Developing Fast Fluorescent Protein Voltage Sensors by Optimizing FRET Interactions

FRET (Förster Resonance Energy Transfer)-based protein voltage sensors can be useful for monitoring neuronal activity in vivo because the ratio of signals between the donor and acceptor pair reduces common sources of noise such as heart beat artifacts. We improved the performance of FRET based genetically encoded Fluorescent Protein (FP) voltage sensors by optimizing the location of donor and acceptor FPs flanking the voltage sensitive domain of the Ciona intestinalis voltage sensitive phosphatase. First, we created 39 different “Nabi1” constructs by positioning the donor FP, UKG, at 8 different locations downstream of the voltage-sensing domain and the acceptor FP, mKO, at 6 positions upstream. Several of these combinations resulted in large voltage dependent signals and relatively fast kinetics. Nabi1 probes responded with signal size up to 11% ΔF/F for a 100 mV depolarization and fast response time constants both for signal activation (~2 ms) and signal decay (~3 ms). We improved expression in neuronal cells by replacing the mKO and UKG FRET pair with Clover (donor FP) and mRuby2 (acceptor FP) to create Nabi2 probes. Nabi2 probes also had large signals and relatively fast time constants in HEK293 cells. In primary neuronal culture, a Nabi2 probe was able to differentiate individual action potentials at 45 Hz.     

Single action potentials and subthreshold electrical events imaged in neurons with a fluorescent protein voltage probe

Monitoring neuronal electrical activity using fluorescent protein-based voltage sensors has been limited by small response magnitudes and slow kinetics of existing probes. Here we report the development of?a fluorescent protein voltage sensor, named ArcLight, and derivative probes that exhibit large changes in fluorescence intensity in response to voltage changes. ArcLight consists of the voltage-sensing domain of Ciona intestinalis voltage-sensitive phosphatase and super ecliptic pHluorin that carries the point mutation A227D. The fluorescence intensity of ArcLight A242 decreases by 35% in response to?a 100mV depolarization when measured in HEK293 cells, which is more than five times larger than the signals from previously reported fluorescent protein voltage sensors. We show that the combination of signal size and response speed of these new probes allows the reliable detection of single action potentials and excitatory potentials in individual neurons and dendrites.     

Mechanistic Studies of the Genetically Encoded Fluorescent Protein Voltage Probe ArcLight

ArcLight, a genetically encoded fluorescent protein voltage probe with a large ΔF/ΔV, is a fusion between the voltage sensing domain of the Ciona instestinalis voltage sensitive phosphatase and super ecliptic pHluorin carrying a single mutation (A227D in the fluorescent protein). Without this mutation the probe produces only a very small change in fluorescence in response to voltage deflections (∼1%). The large signal afforded by this mutation allows optical detection of action potentials and sub-threshold electrical events in single-trials in vitro and in vivo. However, it is unclear how this single mutation produces a probe with such a large modulation of its fluorescence output with changes in membrane potential. In this study, we identified which residues in super ecliptic pHluorin (vs eGFP) are critical for the ArcLight response, as a similarly constructed probe based on eGFP also exhibits large response amplitude if it carries these critical residues. We found that D147 is responsible for determining the pH sensitivity of the fluorescent protein used in these probes but by itself does not result in a voltage probe with a large signal. We also provide evidence that the voltage dependent signal of ArcLight is not simply sensing environmental pH changes. A two-photon polarization microscopy study showed that ArcLight''s response to changes in membrane potential includes a reorientation of the super ecliptic pHluorin. We also explored different changes including modification of linker length, deletion of non-essential amino acids in the super ecliptic pHluorin, adding a farnesylation site, using tandem fluorescent proteins and other pH sensitive fluorescent proteins.     

Enhanced Archaerhodopsin Fluorescent Protein Voltage Indicators

A longstanding goal in neuroscience has been to develop techniques for imaging the voltage dynamics of genetically defined subsets of neurons. Optical sensors of transmembrane voltage would enhance studies of neural activity in contexts ranging from individual neurons cultured in vitro to neuronal populations in awake-behaving animals. Recent progress has identified Archaerhodopsin (Arch) based sensors as a promising, genetically encoded class of fluorescent voltage indicators that can report single action potentials. Wild-type Arch exhibits sub-millisecond fluorescence responses to trans-membrane voltage, but its light-activated proton pump also responds to the imaging illumination. An Arch mutant (Arch-D95N) exhibits no photocurrent, but has a slower, ~40 ms response to voltage transients. Here we present Arch-derived voltage sensors with trafficking signals that enhance their localization to the neural membrane. We also describe Arch mutant sensors (Arch-EEN and -EEQ) that exhibit faster kinetics and greater fluorescence dynamic range than Arch-D95N, and no photocurrent at the illumination intensities normally used for imaging. We benchmarked these voltage sensors regarding their spike detection fidelity by using a signal detection theoretic framework that takes into account the experimentally measured photon shot noise and optical waveforms for single action potentials. This analysis revealed that by combining the sequence mutations and enhanced trafficking sequences, the new sensors improved the fidelity of spike detection by nearly three-fold in comparison to Arch-D95N.     

Excitation-Contraction Coupling: The distinct role of the four voltage sensors of the skeletal CaV1.1 channel in voltage-dependent activation

Initiation of skeletal muscle contraction is triggered by rapid activation of RYR1 channels in response to sarcolemmal depolarization. RYR1 is intracellular and has no voltage-sensing structures, but it is coupled with the voltage-sensing apparatus of Ca_V1.1 channels to inherit voltage sensitivity. Using an opto-electrophysiological approach, we resolved the excitation-driven molecular events controlling both Ca_V1.1 and RYR1 activations, reported as fluorescence changes. We discovered that each of the four human Ca_V1.1 voltage-sensing domains (VSDs) exhibits unique biophysical properties: VSD-I time-dependent properties were similar to ionic current activation kinetics, suggesting a critical role of this voltage sensor in Ca_V1.1 activation; VSD-II, VSD-III, and VSD-IV displayed faster activation, compatible with kinetics of sarcoplasmic reticulum Ca²⁺ release. The prominent role of VSD-I in governing Ca_V1.1 activation was also confirmed using a naturally occurring, charge-neutralizing mutation in VSD-I (R174W). This mutation abolished Ca_V1.1 current at physiological membrane potentials by impairing VSD-I activation without affecting the other VSDs. Using a structurally relevant allosteric model of Ca_V activation, which accounted for both time- and voltage-dependent properties of Ca_V1.1, to predict VSD-pore coupling energies, we found that VSD-I contributed the most energy (~75 meV or ∼3 kT) toward the stabilization of the open states of the channel, with smaller (VSD-IV) or negligible (VSDs II and III) energetic contribution from the other voltage sensors (<25 meV or ∼1 kT). This study settles the longstanding question of how Ca_V1.1, a slowly activating channel, can trigger RYR1 rapid activation, and reveals a new mechanism for voltage-dependent activation in ion channels, whereby pore opening of human Ca_V1.1 channels is primarily driven by the activation of one voltage sensor, a mechanism distinct from that of all other voltage-gated channels.     

Charge movement in a fast twitch skeletal muscle from rat

Voltage-dependent charge movement in the rat omohyoid muscle was investigated using the three microelectrode voltage clamp technique. The charge that moved during a depolarization from the holding potential (-90 mV) to the test potential, V, increased with increasing V, saturating around 0 mV. The charge vs. voltage relationship was well fitted by Q = Q_max/{1 + exp[-(V - V)/k]}, with Q_max = 28.5 nC/μF, V = -34.2 mV, and k = 8.7 mV. Repolarization of the fiber from the test potential back to the holding potential caused an equal but opposite amount of charge to move. The kinetics of ON charge movement could be well described by a model developed for frog muscle by Horowicz and Schneider (1981b), which suggests that rat and frog charge movements are similar. This model failed to describe the kinetics of OFF charge movement for steps in potential from 0 mV to test potentials of -10 to -90 mV. OFF-charge movement rose to a peak more slowly and decayed more slowly than predicted by the theory.     

Two-Photon Lifetime Imaging of Voltage Indicating Proteins as a Probe of Absolute Membrane Voltage

Genetically encoded voltage indicators (GEVIs) can report cellular electrophysiology with high resolution in space and time. Two-photon (2P) fluorescence has been explored as a means to image voltage in tissue. Here, we used the 2P electronic excited-state lifetime to probe absolute membrane voltage in a manner that is insensitive to the protein expression level, illumination intensity, or photon detection efficiency. First, we tested several GEVIs for 2P brightness, response speed, and voltage sensitivity. ASAP1 and a previously described citrine-Arch electrochromic Förster resonance energy transfer sensor (dubbed CAESR) showed the best characteristics. We then characterized the voltage-dependent lifetime of ASAP1, CAESR, and ArcLight under voltage-clamp conditions. ASAP1 and CAESR showed voltage-dependent lifetimes, whereas ArcLight did not. These results establish 2P fluorescence lifetime imaging as a viable means of measuring absolute membrane voltage. We discuss the prospects and improvements necessary for applications in tissue.     

Importance of the Voltage Dependence of Cardiac Na/K ATPase Isozymes

Cardiac cells express more than one isoform of the Na, K-ATPase (NKA), the heteromeric enzyme that creates the Na⁺ and K⁺ gradients across the plasmalemma. Cardiac isozymes contain one catalytic α-subunit isoform (α1, α2, or α3) associated with an auxiliary β-subunit isoform (β1 or β2). Past studies using biochemical approaches have revealed minor kinetic differences between isozymes formed by different α-β isoform combinations; these results make it difficult to understand the physiological requirement for multiple isoforms. In intact cells, however, NKA enzymes operate in a more complex environment, which includes a substantial transmembrane potential. We evaluated the voltage dependence of human cardiac NKA isozymes expressed in Xenopus oocytes, and of native NKA isozymes in rat ventricular myocytes, using normal mammalian physiological concentrations of Na⁺_o and K⁺_o. We demonstrate that although α1 and α3 pumps are functional at all physiologically relevant voltages, α2β1 pumps and α2β2 pumps are inhibited by ∼75% and ∼95%, respectively, at resting membrane potentials, and only activate appreciably upon depolarization. Furthermore, phospholemman (FXYD1) inhibits pump function without significantly altering the pump’s voltage dependence. Our observations provide a simple explanation for the physiological relevance of the α2 subunit (∼20% of total α subunits in rat ventricle): they act as a reserve and are recruited into action for extra pumping during the long-lasting cardiac action potential, where most of the Na⁺ entry occurs. This strong voltage dependence of α2 pumps also helps explain how cardiotonic steroids, which block NKA pumps, can be a beneficial treatment for heart failure: by only inhibiting the α2 pumps, they selectively reduce NKA activity during the cardiac action potential, leading to an increase in systolic Ca²⁺, due to reduced extrusion through the Na/Ca exchanger, without affecting resting Na⁺ and Ca²⁺ concentrations.     

Kinetic diversity of single-channel burst openings underlying persistent Na(+) current in entorhinal cortex neurons

The kinetic diversity of burst openings responsible for the persistent Na⁺ current (I_NaP) in entorhinal cortex neurons was examined by separately analyzing single bursts. Although remarkable kinetic variability was observed among bursts in terms of intraburst opening probability and mean open and closed times, the values of time constants describing intraburst open times (τ_o(b)s) and closed times (τ_c(b)s) were distributed around well-defined peaks. At −40 mV, τ_o(b) peaks were found at ~0.34 (τ_o(b)1) and 0.77 (τ_o(b)2) ms, and major τ_c(b) peaks were found at ~0.24 (τ_c(b)1) and 0.54 (τ_c(b)2) ms. In ~80% of the bursts two preferential gating modes were found that consisted of a combination of either τ_o(b)1 and τ_c(b)2 (“intraburst mode 1”), or τ_o(b)2 and τ_c(b)1 (“intraburst mode 2”). Individual channels could switch between different gating modalities, but normally tended to maintain a specific gating mode for long periods. Mean burst duration also displayed considerable variability. At least three time constants were found to describe burst duration, and the frequencies at which each of the corresponding “bursting states” occurred varied in different channels. Short-lasting bursting states were preferentially associated with intraburst mode 1, whereas very-long-lasting bursts tended to gate according to mode 2 only or other modes that included considerably longer mean open times. These results show that I_NaP channels can generate multiple intraburst open and closed states and bursting states, but these different kinetic states tend to combine in definite ways to produce a limited number of prevalent, well-defined gating modalities. Modulation of distinct gating modalities in individual Na⁺ channels may be a powerful form of plasticity to influence neuronal excitability and function.     

Voltage and Current Clamp Transients with Membrane Dielectric Loss

Transient responses of a space-clamped squid axon membrane to step changes of voltage or current are often approximated by exponential functions of time, corresponding to a series resistance and a membrane capacity of 1.0 μF/cm². Curtis and Cole (1938, J. Gen. Physiol. 21:757) found, however, that the membrane had a constant phase angle impedance z = z₁(jωτ)^-α, with a mean α = 0.85. (α = 1.0 for an ideal capacitor; α < 1.0 may represent dielectric loss.) This result is supported by more recently published experimental data. For comparison with experiments, we have computed functions expressing voltage and current transients with constant phase angle capacitance, a parallel leakage conductance, and a series resistance, at nine values of α from 0.5 to 1.0. A series in powers of t^α provided a good approximation for short times; one in powers of t^-α, for long times; for intermediate times, a rational approximation matching both series for a finite number of terms was used. These computations may help in determining experimental series resistances and parallel leakage conductances from membrane voltage or current clamp data.     

Estimation of the volumetric elastic modulus and membrane hydraulic conductivity of willow sieve tubes

Severed aphid stylets were used to follow the kinetics of sieve tube turgor and osmotic pressure (π) responses following step changes in water potential applied to the cambial surface of willow (Salix exigua Nutt.) bark strips. The kinetics of the turgor response were monitored with a pressure transducer. In separate experiments, the kinetics of the π response were followed by freezing point determinations on stylet exudate. The sieve tube volumetric elastic modulus in the bark strips was about 21 bars, but may be higher in intact stems. The membrane hydraulic conductivity was about 5 × 10⁻³ centimeters per second per bar; several factors make it difficult to estimate its value accurately. Differences in the turgor pressure (P) and π responses, as well as the relatively more rapid initial turgor response to a water potential (ψ) change, suggested a time-dependent component in sieve tube wall elasticity.

Our observations were generally not supportive of the idea that sieve tubes might osmoregulate. However, the bark strip system may not be suitable for addressing that question.

Separate measurements of ψ, P, and π demonstrate that the relationship predicted by the fundamental cell water potential equation, ψ = P − π, is applicable within experimental error (± 0.4 bar) to sieve tube water relations.

     

Distinct roles for CaV1.1’s voltage-sensing domains

Study reveals how a slowly activating calcium channel is able to control rapid excitation–contraction coupling in skeletal muscle.

Skeletal muscle contraction is initiated by action potentials that depolarize the muscle fiber and trigger the rapid release of Ca²⁺ from the SR via RYR1 channels. This process of excitation–contraction coupling depends on voltage-gated Ca_V1.1 channels in the plasma membrane, or sarcolemma, of muscle fibers. But Ca_V1.1 channels are only slowly activated by changes in the sarcolemma membrane potential, and it is therefore unclear how they are able to trigger the much faster activation of RYR1 channels. In this issue of JGP, Savalli et al. reveal that this paradox can be explained by the fact that each of Ca_V1.1’s four voltage-sensing domains (VSDs) have distinct biophysical properties (1).Nicoletta Savalli (left), Riccardo Olcese (center), and colleagues reveal the distinct physical properties of the Ca_V1.1 channel’s four voltage-sensing domains (VSD I–IV, right). VSD-I shows slow activation kinetics and is the main contributor to the opening of Ca_V1.1. The other VSDs activate much faster and may therefore be coupled to RYR1 to mediate the rapid release of Ca²⁺ from the SR during skeletal muscle contraction.RYR1 channels have no voltage-sensing machinery of their own and therefore rely on a physical connection to Ca_V1.1 channels to release Ca²⁺ and initiate muscle contraction in response to muscle fiber depolarization. But RYR1 channels open ∼25 times faster than Ca_V1.1 channels. “So, how can these slowly activating Ca_V1.1 channels trigger the rapid release of Ca²⁺ from the SR?” asks Riccardo Olcese, a professor at the David Geffen School of Medicine, UCLA.Olcese and colleagues, including Assistant Project Scientist Nicoletta Savalli, suspected that the answer might lie in the fact that, like many other voltage-gated ion channels, Ca_V1.1 has four VSDs that alter their conformation in response to voltage changes. These domains are similar, but not identical, to each other, potentially enabling them to have distinct biophysical properties and perform distinct functions. Indeed, Olcese and colleagues previously demonstrated that, in the closely related channel Ca_V1.2, only VSDs II and III are involved in pore opening (2, 3).Savalli et al. used voltage-clamp fluorometry to compare the properties of Ca_V1.1’s VSDs, expressing the channel in Xenopus oocytes and labeling each of its VSDs in turn with an environmentally sensitive fluorophore to report voltage-dependent changes in their conformation (1). “We found that the four VSDs were very heterogenous in both their kinetics and voltage dependencies,” says Olcese. “VSD-I had very slow kinetics, compatible with the slow activation of the Ca_V1.1 pore. The other three VSDs had much faster kinetics and could, therefore, be good candidates to be the voltage sensors for RYR1 activation.”Olcese and colleagues confirmed the importance of VSD-I for Ca_V1.1 activation by analyzing a naturally occurring, charge-neutralizing mutation in this domain, R174W, that is linked to malignant hyperthermia (4). The team found that this mutation reduced the voltage-sensitivity of VSD-I and abolished the ability of Ca_V1.1 to conduct Ca²⁺ at physiological membrane potentials, but had no effect on the behavior of the other three VSDs.Finally, Savalli et al. applied their data on both the wild-type and mutant VSDs to an allosteric model of Ca_V activation (2, 3), which predicted that VSD-I contributes most of the energy required to stabilize the open state of Ca_V1.1, while the other VSDs contribute little to nothing.Thus, Ca_V1.1 activation is mainly driven by a single VSD—a mechanism that hasn’t been seen in any other voltage-gated ion channel—leaving the other VSDs free to perform other functions, such as the rapid activation of RYR1. Olcese and colleagues now want to pinpoint exactly which VSD(s) are coupled to RYR1 and determine how they trigger rapid Ca²⁺ release from the SR.     

Reversible thermal denaturation of a 60-kDa genetically engineered beta-sheet polypeptide

A de novo 687-amino-acid residue polypeptide with a regular 32-amino-acid repeat sequence, (GA)₃GY(GA)₃GE(GA)₃GH(GA)₃GK, forms large β-sheet assemblages that exhibit remarkable folding properties and, as well, form fibrillar structures. This construct is an excellent tool to explore the details of β-sheet formation yielding intimate folding information that is otherwise difficult to obtain and may inform folding studies of naturally occurring materials. The polypeptide assumes a fully folded antiparallel β-sheet/turn structure at room temperature, and yet is completely and reversibly denatured at 125°C, adopting a predominant polyproline II conformation. Deep ultraviolet Raman spectroscopy indicated that melting/refolding occurred without any spectroscopically distinct intermediates, yet the relaxation kinetics depend on the initial polypeptide state, as would be indicative of a non-two-state process. Thermal denaturation and refolding on cooling appeared to be monoexponential with characteristic times of ~1 and ~60 min, respectively, indicating no detectable formation of hairpin-type nuclei in the millisecond timescale that could be attributed to nonlocal “nonnative” interactions. The polypeptide folding dynamics agree with a general property of β-sheet proteins, i.e., initial collapse precedes secondary structure formation. The observed folding is much faster than expected for a protein of this size and could be attributed to a less frustrated free-energy landscape funnel for folding. The polypeptide sequence suggests an important balance between the absence of strong nonnative contacts (salt bridges or hydrophobic collapse) and limited repulsion of charged side chains.     

Phe-Met-Arg-Phe-amide Activates a Novel Voltage-dependent K+ Current through a Lipoxygenase Pathway in Molluscan Neurones

The neuropeptide Phe-Met-Arg-Phe-amide (FMRFa) dose dependently (ED₅₀ = 23 nM) activated a K⁺ current in the peptidergic caudodorsal neurones that regulate egg laying in the mollusc Lymnaea stagnalis. Under standard conditions ([K⁺]_o = 1.7 mM), only outward current responses occurred. In high K⁺ salines ([K⁺]_o = 20 or 57 mM), current reversal occurred close to the theoretical reversal potential for K⁺. In both salines, no responses were measured below −120 mV. Between −120 mV and the K⁺ reversal potential, currents were inward with maximal amplitudes at ∼−60 mV. Thus, U-shaped current–voltage relations were obtained, implying that the response is voltage dependent. The conductance depended both on membrane potential and extracellular K⁺ concentration. The voltage sensitivity was characterized by an e-fold change in conductance per ∼14 mV at all [K⁺]_o. Since this result was also obtained in nearly symmetrical K⁺ conditions, it is concluded that channel gating is voltage dependent. In addition, outward rectification occurs in asymmetric K⁺ concentrations. Onset kinetics of the response were slow (rise time ∼650 ms at −40 mV). However, when FMRFa was applied while holding the cell at −120 mV, to prevent activation of the current but allow activation of the signal transduction pathway, a subsequent step to −40 mV revealed a much more rapid current onset. Thus, onset kinetics are largely determined by steps preceding channel activation. With FMRFa applied at −120 mV, the time constant of activation during the subsequent test pulse decreased from ∼36 ms at −60 mV to ∼13 ms at −30 mV, confirming that channel opening is voltage dependent. The current inactivated voltage dependently. The rate and degree of inactivation progressively increased from −120 to −50 mV. The current is blocked by internal tetraethylammonium and by bath- applied 4-aminopyridine, tetraethylammonium, Ba²⁺, and, partially, Cd²⁺ and Cs⁺. The response to FMRFa was affected by intracellular GTPγS. The response was inhibited by blockers of phospholipase A₂ and lipoxygenases, but not by a cyclo-oxygenase blocker. Bath-applied arachidonic acid induced a slow outward current and occluded the response to FMRFa. These results suggest that the FMRFa receptor couples via a G-protein to the lipoxygenase pathway of arachidonic acid metabolism. The biophysical and pharmacological properties of this transmitter operated, but voltage-dependent K⁺ current distinguish it from other receptor-driven K⁺ currents such as the S-current- and G-protein-dependent inward rectifiers.     

Different pathways for activation and deactivation in CaV1.2: a minimal gating model

Point mutations in pore-lining S6 segments of Ca_V1.2 shift the voltage dependence of activation into the hyperpolarizing direction and significantly decelerate current activation and deactivation. Here, we analyze theses changes in channel gating in terms of a circular four-state model accounting for an activation R–A–O and a deactivation O–D–R pathway. Transitions between resting-closed (R) and activated-closed (A) states (rate constants x(V) and y(V)) and open (O) and deactivated-open (D) states (u(V) and w(V)) describe voltage-dependent sensor movements. Voltage-independent pore openings and closures during activation (A–O) and deactivation (D–R) are described by rate constants α and β, and γ and δ, respectively. Rate constants were determined for 16-channel constructs assuming that pore mutations in IIS6 do not affect the activating transition of the voltage-sensing machinery (x(V) and y(V)). Estimated model parameters of 15 Ca_V1.2 constructs well describe the activation and deactivation processes. Voltage dependence of the “pore-releasing” sensor movement ((x(V)) was much weaker than the voltage dependence of “pore-locking” sensor movement (y(V)). Our data suggest that changes in membrane voltage are more efficient in closing than in opening Ca_V1.2. The model failed to reproduce current kinetics of mutation A780P that was, however, accurately fitted with individually adjusted x(V) and y(V). We speculate that structural changes induced by a proline substitution in this position may disturb the voltage-sensing domain.     

Single-Channel Kinetics,Inactivation, and Spatial Distribution of Inositol Trisphosphate (IP3) Receptors in Xenopus Oocyte Nucleus

Single-channel properties of the Xenopus inositol trisphosphate receptor (IP₃R) ion channel were examined by patch clamp electrophysiology of the outer nuclear membrane of isolated oocyte nuclei. With 140 mM K⁺ as the charge carrier (cytoplasmic [IP₃] = 10 μM, free [Ca²⁺] = 200 nM), the IP₃R exhibited four and possibly five conductance states. The conductance of the most-frequently observed state M was 113 pS around 0 mV and ∼300 pS at 60 mV. The channel was frequently observed with high open probability (mean P _o = 0.4 at 20 mV). Dwell time distribution analysis revealed at least two kinetic states of M with time constants τ < 5 ms and ∼20 ms; and at least three closed states with τ ∼1 ms, ∼10 ms, and >1 s. Higher cytoplasmic potential increased the relative frequency and τ of the longest closed state. A novel “flicker” kinetic mode was observed, in which the channel alternated rapidly between two new conductance states: F₁ and F₂. The relative occupation probability of the flicker states exhibited voltage dependence described by a Boltzmann distribution corresponding to 1.33 electron charges moving across the entire electric field during F₁ to F₂ transitions. Channel run-down or inactivation (τ ∼ 30 s) was consistently observed in the continuous presence of IP₃ and the absence of change in [Ca²⁺]. Some (∼10%) channel disappearances could be reversed by an increase in voltage before irreversible inactivation. A model for voltage-dependent channel gating is proposed in which one mechanism controls channel opening in both the normal and flicker modes, whereas a separate independent mechanism generates flicker activity and voltage- reversible inactivation. Mapping of functional channels indicates that the IP₃R tends to aggregate into microscopic (<1 μm) as well as macroscopic (∼10 μm) clusters. Ca²⁺-independent inactivation of IP₃R and channel clustering may contribute to complex [Ca²⁺] signals in cells.     

Components of gating charge movement and S4 voltage-sensor exposure during activation of hERG channels

The human ether-á-go-go–related gene (hERG) K⁺ channel encodes the pore-forming α subunit of the rapid delayed rectifier current, I_Kr, and has unique activation gating kinetics, in that the α subunit of the channel activates and deactivates very slowly, which focuses the role of I_Kr current to a critical period during action potential repolarization in the heart. Despite its physiological importance, fundamental mechanistic properties of hERG channel activation gating remain unclear, including how voltage-sensor movement rate limits pore opening. Here, we study this directly by recording voltage-sensor domain currents in mammalian cells for the first time and measuring the rates of voltage-sensor modification by [2-(trimethylammonium)ethyl] methanethiosulfonate chloride (MTSET). Gating currents recorded from hERG channels expressed in mammalian tsA201 cells using low resistance pipettes show two charge systems, defined as Q₁ and Q₂, with V_1/2’s of −55.7 (equivalent charge, z = 1.60) and −54.2 mV (z = 1.30), respectively, with the Q₂ charge system carrying approximately two thirds of the overall gating charge. The time constants for charge movement at 0 mV were 2.5 and 36.2 ms for Q₁ and Q₂, decreasing to 4.3 ms for Q₂ at +60 mV, an order of magnitude faster than the time constants of ionic current appearance at these potentials. The voltage and time dependence of Q₂ movement closely correlated with the rate of MTSET modification of I521C in the outermost region of the S4 segment, which had a V_1/2 of −64 mV and time constants of 36 ± 8.5 ms and 11.6 ± 6.3 ms at 0 and +60 mV, respectively. Modeling of Q₁ and Q₂ charge systems showed that a minimal scheme of three transitions is sufficient to account for the experimental findings. These data point to activation steps further downstream of voltage-sensor movement that provide the major delays to pore opening in hERG channels.     

Single-channel analysis of the anion channel-forming protein from the plant pathogenic bacterium Clavibacter michiganense ssp. nebraskense

The anion channel protein from Clavibacter michiganense ssp. nebraskense (Schürholz, Th. et al. 1991, J. Membrane Biol. 123: 1-8) was analyzed at different concentrations of KCl and KF. At 0.8 M KCl the conductance G(V_m) increases exponentially from 21 pS at 50 mV up to 53 pS at V_m = 200 mV, 20°C. The concentration dependence of G(V_m) corresponds to a Michaelis-Menten type saturation function at all membrane voltage values applied (0-200 mV). The anion concentration K_0.5, where G(V_m) has its half-maximum value, increases from 0.12 M at 50 mV to 0.24 M at 175 mV for channels in a soybean phospholipid bilayer. The voltage dependence of the single channel conductance, which is different for charged and neutral lipid bilayers, can be described either by a two-state flicker (2SF) model and the Nernst-Planck continuum theory, or by a two barrier, one-site (2B1S) model with asymmetric barriers. The increase in the number of open channels after a voltage jump from 50 mV to 150 mV has a time constant of 0.8 s. The changes of the single-channel conductance are much faster (<1 ms). The electric part of the gating process is characterized by the (reversible) molar electrical work ΔG^θ_el = ρZ_gFV_m ≈ -1.3 RT, which corresponds to the movement of one charge of the gating charge number |Z_g| = 1 across the fraction ρ = ΔV_m/V_m = 0.15 of the membrane voltage V_m = 200 mV. Unlike with chloride, the single channel conductance of fluoride has a maximum at about 150 mV in the presence of the buffer PIPES (≥5 mM, pH 6.8) with K_0.5 ≈ 1 M. It is shown that the decrease in conductance is due to a blocking of the channel by the PIPES anion. In summary, the results indicate that the anion transport by the Clavibacter anion channel (CAC) does not require a voltage dependent conformation change of the CAC.     

Activation and Two Modes of Blockade by Strontium of Ca2+-activated K+ Channels in Goldfish Saccular Hair Cells

Effects of internal Sr²⁺ on the activity of large-conductance Ca²⁺-activated K⁺ channels were studied in inside-out membrane patches from goldfish saccular hair cells. Sr²⁺ was approximately one-fourth as potent as Ca²⁺ in activating these channels. Although the Hill coefficient for Sr²⁺ was smaller than that for Ca²⁺, maximum open-state probability, voltage dependence, steady state gating kinetics, and time courses of activation and deactivation of the channel were very similar under the presence of equipotent concentrations of Ca²⁺ and Sr²⁺. This suggests that voltage-dependent activation is partially independent of the ligand. Internal Sr²⁺ at higher concentrations (>100 μM) produced fast and slow blockade both concentration and voltage dependently. The reduction in single-channel amplitude (fast blockade) could be fitted with a modified Woodhull equation that incorporated the Hill coefficient. The dissociation constant at 0 mV, the Hill coefficient, and zd (a product of the charge of the blocking ion and the fraction of the voltage difference at the binding site from the inside) in this equation were 58–209 mM, 0.69–0.75, 0.45–0.51, respectively (n = 4). Long shut events (slow blockade) produced by Sr²⁺ lasted ∼10–200 ms and could be fitted with single-exponential curves (time constant, τ_l−s) in shut-time histograms. Durations of burst events, periods intercalated by long shut events, could also be fitted with single exponentials (time constant, τ_b). A significant decrease in τ_b and no large changes in τ_l−s were observed with increased Sr²⁺ concentration and voltage. These findings on slow blockade could be approximated by a model in which single Sr²⁺ ions bind to a blocking site within the channel pore beyond the energy barrier from the inside, as proposed for Ba²⁺ blockade. The dissociation constant at 0 mV and zd in the Woodhull equation for this model were 36–150 mM and 1–1.8, respectively (n = 3).     

Permeation and Gating of an Inwardly Rectifying Potassium Channel : Evidence for a Variable Energy Well

Permeation, gating, and their interrelationship in an inwardly rectifying potassium (K⁺) channel, ROMK2, were studied using heterologous expression in Xenopus oocytes. Patch-clamp recordings of single channels were obtained in the cell-attached mode. The gating kinetics of ROMK2 were well described by a model having one open and two closed states. One closed state was short lived (∼1 ms) and the other was longer lived (∼40 ms) and less frequent (∼1%). The long closed state was abolished by EDTA, suggesting that it was due to block by divalent cations. These closures exhibit a biphasic voltage dependence, implying that the divalent blockers can permeate the channel. The short closures had a similar biphasic voltage dependence, suggesting that they could be due to block by monovalent, permeating cations. The rate of entering the short closed state varied with the K⁺ concentration and was proportional to current amplitude, suggesting that permeating K⁺ ions may be related to the short closures. To explain the results, we propose a variable intrapore energy well model in which a shallow well may change into a deep one, resulting in a normally permeant K⁺ ion becoming a blocker of its own channel.