11 research outputs found
Near-IR Resonance Raman Spectroscopy of Archaerhodopsin 3: Effects of Transmembrane Potential
Archaerhodopsin 3 (AR3) is a light driven proton pump
from <i>Halorubrum sodomense</i> that has been used as a
genetically
targetable neuronal silencer and an effective fluorescent sensor of
transmembrane potential. Unlike the more extensively studied bacteriorhodopsin
(BR) from <i>Halobacterium salinarum</i>, AR3 readily incorporates
into the plasma membrane of both <i>E. coli</i> and mammalian
cells. Here, we used near-IR resonance Raman confocal microscopy to
study the effects of pH and membrane potential on the AR3 retinal
chromophore structure. Measurements were performed both on AR3 reconstituted
into <i>E. coli</i> polar lipids and <i>in vivo</i> in <i>E. coli</i> expressing AR3 in the absence and presence
of a negative transmembrane potential. The retinal chromophore structure
of AR3 is in an all-trans configuration almost identical to BR over
the entire pH range from 3 to 11. Small changes are detected in the
retinal ethylenic stretching frequency and Schiff Base (SB) hydrogen
bonding strength relative to BR which may be related to a different
water structure near the SB. In the case of the AR3 mutant D95N, at
neutral pH an all-trans retinal O-like species (O<sup>all‑trans</sup>) is found. At higher pH a second 13-cis retinal N-like species (N<sup>13‑cis</sup>) is detected which is attributed to a slowly
decaying intermediate in the red-light photocycle of D95N. However,
the amount of N<sup>13‑cis</sup> detected is less in <i>E. coli</i> cells but is restored upon addition of carbonyl
cyanide <i>m</i>-chlorophenyl hydrazone (CCCP) or sonication,
both of which dissipate the normal negative membrane potential. We
postulate that these changes are due to the effect of membrane potential
on the N<sup>13‑cis</sup> to M<sup>13‑cis</sup> levels
accumulated in the D95N red-light photocycle and on a molecular level
by the effects of the electric field on the protonation/deprotonation
of the cytoplasmic accessible SB. This mechanism also provides a possible
explanation for the observed fluorescence dependence of AR3 and other
microbial rhodopsins on transmembrane potential
Patch clamp characterization of Arch(D95Y) and D95H.
<p>A) Fluorescence response to a step in membrane voltage from -70 to +30Â mV. Each trace is the average of 38 steps. Fits to a bi-exponential are shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0085221#pone-0085221-t001" target="_blank">Table 1</a>. B) Fluorescence as a function of membrane voltage. Both proteins showed hysteresis at slow sweep speeds (0.5 Hz), indicating multiple stable states. Each trace shows the fluorescence as the voltage was cycled three times in the direction indicated by the arrows. Raw fluorescence was corrected for photobleaching of the baseline. C) and D) Images of Arch fluorescence in the cells measured in A and B. E) The relative brightness, defined as the ratio of Arch fluorescence to fluorescence from a covalently bound GFP, for key Arch variants at a 640 nm illumination intensity of 440 W/cm<sup>2</sup>. The brightness for each mutant was normalized to WT brightness; error bars show SEM from ~15 cells per mutant.</p
Spiking HEK cells report sensitivity and approximate speed of voltage indicators.
<p>A) Voltage-sensitive dyes showed fluorescence sensitive to electrical spikes. For VF2.1.Cl excitation was at 488 nm and emission was collected from 525- 575 nm. For di-8-ANEPPS, excitation was at 488 nm, fluorescence for the positive-going signal was collected from 525-575 nm and negative-going signal was collected between 660 and 740 nm. For RH237 excitation was at 532 nm and emission was 660 - 740 nm. The images (40 µm across) show staining efficiency. B) Representative fluorescence waveforms of genetically encoded voltage indicators. The Arch mutants were excited at 640 nm (~500 W/cm<sup>2</sup>), with fluorescence emission collected from 660-740 nm. ArcLight-A was excited at 488 nm with fluorescence emission collected from 525- 575 nm. C) Sensitivity and speed of fluorescent voltage indicators. The speeds plotted for the genetically encoded reporters represent the apparent speed, determined by convolution of the upswing of the action potential, the camera exposure, and the underlying speed of the reporter. Thus the apparent speeds of Arch(D95E) and Arch WT were slower than their true speeds. The VSDs are known to be significantly faster than 4 ms, so no effort was made to measure their speeds optically. The sign of response of ArcLight-A, RH237, and the negative-going signal of di-8-ANEPPS have been inverted to facilitate comparison. Error bars represent s.e.m. of n = 7 - 38 single-cell measurements.</p
Reproducibility of spiking.
<p>A) Patch clamp voltage recordings of spontaneously spiking HEK cells at different levels of confluence. Although the beat rate varied with cell density, the rising edge was consistently fast (2-3 ms) and the variation in the voltage swing was typically ~10% between dishes. B) Representative fluorescence traces from eight different dishes treated with the voltage sensitive dye VF2.1.Cl. Fluorescence was recorded at a 1 kHz frame rate. C) Histogram of fluorescent spike amplitudes. D) Histogram of spike rise time (40% to 70% depolarization) as recorded optically. Histograms show the aggregate results from 13 dishes of cells and 15 locations within each dish.</p
HEK cells expressing Na<sub>V</sub> 1.3 and K<sub>IR</sub> 2.1 generate spontaneous electrical spikes.
<p>A) Cartoon showing ion channels whose expression is sufficient to induce electrical spiking in a syncytial monolayer. B) Image of spiking HEK cells. A patch pipette is also visible. C) Patch clamp recording of membrane voltage in a single spiking HEK cell. D) Voltage-sensitive dye images showing electrical wave propagation in a culture of spiking HEK cells. E) Waves originated as self-reinforcing spirals. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0085221#pone.0085221.s001" target="_blank">Videos S1-S4</a> show more propagation patterns in spiking HEK cells.</p
Spiking HEK sensitivity and speed correlate with patch clamp results.
<p>A) Sensitivity per 100 mV (-70 mV to +30 mV) as measured by manual patch clamp and spiking HEKs. Literature values are used for RH237 [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0085221#B19" target="_blank">19</a>], ArcLight-A (Q239) [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0085221#B20" target="_blank">20</a>], VF2.1.Cl [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0085221#B14" target="_blank">14</a>], and di-8-ANEPPS [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0085221#B14" target="_blank">14</a>]. B) Effective rising edge time constants measured with patch clamp and with spiking HEKs. For patch clamp data, the time constant is a weighted average of the time constants from a bi-exponential fit.</p