ultrafast fluorescence resonance energy transfer in a catanionic vesicle.

Ultrafast fluorescence resonance energy transfer (FRET) in a catanionic [sodium dodecyl sulfate (SDS)-dodecyltrimethyl ammonium bromide (DTAB)] vesicle is studied by femtosecond up-conversion. The vesicles (diameter ∼400 nm for SDS-rich and ∼250 nm for DTAB-rich vesicles) are much larger than the SDS and DTAB micelles (diameter ∼4 nm). In both micelle and vesicles, FRET occurs in multiple time scales and the time scales of FRET correspond to a donor-acceptor distance varying between 12 and 36 Å.

Diffusion of organic dyes in ionic liquid and giant micron sized ionic liquid mixed micelle: fluorescence correlation spectroscopy.

Diffusion of organic dyes in neat room temperature ionic liquid (RTIL) and RTIL-mixed micelle has been studied by fluorescence correlation spectroscopy (FCS). We have selected two RTILs, 3-pentyl-1-methyl imidazolium bromide ([C5C1Im][Br]) and the corresponding tetra-fluoroborate ([C5C1Im][BF(4)]). Diffusion coefficients (D(t)) of three organic dyes--DCM (neutral), C480 (neutral), and C343 (anionic)--in these RTILs are ∼100 times slower compared to water. This indicates very high viscosity of the RTILs. In contrast to water, the D(t) in RTIL exhibits a wide distribution which suggests the presence of heterogeneity (nanoscale organization). The presence of ions in the RTILs markedly affects diffusion in the RTILs. D(t)'s of C480 (neutral) and C343 (anionic) are very similar in water but in RTILs the ionic dye C343 diffuses 1.7 times slower than neutral C480. This is attributed to the electrostatic force exerted by the ions in the RTILs. In the giant (∼2-4 µm) [C5C1Im][Br]-triblock copolymer (P123) mixed micelle D(t) of DCM, C480, and C343 are found to be 7, 15, and 7 µm(2) s(-1), respectively. The results are compared with those in P123 micelle and gel.

Excited state proton transfer in ionic liquid mixed micelles.

Excited state proton transfer (ESPT) of pyranine (8-hydroxypyranine-1,3,6-trisulfonate, HPTS) in room temperature ionic liquid (RTIL) mixed micelles is studied by femtosecond up-conversion. The mixed micelle consists of a triblock copolymer, (PEO)(20)-(PPO)(70)-(PEO)(20) (Pluronic P123), and one of the two RTILs, 1-pentyl-3-methyl-imidazolium bromide ([pmim][Br]) and 1-pentyl-3-methyl-imidazolium tetra-fluoroborate ([pmim][BF(4)]). The size and structure of the mixed micelle vary with the relative amount of the RTIL. For [pmim][Br], the hydrodynamic diameter of the mixed micelle is 26 nm in 0.3 M RTIL and 3500 nm in 3.0 M RTIL. The time constant of initial proton transfer (τ(PT)) in P123 micelle (65 ps) is 10 times slower than that (5 ps) in water, while the time constants of recombination (τ(rec)) and dissociation (τ(diss)) are 2-3 times slower in P123 micelle. On addition of the RTIL, the rate of ESPT is markedly modified. In 0.3 M RTIL-P123 mixed micelle, τ(PT) is shorter than that in P123 micelle. In the mixed micelle, τ(PT) increases with an increase in the concentration of the RTIL (230 ps in 3 M [pmim][Br] and 55 ps in 0.9 M [pmim][BF(4)]). This is attributed to large scale penetration of the P123 micelle by RTIL replacing water molecules. The time constants of proton transfer (τ(PT), τ(rec), and τ(diss)) are faster than the slowest component (200-500 ps) of solvation dynamics. It seems that the ultrafast component of solvation (<0.3 ps and <5 ps) is enough for inducing proton transfer. The time constant of the proton transfer (τ(PT)) in [pmim][BF(4)]-P123 mixed micelle is longer (∼20%) than that in [pmim][Br]-P123 mixed micelle for the same concentration of RTIL. The counterion dependence of ESPT is attributed to the difference in the structure and greater hydrophobicity of the [pmim][BF(4)].

Deuterium isotope effect on femtosecond solvation dynamics in an ionic liquid microemulsion: an excitation wavelength dependence study.

The deuterium isotope effect on the solvation dynamics and the anisotropy decay of coumarin 480 (C480) in a room temperature ionic liquid (RTIL) microemulsion is studied by femtosecond up-conversion. The microemulsion consists of the RTIL 1-pentyl-3-methyl-imidazolium tetra-fluoroborate ([pmim][BF(4)]) in triton X-100 (TX-100)/benzene. Replacement of H(2)O by D(2)O in the microemulsion causes retardation of solvation dynamics. The average solvation time of C480 (tau(s)) in RTIL microemulsion with 5 wt % D(2)O is approximately 1.5-1.7 times slower compared to that in the H(2)O containing RTIL microemulsion. This suggests that the main species in the microemulsion responsible for solvation is the water molecules. In both D(2)O and H(2)O containing RTIL microemulsion, the solvation dynamics exhibits marked dependence on the excitation wavelength (lambda(ex)) and becomes about 15 times faster as lambda(ex) increases from 375 to 435 nm. This is ascribed to the structural heterogeneity in the RTIL microemulsion. For lambda(ex) = 375 nm, the region near the TX-100 surfactant is probed where bound water molecules cause slow solvation dynamics. At 435 nm, the RTIL pool is selected where the water molecules are more mobile and hence gives rise to faster solvation. The average time constant of anisotropy decay shows opposite dependence on lambda(ex) and increases about 2.5-fold from 180 ps at lambda(ex) = 375 nm to 500 ps at lambda(ex) = 435 nm for D(2)O containing RTIL microemulsion. The slower anisotropy decay at lambda(ex) = 435 nm is ascribed to the higher viscosity of RTIL which causes greater friction at the core.

Deuterium isotope effect on femtosecond solvation dynamics in methyl beta-cyclodextrins.

Deuterium isotope effect on the solvation dynamics and fluorescence anisotropy decay of coumarin 153 (C153) bound to dimethyl beta-cyclodextrin (DMB) and trimethyl beta-cyclodextrin (TMB) is studied using femtosecond upconversion. In D(2)O, there is a marked increase in the steady state emission quantum yield and fluorescence lifetime of C153 bound to DMB and TMB. This suggests strong coupling between C153 and D(2)O inside the cyclodextrin cavity. In D(2)O, average solvation time of C153 in DMB is about 1.7 times slower compared to that in water. For TMB in D(2)O, solvation is 1.5 times slower. The deuterium isotope effect on solvation dynamics at long time arises mainly from the longer excited state lifetime. The longest components of solvation dynamics are ascribed to self-diffusion of C153 out of the cyclodextrin cavity. The nearly 1.5 times slower anisotropy decay of C153 bound to DMB and TMB in D(2)O (compared to H(2)O) is attributed to higher viscosity of D(2)O.

Ultrafast FRET in a room temperature ionic liquid microemulsion: a femtosecond excitation wavelength dependence study.

Fluorescence resonance energy transfer (FRET) from coumarin 480 (C480) to rhodamine 6G (R6G) is studied in a room temperature ionic liquid (RTIL) microemulsion by picosecond and femtosecond emission spectroscopy. The microemulsion is comprised of the RTIL 1-pentyl-3-methylimidazolium tetraflouroborate, [pmim][BF4], in TX-100/ benzene. We have studied the microemulsion with and without water. The time constants of FRET were obtained from the risetime of the acceptor (R6G) emission. In the RTIL microemulsion, FRET occurs on multiple time scales: 1, 250, and 3900 ps. In water containing RTIL microemulsion, the rise components are 1.5, 250, and 3900 ps. The 1 and 1.5 ps components are assigned to FRET at a close contact of donor and acceptor (RDA approximately 12 A). This occurs within the highly polar (RTIL/water) pool of the microemulsion. With increase in the excitation wavelength (lambdaex) from 375 to 435 nm, the relative contribution of the ultrafast component of FRET (1 ps) increases from 4% to 100% in the RTIL microemulsion and 12% to 100% in the water containing RTIL microemulsion. It is suggested that at lambdaex = 435 nm, mainly the highly polar RTIL pool is probed where FRET is very fast due to the close proximity of the donor and the acceptor. The very long 3900 ps (RDA approximately 45 A) component may arise from FRET from a donor in the outer periphery of the microemulsion to an acceptor in the polar RTIL pool. The 250 ps component (RDA approximately 29 A) is assigned to FRET from a donor inside the surfactant chains.

Femtosecond solvation dynamics in a micron-sized aggregate of an ionic liquid and P123 triblock copolymer.

Dynamic light scattering studies indicate that addition of a room temperature ionic liquid (RTIL, [pmim][Br]), to a triblock copolymer (P123) micelle leads to the formation of giant P123-RTIL clusters of size (diameter) 40 nm in 0.9 M and 3500 nm (3.5 microm) in 3 M RTIL. They are much larger than a P123 micelle ( approximately 18 nm) or [pmim][Br] (1.3 nm). Dynamics in different regions of the P123-RTIL aggregate is probed by variation of the excitation wavelength (lambda(ex)) using femtosecond up-conversion. For lambda(ex) = 375 nm, the nonpolar core of the P123-RTIL aggregate is preferentially excited while lambda(ex) = 435 nm selects the polar corona region. Solvation dynamics and anisotropy decay of coumarin 480 (C480) in a P123-RTIL giant aggregate are markedly different from those in either P123 micelle or those in an aqueous solution of the RTIL. For lambda(ex) = 405 nm in 5 wt % P123 and 0.9 M RTIL average rotational time, ( = 1350 ps) of C480 is approximately 7 times longer than that (200 ps) in an aqueous solution of the RTIL in the absence of P123 and is shorter than that (3000 ps) in a P123 micelle. In 0.9 M RTIL and 5 wt % P123, solvation dynamics in the corona region (lambda(ex) = 435 nm, = 75 ps) is approximately 25 times faster than that at the core region (at lambda(ex) = 375 nm, = 1900 ps). The solvation dynamics in the core of the P123-RTIL aggregate is faster than that in P123 micelle (3550 ps in the core) and is much slower than that (130 ps) in an aqueous solution containing 0.9 M RTIL. In the 3.5 microm sized aggregate (3 M RTIL and P123), the solvation dynamics in the core ( = 500 ps) is approximately 4 times faster than that in 0.9 M RTIL.

A femtosecond study of solvation dynamics and anisotropy decay in a catanionic vesicle: excitation-wavelength dependence.

The structure and dynamics of a catanionic vesicle are studied by means of femtosecond up-conversion and dynamic light scattering (DLS). The catanionic vesicle is composed of dodecyl-trimethyl-ammonium bromide (DTAB) and sodium dodecyl sulphate (SDS). The DLS data suggest that 90 % of the vesicles have a diameter of about 400 nm, whereas the diameter of the other 10 % is about 50 nm. The dynamics in the catanionic vesicle are compared with those in pure SDS and DTAB micelles. We also study the dynamics in different regions of the micelle/vesicle by varying the excitation wavelength (lambda(ex)) from 375 to 435 nm. The catanionic vesicle is found to be more heterogeneous than the SDS or DTAB micelles, and hence, the lambda(ex)-dependent variation of the solvation dynamics is more prominent in the first case. The solvation dynamics in the vesicle and the micelles display an ultraslow component (2 and 300 ps, respectively), which arises from the quasibound, confined water inside the micelle, and an ultrafast component (<0.3 ps), which is due to quasifree water at the surface/exposed region. With an increase in lambda(ex), the solvation dynamics become faster. This is manifested in a decrease in the total dynamic solvent shift and an increase in the contribution of the ultrafast component (<0.3 ps). At a long lambda(ex) (435 nm), the surface (exposed region) of a micelle/vesicle is probed, where the solvation dynamics of the water molecules are faster than those in a buried location of the vesicle and the micelles. The time constant of anisotropy decay becomes longer with increasing lambda(ex), in both the catanionic vesicle and the ordinary micelles (SDS and DTAB). The slow rotational dynamics (anisotropy decay) in the polar region (at long lambda(ex)) may be due to the presence of ionic head groups and counter ions.