Electromagnetic energy (670 nm) stimulates vasodilation through activation of the large conductance potassium channel (BKCa).

INTRODUCTION: Peripheral artery disease (PAD) is a highly morbid condition in which impaired blood flow to the limbs leads to pain and tissue loss. Previously we identified 670 nm electromagnetic energy (R/NIR) to increase nitric oxide levels in cells and tissue. NO elicits relaxation of smooth muscle (SMC) by stimulating potassium efflux and membrane hyperpolarization. The actions of energy on ion channel activity have yet to be explored. Here we hypothesized R/NIR stimulates vasodilation through activation of potassium channels in SMC. METHODS: Femoral arteries or facial arteries from C57Bl/6 and Slo1-/- mice were isolated, pressurized to 60 mmHg, pre-constricted with U46619, and irradiated twice with energy R/NIR (10 mW/cm2 for 5 min) with a 10 min dark period between irradiations. Single-channel K+ currents were recorded at room temperature from cell-attached and excised inside-out membrane patches of freshly isolated mouse femoral arterial muscle cells using the patch-clamp technique. RESULTS: R/NIR stimulated vasodilation requires functional activation of the large conductance potassium channels. There is a voltage dependent outward current in SMC with light stimulation, which is due to increases in the open state probability of channel opening. R/NIR modulation of channel opening is eliminated pharmacologically (paxilline) and genetically (BKca α subunit knockout). There is no direct action of light to modulate channel activity as excised patches did not increase the open state probability of channel opening. CONCLUSION: R/NIR vasodilation requires indirect activation of the BKca channel.

Changing the firing threshold for normal optic nerve axons by the application of infra-red laser light.

Normal optic nerve axons exhibit a temperature dependence, previously explained by a membrane potential hyperpolarization on warming. We now report that near infra-red laser light, delivered via a fibre optic light guide, also affects axonal membrane potential and threshold, at least partly through a photo-thermal effect. Application of light to optic nerve, at the recording site, gave rise to a local membrane potential hyperpolarization over a period of about a minute, and increased the size of the depolarizing after potential. Application near the site of electrical stimulation reversibly raised current-threshold, and the change in threshold recorded over minutes of irradiation was significantly increased by the application of the Ih blocker, ZD7288 (50 µM), indicating Ih limits the hyperpolarizing effect of light. Light application also had fast effects on nerve behaviour, increasing threshold without appreciable delay (within seconds), probably by a mechanism independent of kinetically fast K+ channels and Na+ channel inactivation, and hypothesized to be caused by reversible changes in myelin function.

Mechanism behind the neuronal ephaptic coupling during synchronizing by specific brain-triggered wave as neuronal motor toolkit.

Probable mechanism behind the neuronal ephaptic coupling is investigated based on the introduction of "Brain"-triggered potential excitation signal smartly with a specific very low frequency (VLF) waves as a neuronal motor toolkit. Detection of this electric motor toolkit is attributed to in-vitro precise analyses of a neural network of snail, along to the disconnected snail's neuronal network as a control. This is achieved via rapid (real-time) electrical signals acquisition by blind patch-clamp method during micro-electrode implanting in the neurons at the gigaseal conditions by the surgery operations. This process is based on its waveform (potential excitation signal) detection by mathematical curve fitting process. The characterized waveform of this electrical signal is "Saw Tooth" that is smartly stimulated, alternatively, by the brain during triggering the action potential's (AP's) hyperpolarization zone at a certain time interval at the several µs levels. Triggering the neuron cells results in (1) observing a positive shift (10.0%, depending on the intensity of the triggering wave), and (2) major promotion in the electrical current from sub nano (n) to micro (µ) amper (nA, µA) levels. Direct tracing the time domain (i.e., electrical signal vs. time) and estimation of the frequency domain (diagram of electrical response vs. the applied electrical frequencies) by the "Discrete Fast Fourier Transform" algorithm approve the presence of bilateral and reversible electrical currents between axon and dendrite. This mechanism therefore opens a novel view about the neuronal motor toolkit mechanism, versus the general knowledge about the unilateral electrical current flow from axon to dendrite operations in as neural network. The reliability of this mechanism is evaluated via (1) sequential modulation and demodulation of the snail's neuron network by a simulation electrical functions and sequentially evaluation of the neuronal current sensitivity between pA and nA (during the promotion of the signal-to-noise ratio, via averaging of 30 ± 1 (n = 15) and recycling the electrical cycles before any neuronal response); and (2) operation of the process on the differentiated stem cells. The interstice behavior is attributed to the effective role of Ca2+ channels (besides Na+ and K+ ionic pumping), during hyper/hypo calcium processes, evidenced by inductively coupled plasma as the selected analytical method. This phenomenon is also modeled during proposing quadrupole well potential levels in the neuron systems. This mechanism therefore points to the microprocessor behavior of neuron networks. Stimulation of the neuronal system based on this mechanism, not only controls the sensitivity of neuron electrical stimulation, but also would open a light window for more efficient operating the neuronal connectivity during providing interruptions by phenomena such as neurolysis as well as an efficient treatment of neuron-based disorders.

Genetically Encoded Voltage Indicators.

Optogenetic approaches combine the power to allocate optogenetic tools (proteins) to specific cell populations (defined genetically or functionally) and the use of light-based interfaces between biological wetware (cells and tissues) and hardware (controllers and recorders). The optogenetic toolbox contains two main compartments: tools to interfere with cellular processes and tools to monitor cellular events. Among the latter are genetically encoded voltage indicators (GEVIs). This chapter outlines the development, current state of the art and prospects of emerging optical GEVI imaging technologies.

Charge Transport by Light-Activated Rhodopsins Determined by Electrophysiological Recordings.

Electrophysiological experiments are required to determine the ion transport properties of light-activated currents from microbial rhodopsin expressing cells. The recordings set the quantitative basis for correlation with spectroscopic data and for understanding of channel gating, ion transport vectoriality, or ion selectivity. This chapter focuses on voltage-clamp recordings of channelrhodopsin-2-expressing cells, and it will describe different illumination protocols that reveal the kinetic properties of gating. While the opening and closing reaction is determined from a single turnover upon a short laser flash, desensitization of the light-gated currents is studied under continuous illumination. Recovery from the desensitized state is probed after prolonged illumination with a subsequent light activation upon different dark intervals. Compiling the experimental data will define a minimum number of states in kinetic schemes used to describe the light-gated currents in channelrhodopsins, and emphasis will be given on how to correlate the results with the different time-resolved spectroscopic experiments.

TRESK is a key regulator of nocturnal suprachiasmatic nucleus dynamics and light adaptive responses.

The suprachiasmatic nucleus (SCN) is a complex structure dependent upon multiple mechanisms to ensure rhythmic electrical activity that varies between day and night, to determine circadian adaptation and behaviours. SCN neurons are exposed to glutamate from multiple sources including from the retino-hypothalamic tract and from astrocytes. However, the mechanism preventing inappropriate post-synaptic glutamatergic effects is unexplored and unknown. Unexpectedly we discovered that TRESK, a calcium regulated two-pore potassium channel, plays a crucial role in this system. We propose that glutamate activates TRESK through NMDA and AMPA mediated calcium influx and calcineurin activation to then oppose further membrane depolarisation and rising intracellular calcium. Hence, in the absence of TRESK, glutamatergic activity is unregulated leading to membrane depolarisation, increased nocturnal SCN firing, inverted basal calcium levels and impaired sensitivity in light induced phase delays. Our data reveals TRESK plays an essential part in SCN regulatory mechanisms and light induced adaptive behaviours.

Fine Tuning of Calcium Constitutive Entry by Optogenetically-Controlled Membrane Polarization: Impact on Cell Migration.

Anomalies in constitutive calcium entry (CCE) have been commonly attributed to cell dysfunction in pathological conditions such as cancer. Calcium influxes of this type rely on channels, such as transient receptor potential (TRP) channels, to be constitutively opened and strongly depend on membrane potential and a calcium driving force. We developed an optogenetic approach based on the expression of the halorhodopsin chloride pump to study CCE in non-excitable cells. Using C2C12 cells, we found that halorhodopsin can be used to achieve a finely tuned control of membrane polarization. Escalating the membrane polarization by incremental changes in light led to a concomitant increase in CCE through transient receptor potential vanilloid 2 (TRPV2) channels. Moreover, light-induced calcium entry through TRPV2 channels promoted cell migration. Our study shows for the first time that by modulating CCE and related physiological responses, such as cell motility, halorhodopsin serves as a potentially powerful tool that could open new avenues for the study of CCE and associated cellular behaviors.

Predicting electrotransfer in ultra-high frequency sub-microsecond square wave electric fields.

Measurement of cell transmembrane potential (TMP) is a complex methodology involving patch-clamp methods or fluorescence-based potentiometric markers, which have limited to no applicability during ultrafast charging and relaxation phenomena. In such a case, analytical methods are applied for evaluation of the voltage potential changes in biological cells. In this work, the TMP-based electrotransfer mechanism during ultra-high frequency (≥1 MHz) electric fields is studied and the phenomenon of rapid membrane charge accumulation, which is non-occurrent during conventional low-frequency electroporation is simulated using finite element method (FEM). The influence of extracellular medium conductivity (0.1, 1.5 S/m) and pulse rise/fall times (10-50 ns) TMP generation are presented. It is shown that the medium conductivity has a dramatic influence on the electroporation process in the high-frequency range of applied pulsed electric fields (PEF). The applied model allowed to grasp the differences in polarization between 100 and 900 ns PEF and enabled successful prediction of the experimental outcome of propidium iodide electrotransfer into CHO-K1 cells and the conductivity-dependent patterns of MHz range PEF-triggered electroporation were determined. The results of this study form recommendations for development and pre-evaluation of future PEF protocols and generators based on ultra-high frequency electroporation for anticancer and gene therapies.

Desensitization and Recovery of Crayfish Photoreceptors Upon Delivery of a Light Stimulus.

A method to study desensitization and recovery of crayfish photoreceptors is presented. We performed intracellular electrical recordings of photoreceptor cells in isolated eyestalks using the discontinuous single electrode-switched voltage-clamp configuration. First, with a razor blade we made an opening in the dorsal cornea to get access to the retina. Thereafter, we inserted a glass electrode through the opening, and penetrated a cell as reported by the recording of a negative potential. Membrane potential was clamped at the photoreceptor's resting potential and a light-pulse was applied to activate currents. Finally, the two light-flash protocol was employed to measure current desensitization and recovery. The first light-flash triggers, after a lag period, the transduction ionic current, which after reaching a peak amplitude decays towards a desensitized state; the second flash, applied at varying time intervals, assesses the state of the light-activated conductance. To characterize the light-elicited current, three parameters were measured: 1) latency (the time elapsed between light flash delivery and the moment in which current achieves 10% of its maximum value); 2) peak current; and 3) desensitization time constant (exponential time constant of the current decay phase). All parameters are affected by the first pulse. To quantify recovery from desensitization, the ratio p2/p1 was employed versus time between pulses. p1 is the peak current evoked by the first light-pulse, and p2 is the peak current evoked by the second pulse. These data were fitted to a sum of exponential functions. Finally, these measurements were carried out as function of circadian time.

Modulation of cardiac optogenetics by vitamin A.

Cardiac optogenetics is an emergent research area and refers to the delivery of light-activated proteins to excitable heart tissue and the subsequent use of light for controlling the electrical function with high spatial and temporal resolution. Channelrhodopsin-2 (ChR2) is a light-sensitive ion channel with the chromophore, all trans retinal, derived from vitamin A (all-trans-retinol; retinol). In this study, we explored whether exogenous vitamin A can be a limiting factor in the light responsiveness of cardiomyocytes-expressing ChR2. We showed that in cardiomyocytes virally transduced with ChR2 (H134R)-enhanced yellow fluorescent protein, vitamin A supplements lower than 10 µM significantly increased ChR2 expression. Adding 1 µM vitamin A changed light-induced transmembrane potential difference significantly, whereas 5 µM dramatically induced membrane depolarization and triggered intracellular calcium elevation. We concluded that vitamin A supplementation can modulate the efficiency of ChR2 and provide a complementary strategy for improving the performance of optogenetic tools.

Stimulation or Cancellation of Ca2+ Influx by Bipolar Nanosecond Pulsed Electric Fields in Adrenal Chromaffin Cells Can Be Achieved by Tuning Pulse Waveform.

Exposing adrenal chromaffin cells to single 150 to 400 ns electric pulses triggers a rise in intracellular Ca2+ ([Ca2+]i) that is due to Ca2+ influx through voltage-gated Ca2+ channels (VGCC) and plasma membrane electropores. Immediate delivery of a second pulse of the opposite polarity in which the duration and amplitude were the same as the first pulse (a symmetrical bipolar pulse) or greater than the first pulse (an asymmetrical bipolar pulse) had a stimulatory effect, evoking larger Ca2+ responses than the corresponding unipolar pulse. Progressively decreasing the amplitude of the opposite polarity pulse while also increasing its duration converted stimulation to attenuation, which reached a maximum of 43% when the positive phase was 150 ns at 3.1 kV/cm, and the negative phase was 800 ns at 0.2 kV/cm. When VGCCs were blocked, Ca2+ responses evoked by asymmetrical and even symmetrical bipolar pulses were significantly reduced relative to those evoked by the corresponding unipolar pulse under the same conditions, indicating that attenuation involved mainly the portion of Ca2+ influx attributable to membrane electropermeabilization. Thus, by tuning the shape of the bipolar pulse, Ca2+ entry into chromaffin cells through electropores could be attenuated while preserving Ca2+ influx through VGCCs.

Membrane potential manipulation with visible flash lamp illumination of targeted microbeads.

The electrical membrane potential (Vm) is a key dynamical variable of excitable membranes. Despite the tremendous success of optogenetic methods to modulate Vm with light, there are some shortcomings, such as the need of genetic manipulation and limited time resolution. Direct optical stimulation of gold nanoparticles targeted to cells is an attractive alternative because the absorbed energy heats the membrane and, thus, generates capacitive current sufficient to trigger action potentials [1, Carvalho-de-Souza et al., 2015]. However, focused laser light is required and precise location and binding of the nanoparticles cannot be assessed with a conventional microscope. We therefore examined a complementary method to manipulate Vm in a spatio-temporal fashion by non-focused visible flashlight stimulation (Xenon discharge lamp, 385-485 nm, ∼500 µs) of superparamagnetic microbeads. Flashlight stimulation of single beads targeted to cells resulted in transient inward currents under whole-cell patch-clamp control. The waveform of the current reflected the first time derivative of the local temperature induced by the absorbed light and subsequent heat dissipation. The maximal peak current as well as the temperature excursion scaled with the proximity to the plasma membrane. Transient illumination of light-absorbing beads, targeted to specific cellular sites via protein-protein interaction or direct micromanipulation, may provide means of rapid and spatially confined heating and electrical cell stimulation.

Effect of pulsed millisecond current magnetic field on the proliferation of C6 rat glioma cells.

In recent years, using electromagnetic fields as a targeted therapy for tumors has become a new idea. This paper aims to study the response of rat glioma cells (C6) when the external electromagnetic field parameters change and to obtain a complete working range of magnetic field parameters. Four-day, 4-h daily millisecond magnetic field exposure experiments were performed with C6 cells. The peak values of magnetic field intensity were 260 mT, 90 mT, 19 mT and 6 mT. Each day after exposure, cell morphology and cell viability assay (MTT method) were measured. The response of C6 cells shows a significant window effect and time cumulative effect on the cell, and it is non-destructive. The working inhibited magnetic field range of magnetic field increase rate dB/dt (T/s) is [34, 119.5] and [166.75, 527.25], the magnetic field amplitude B (mT) is [6, 260], the magnetic field integral Bt (mT·s) is [0.1649, 0.8085] and the energy integral B2t (mT2·s) is [2.317, 53.328]. Our findings provide the theoretical and experimental basis for clinical applications of electromagnetic fields.

Resting-state fMRI study of brain activation using low-intensity repetitive transcranial magnetic stimulation in rats.

Repetitive transcranial magnetic stimulation (rTMS) is a non-invasive neuromodulation technique used to treat many neuropsychiatric conditions. However, the mechanisms underlying its mode of action are still unclear. This is the first rodent study using resting-state functional MRI (rs-fMRI) to examine low-intensity (LI) rTMS effects, in an effort to provide a direct means of comparison between rodent and human studies. Using anaesthetised Sprague-Dawley rats, rs-fMRI data were acquired before and after control or LI-rTMS at 1 Hz, 10 Hz, continuous theta burst stimulation (cTBS) or biomimetic high-frequency stimulation (BHFS). Independent component analysis revealed LI-rTMS-induced changes in the resting-state networks (RSN): (i) in the somatosensory cortex, the synchrony of resting activity decreased ipsilaterally following 10 Hz and bilaterally following 1 Hz stimulation and BHFS, and increased ipsilaterally following cTBS; (ii) the motor cortex showed bilateral changes following 1 Hz and 10 Hz stimulation, a contralateral decrease in synchrony following BHFS, and an ipsilateral increase following cTBS; and (iii) hippocampal synchrony decreased ipsilaterally following 10 Hz, and bilaterally following 1 Hz stimulation and BHFS. The present findings demonstrate that LI-rTMS modulates functional links within the rat RSN with frequency-specific outcomes, and the observed changes are similar to those described in humans following rTMS.

Persistent nature of alterations in cognition and neuronal circuit excitability after exposure to simulated cosmic radiation in mice.

Of the many perils associated with deep space travel to Mars, neurocognitive complications associated with cosmic radiation exposure are of particular concern. Despite these realizations, whether and how realistic doses of cosmic radiation cause cognitive deficits and neuronal circuitry alterations several months after exposure remains unclear. In addition, even less is known about the temporal progression of cosmic radiation-induced changes transpiring over the duration of a time period commensurate with a flight to Mars. Here we show that rodents exposed to the second most prevalent radiation type in space (i.e. helium ions) at low, realistic doses, exhibit significant hippocampal and cortical based cognitive decrements lasting 1 year after exposure. Cosmic-radiation-induced impairments in spatial, episodic and recognition memory were temporally coincident with deficits in cognitive flexibility and reduced rates of fear extinction, elevated anxiety and depression like behavior. At the circuit level, irradiation caused significant changes in the intrinsic properties (resting membrane potential, input resistance) of principal cells in the perirhinal cortex, a region of the brain implicated by our cognitive studies. Irradiation also resulted in persistent decreases in the frequency and amplitude of the spontaneous excitatory postsynaptic currents in principal cells of the perirhinal cortex, as well as a reduction in the functional connectivity between the CA1 of the hippocampus and the perirhinal cortex. Finally, increased numbers of activated microglia revealed significant elevations in neuroinflammation in the perirhinal cortex, in agreement with the persistent nature of the perturbations in key neuronal networks after cosmic radiation exposure. These data provide new insights into cosmic radiation exposure, and reveal that even sparsely ionizing particles can disrupt the neural circuitry of the brain to compromise cognitive function over surprisingly protracted post-irradiation intervals.

Kinetic profiles of photocurrents in cells expressing two types of channelrhodopsin genes.

Channelrhodopsin-2 (ChR2), a light-activated cation-selective ion channel, has been widely used as a tool in optogenetic research. ChR2 is specifically sensitive to wavelengths less than 550 nm. One of the methods to expand the sensitivity of a channelrhodopsin to a wider range of wavelengths is to express another channelrhodopsin in the cells by the transduction of an additional gene. Here, we report the characteristic features of cells expressing two types of channelrhodopsins, each having different wavelength sensitivities. In HEK293 cells stably expressing ChR2, photocurrents were elicited at stimuli of 400-550 nm, and the wavelength sensitivity range was expanded by the additional transduction of the modified Volvox channelrhodopsin-1 (mVChR1) gene, which has broad wavelength sensitivities, ranging from 400 to 600 nm. However, the photocurrent at 550 nm was lower than that of the mVChR1-expressing cell; moreover, the turning-on and turning-off constants were delayed, and the deactivation rates were decreased. Meanwhile, the response to lower light intensity was improved by the additional gene. Thus, the transduction of an additional gene is a useful method to improve the light and wavelength sensitivities, as well as photocurrent kinetic profiles, of channelrhodopsins.

Quantifying the effect of light activated outer and inner retinal inhibitory pathways on glutamate release from mixed bipolar cells.

Inhibition mediated by horizontal and amacrine cells in the outer and inner retina, respectively, are fundamental components of visual processing. Here, our purpose was to determine how these different inhibitory processes affect glutamate release from ON bipolar cells when the retina is stimulated with full-field light of various intensities. Light-evoked membrane potential changes (ΔVm ) were recorded directly from axon terminals of intact bipolar cells receiving mixed rod and cone inputs (Mbs) in slices of dark-adapted goldfish retina. Inner and outer retinal inhibition to Mbs was blocked with bath applied picrotoxin (PTX) and NBQX, respectively. Then, control and pharmacologically modified light responses were injected into axotomized Mb terminals as command potentials to induce voltage-gated Ca2+ influx (QCa ) and consequent glutamate release. Stimulus-evoked glutamate release was quantified by the increase in membrane capacitance (ΔCm ). Increasing depolarization of Mb terminals upon removal of inner and outer retinal inhibition enhanced the ΔVm /QCa ratio equally at a given light intensity and inhibition did not alter the overall relation between QCa and ΔCm . However, relative to control, light responses recorded in the presence of PTX and PTX + NBQX increased ΔCm unevenly across different stimulus intensities: at dim stimulus intensities predominantly the inner retinal GABAergic inhibition controlled release from Mbs, whereas the inner and outer retinal inhibition affected release equally in response to bright stimuli. Furthermore, our results suggest that non-linear relationship between QCa and glutamate release can influence the efficacy of inner and outer retinal inhibitory pathways to mediate Mb output at different light intensities.

Modeling of Electroporation Induced by Pulsed Electric Fields in Irregularly Shaped Cells.

During the past decades, the poration of cell membrane induced by pulsed electric fields has been widely investigated. Since the basic mechanisms of this process have not yet been fully clarified, many research activities are focused on the development of suitable theoretical and numerical models. To this end, a nonlinear, nonlocal, dispersive, and space-time numerical algorithm has been developed and adopted to evaluate the transmembrane voltage and pore density along the perimeter of realistic irregularly shaped cells. The presented model is based on the Maxwell's equations and the asymptotic Smoluchowski's equation describing the pore dynamics. The dielectric dispersion of the media forming the cell has been modeled by using a general multirelaxation Debye-based formulation. The irregular shape of the cell is described by using the Gielis' superformula. Different test cases pertaining to red blood cells, muscular cells, cell in mitosis phase, and cancer-like cell have been investigated. For each type of cell, the influence of the relevant shape, the dielectric properties, and the external electric pulse characteristics on the electroporation process has been analyzed. The numerical results demonstrate that the proposed model is an efficient numerical tool to study the electroporation problem in arbitrary-shaped cells.

Detergent-resistant oligomeric Leptosphaeria rhodopsin is a promising bio-nanomaterial and an alternative to bacteriorhodopsin.

Bacteriorhodopsin has attracted remarkable attention as a photoactive bio-nanomaterial in the last decades. However, its instability in the presence of detergents has restricted the extent to which bacteriorhodopsin may be applied. In this study, we investigated the oligomerization of a eukaryotic light-driven H+-pump, Leptosphaeria rhodopsin, using circular dichroism spectroscopy and other biophysical and biochemical methods. Our findings revealed that Leptosphaeria rhodopsin assembled into oligomers in the cell membrane and also in 0.05% DDM detergent micelles. Moreover, unlike bacteriorhodopsin in purple membrane, Leptosphaeria rhodopsin retained its oligomeric structure in 1% Triton X-100 and demonstrated strong resistance to other common detergents. A maximal photocurrent density of ∼85 nA/cm2 was consistently generated, which was substantially larger than that of solubilized bacteriorhodopsin (∼10 nA/cm2). Therefore, oligomeric Leptosphaeria rhodopsin may be a promising bio-nanomaterial, and an alternative to bacteriorhodopsin, especially with the use of detergents.

Photo-Cross-Linking of IKs Demonstrates State-Dependent Interactions between KCNE1 and KCNQ1.

The slow delayed rectifier potassium current (IKs) is a key repolarizing current during the cardiac action potential. It consists of four KCNQ1 α-subunits and up to four KCNE1 ß-subunits, which are thought to reside within external clefts of the channel. The interaction of KCNE1 with KCNQ1 dramatically delays opening of the channel but the mechanisms by which this occur are not yet fully understood. Here, we have used unnatural amino acid photo-cross-linking to investigate the dynamic interactions that occur between KCNQ1 and KCNE1 during activation gating. The unnatural amino acid p-Benzoylphenylalanine was successfully incorporated into two residues within the transmembrane domain of KCNE1: F56 and F57. UV-induced cross-linking suggested that F56Bpa interacts with KCNQ1 in the open state, whereas F57Bpa interacts predominantly in resting channel conformations. When UV was applied at progressively more depolarized preopen holding potentials, cross-linking of F57Bpa with KCNQ1 was slowed, which indicates that KCNE1 is displaced within the channel's cleft early during activation, or that conformational changes in KCNQ1 alter its interaction with KCNE1. In E1R/R4E KCNQ1, a mutant with constitutively activated voltage sensors, F56Bpa and F57Bpa KCNE1 were cross-linked in open and closed states, respectively, which suggests that their actions are mediated mainly by modulation of KCNQ1 pore function.