EPR approaches to ion channel structure and function.

The fundamental processes that underlie ion channel function are permeation/ selectivity and gating. In an effort to understand ion channel gating, we have used an approach that combines reporter-group spectroscopic techniques (spin labelling/ electron paramagnetic resonance, EPR) and electrophysiological methods with classical biochemical and molecular biological procedures. As an ideal test channel, we have focused our attention on the K+ channel from Streptomyces lividans, KcsA. Through site-directed spin labelling, cysteine chemistry was used to introduce nitroxide radicals into specific sites within KcsA with high reactivity and specificity. EPR spectroscopy analysis of the spin labelled mutants yields two types of structural information: (1) mobility and solvent accessibility of the attached nitroxide through collisional relaxation methods and (2) distances between pairs of nitroxides through dipole-dipole interactions. Using this approach, we analysed the correlation between KcsA crystal structure and the EPR data, extend it to derive low-resolution folds of full-length KcsA and apply it in the determination of the molecular rearrangements responsible for pH-dependent gating.

Calculation of rigid-body conformational changes using restraint-driven Cartesian transformations.

We present an approach for calculating conformational changes in membrane proteins using limited distance information. The method, named restraint-driven Cartesian transformations, involves 1) the use of relative distance changes; 2) the systematic sampling of rigid body movements in Cartesian space; 3) a penalty evaluation; and 4) model refinement using energy minimization. As a test case, we have analyzed the structural basis of activation gating in the Streptomyces lividans potassium channel (KcsA). A total of 10 pairs of distance restraints derived from site-directed spin labeling and electron paramagnetic resonance (SDSL-EPR) spectra were used to calculate the open conformation of the second transmembrane domains of KcsA (TM2). The SDSL-EPR based structure reveals a gating mechanism consistent with a scissoring-type motion of the TM2 segments that includes a pivot point near middle of the helix. The present approach considerably reduces the amount of time and effort required to establish the overall nature of conformational changes in membrane proteins. It is expected that this approach can be implemented into restrained molecular dynamics protocol to calculate the structure and conformational changes in a variety of membrane protein systems.

Structure of the KcsA channel intracellular gate in the open state.

Ion channels catalyze the selective transfer of ions across the membrane in response to a variety of stimuli. These channels gate by controlling the access of ions to a centrally located water-filled pore. The crystal structure of the Streptomyces lividans potassium channel (KcsA) has allowed a molecular exploration of this mechanism. Electron paramagnetic resonance (EPR) studies have uncovered significant conformational changes at the intracellular end of the second transmembrane helix (TM2) upon gating. We have used site-directed spin labeling (SDSL) and EPR spectroscopy in an attempt to quantify the structural rearrangements of the KcsA TM2 bundle underlying the transition from the closed to the open state. Under conditions favoring the closed and open conformations, 10 intersubunit distances were obtained across TM2 segments from tandem dimer constructs. Analysis of these data points to a mechanism in which each TM2 helix tilts away from the permeation pathway, towards the membrane plane, and rotates about its helical axis, supporting a scissoring-type motion with a pivot point near residues 107-108. These movements are accompanied by a large increase in the diameter of the vestibule below the central water-filled cavity.

Site-directed spin-labeling analysis of reconstituted Mscl in the closed state.

The mechanosensitive channel from Escherichia coli (Eco-MscL) responds to membrane lateral tension by opening a large, water-filled pore that serves as an osmotic safety valve. In an attempt to understand the structural dynamics of MscL in the closed state and under physiological conditions, we have performed a systematic site-directed spin labeling study of this channel reconstituted in a membrane bilayer. Structural information was derived from an analysis of probe mobility, residue accessibility to O(2) or NiEdda and overall intersubunit proximity. For the majority of the residues studied, mobility and accessibility data showed a remarkable agreement with the Mycobacterium tuberculosis crystal structure, clearly identifying residues facing the large water-filled vestibule at the extracellular face of the molecule, the narrowest point along the permeation pathway (residues 21-26 of Eco-MscL), and the lipid-exposed residues in the peripheral transmembrane segments (TM2). Overall, the present dataset demonstrates that the transmembrane regions of the MscL crystal structure (obtained in detergent and at low pH) are, in general, an accurate representation of its structure in a membrane bilayer under physiological conditions. However, significant differences between the EPR data and the crystal structure were found toward the COOH-terminal end of TM2.

Molecular architecture of full-length KcsA: role of cytoplasmic domains in ion permeation and activation gating.

The molecular architecture of the NH(2) and COOH termini of the prokaryotic potassium channel KcsA has been determined using site-directed spin-labeling methods and paramagnetic resonance EPR spectroscopy. Cysteine mutants were generated (residues 5-24 and 121-160) and spin labeled, and the X-band CW EPR spectra were obtained from liposome-reconstituted channels at room temperature. Data on probe mobility (DeltaHo(-1)), accessibility parameters (PiO(2) and PiNiEdda), and inter-subunit spin-spin interaction (Omega) were used as structural constraints to build a three-dimensional folding model of these cytoplasmic domains from a set of simulated annealing and restrained molecular dynamics runs. 32 backbone structures were generated and averaged using fourfold symmetry, and a final mean structure was obtained from the eight lowest energy runs. Based on the present data, together with information from the KcsA crystal structure, a model for the three-dimensional fold of full-length KcsA was constructed. In this model, the NH(2) terminus of KcsA forms an alpha-helix anchored at the membrane-water interface, while the COOH terminus forms a right-handed four-helix bundle that extend some 40-50 A towards the cytoplasm. Functional analysis of COOH-terminal deletion constructs suggest that, while the COOH terminus does not play a substantial role in determining ion permeation properties, it exerts a modulatory role in the pH-dependent gating mechanism.

Structural rearrangements underlying K+-channel activation gating.

The intramembrane molecular events underlying activation gating in the Streptomyces K+ channel were investigated by site-directed spin-labeling methods and electron paramagnetic resonance spectroscopy. A comparison of the closed and open conformations of the channel revealed periodic changes in spin-label mobility and intersubunit spin-spin interaction consistent with rigid-body movements of the two transmembrane helices TM1 and TM2. These changes involve translations and counterclockwise rotations of both helices relative to the center of symmetry of the channel. The movement of TM2 increases the diameter of the permeation pathway along the point of convergence of the four subunits, thus opening the pore. Although the extracellular residues flanking the selectivity filter remained immobile during gating, small movements were detected at the C-terminal end of the pore helix, with possible implications to the gating mechanism.

Three-dimensional architecture and gating mechanism of a K+ channel studied by EPR spectroscopy.

The transmembrane organization of a potassium channel from Streptomyces lividans has been studied using site-directed spin labeling techniques and electron paramagnetic resonance spectroscopy. In the tetrameric channel complex, two alpha-helices were identified per monomer and assigned to the amino acid sequence. Probe mobility and accessibility data clearly establish that the first helix (TM1) is located in the perimeter of the channel, showing extensive protein-lipid contacts, while the second helix (TM2) is closer to the four-fold symmetric axis of the channel, lining the intracellular vestibule. A large conformational change in the C-terminal end of TM2 was measured when comparing conditions that favor either the open or closed states. The present data suggest that the diameter of the internal vestibule increases with channel opening.

pH-dependent gating in the Streptomyces lividans K+ channel.

Because of its size, high levels of expression, and unusual detergent stability, the small K+ channel from Streptomyces lividans (SKC1) is considered to be an ideal candidate for detailed structural analysis. In this paper, we have used planar lipid bilayers and radiotracer uptake experiments to study purified and reconstituted SKC1, in an attempt to develop a bulk assay for its functional characterization. In channels reconstituted into liposomes with external pH 3.5 and intravesicular pH 7.5, a time-dependent SKC1-catalyzed 86Rb+ uptake was observed. This cationic influx was blocked by Ba2+ ions with a Ki (external) of 0.4 mM and was shown to have the following selectivity sequence: K+ > Rb+ > NH4+ >> Na+ > Li+. In experiments with external pH 7.5 or in liposomes containing no channels, no 86Rb+ uptake was detected. When SKC1 was incorporated into planar lipid bilayers, we failed to observe significant single-channel activity at neutral pH but detected frequent multiple-channel openings a pH < 5.0. These results indicate that under these experimental conditions SKC1 behaves as a pH-gated K+ channel in which protonation of one or more residues promotes channel opening. At acidic pH and symmetrical 200 mM KCl solutions, SKC1 showed numerous brief openings with a main single-channel conductance of 135 pS and a subconductance state of 70 pS. Channel open probability showed a slight voltage dependence, with higher activities observed at negative potentials, a fact which may suggest that the protonation site lies within the transmembrane electrical field. Attempts to determine the pKa of channel activation were obscured by intrinsic limitations of the 86Rb+ flux assay. However, it appears to be lower than pH 4.0. Limited proteolysis experiments demonstrated that SKC1 reconstitutes vectorially, almost exclusively in the right-side-out configuration, indicating that the protonation site responsible for channel opening is located at the extracellular face of the channel. These results point toward a potentially novel gating mechanism for SKC1 and open the possibility of using transmembrane-driven radiotracer influx experiments as a reliable bulk functional assay for reconstituted SKC1.

Structural dynamics of the Streptomyces lividans K+ channel (SKC1): secondary structure characterization from FTIR spectroscopy.

Fourier transform infrared (FTIR) spectroscopy was used to probe the secondary structure, orientation, and the kinetics of amide hydrogen-deuterium exchange (HX) of the small K+ channel from Streptomyces lividans. Frequency component analysis of the amide I band showed that SKC1 is composed of 44-46% alpha-helix, 21-24% beta-sheet, 10-12% turns and 18-20% unordered structures. The order parameter S of the helical component of SKC1 was between 0.60 and 0.69. Close to 80% of SKC1 amide protons exchange within approximately 3 h of D2O exposure, suggesting that the channel is largely accessible to solvent exchange. These results are consistent with a model of SKC1 in which helices slightly tilted from the membrane normal line the water-filled vestibules that flank the K+ selectivity filter.

Structural dynamics of the Streptomyces lividans K+ channel (SKC1): oligomeric stoichiometry and stability.

SKC1, a 160-residue potassium channel with two putative transmembrane (TM) segments was recently identified from Streptomyces lividans. Its high levels of expression, small size, and ease of purification make SKC1 an ideal candidate for high-resolution structural studies. We have initiated the structural characterization of this channel by assessing its oligomeric behavior, stability in detergent, general hydrodynamic properties, and preliminary secondary structure content. SKC1 was readily expressed and purified to homogeneity by sequential metal-chelate and gel filtration chromatography. Standard SDS-PAGE, together with chemical cross-linking analysis indicated that SKC1 behaves as a tightly associated tetramer even in the presence of SDS. Using a gel shift assay to assess its oligomeric state, we determined that SKC1 is stable as a tetramer in most detergents and can be maintained in nonionic detergent solutions for extended periods of time. The tetramer is also stable at relatively high temperatures, with an oligomer-to-monomer transition occurring at approximately 65 degrees C. The Stokes radius of the micellar complex is 5 nm as determined from gel filtration chromatography of SKC1 in dodecyl maltoside. Preliminary estimations of secondary structure from CD spectroscopy showed that the channel exists mostly in alpha-helical conformation, with more than 50% alpha-helical, close to 20% beta-sheet, 10% beta-turn, and about 15% unassigned or random coil. These results are consistent with the idea that a bundle of alpha-helices forming a tetramer around the ion-conductive pathway is the common structural motif for members of the voltage-dependent channel superfamily.

Chemical modification of squid axon K+ channel -SH groups with the organic mercurial compound p-hydroxymercuriphenylsulfonic acid (PHMPS).

In internally dialyzed voltage-clamped squid axons, intracellular or extracellular addition of the sulfhydryl group (-SH) specific reagent p-hydroxymercuriphenylsulfonic acid (PHMPS), causes major modifications in the magnitude and kinetic parameters of the delayed rectifier K+ current. PHMPS produces a dramatic slow-down of the macroscopic current activation kinetics with a simultaneous reduction in its amplitude. In addition, it causes a marked increase in the delay of the macroscopic current at various pre-pulse potentials (Cole-Moore shift). The main effect of PHMPS at the single channel level is a sharp decrease in the open probability (4- to 5-fold). There is, however, a small reduction in single channel conductance (20%). Gating current experiments indicate that PHMPS causes a reduction in the voltage dependence of the activation process as well as a shift of the charge/voltage relationship towards more positive potentials. This, together with an increase in the mean open time, suggests that the open state has been destabilized. The results indicate that the reaction of -SH groups with PHMPS differentially affects the gating process. All the above mentioned effects are partially reversed by either dithiotreitol or beta-mercaptoethanol, -SH group reducing agents.

Removal of transducer HtrI allows electrogenic proton translocation by sensory rhodopsin I.

Sensory rhodopsin I (sR-I) is a phototaxis receptor in halobacteria, which is closely related to the light-driven proton pump bacteriorhodopsin and the chloride pump halorhodopsin found in the same organisms. The three pigments undergo similar cyclic photoreactions, in spite of their different functions. In intact cells or isolated membranes sR-I is complexed with protein HtrI, the next link in the signal transduction chain, and does not function as an electrogenic ion pump. However, illumination of sR-I in membranes lacking HtrI causes pH changes in the medium, and its photoreaction kinetics become pH-dependent. We show here that in closed vesicles, near neutral pH it functions as an electrogenic proton pump capable of generating at least -80 mV transmembrane potential. The action spectrum shows a maximum 37 nm below the 587-nm absorption maximum of the native pigment. This apparent discrepancy occurs because the 587-nm form of HtrI-free sR-I exists in a pH-dependent equilibrium with a 550-nm absorbing species generated through deprotonation of one group with a pKa of 7.2, which we have tentatively identified as Asp-76. We interpret the results in terms of a general model for ion translocation by the bacterial rhodopsins.

Gating of Shaker K+ channels: II. The components of gating currents and a model of channel activation.

Steady-state and kinetic properties of gating currents of the Shaker K+ channels were studied in channels expressed in Xenopus oocytes and recorded with the cut-open oocyte voltage clamp. The charge versus potential (Q-V) curve reveals at least two components of charge, the first moving in the hyperpolarized region (V1/2 = -63 mV) and the second, with a larger apparent valence, moving in the more depolarized region (V1/2 = -44 mV). The kinetic analysis of gating currents revealed also two exponential decaying components that corresponded in their voltage dependence with the charge components described in the steady-state. The first component was found to correlate with the effects of prepulses that produce the Cole-Moore shift of the ionic and gating currents and seems to be occurring completely within closed conformations of the channel. The second component seems to be related to the events occurring between the closed states just preceding, but not including, the transition to the open state. The ON and OFF gating currents exhibit a pronounced rising phase at potentials at which the second component becomes important, and this region corresponds to the potential range where the channel opens. The results could not be explained with simple parallel models, but the data can be fitted to a sequential model that could be related to a first rearrangement of the putative four subunits in cooperative fashion, followed by a concerted charge movement that leads to the open channel. The first series of charge movements are produced by transitions between several closed states carrying less than two electronic charges per step, while a step carrying about 3.5 electronic charges can explain the second component. This step is followed by the transition to the open state carrying less than 0.5 electronic charges. This model is able to reproduce all the kinetic and steady-state properties of the gating currents and predicts many of the properties of the ionic currents.

Gating of Shaker K+ channels: I. Ionic and gating currents.

Ionic and gating currents from noninactivating Shaker B K+ channels were studied with the cut-open oocyte voltage clamp technique and compared with the macropatch clamp technique. The performance of the cut-open oocyte voltage clamp technique was evaluated from the electrical properties of the clamped upper domus membrane, K+ tail current measurements, and the time course of K+ currents after partial blockade. It was concluded that membrane currents less than 20 microA were spatially clamped with a time resolution of at least 50 microseconds. Subtracted, unsubtracted gating currents with the cut-open oocyte voltage clamp technique and gating currents recorded in cell attached macropatches had similar properties and time course, and the charge movement properties directly obtained from capacity measurements agreed with measurements of charge movement from subtracted records. An accurate estimate of the normalized open probability Po(V) was obtained from tail current measurements as a function of the prepulse V in high external K+. The Po(V) was zero at potentials more negative than -40 mV and increased sharply at this potential, then increased continuously until -20 mV, and finally slowly increased with voltages more positive than 0 mV. Deactivation tail currents decayed with two time constants and external potassium slowed down the faster component without affecting the slower component that is probably associated with the return between two of the closed states near the open state. In correlating gating currents and channel opening, Cole-Moore type experiments showed that charge moving in the negative region of voltage (-100 to -40 mV) is involved in the delay of the conductance activation but not in channel opening. The charge moving in the more positive voltage range (-40 to -10 mV) has a similar voltage dependence to the open probability of the channel, but it does not show the gradual increase with voltage seen in the Po(V).

S4 mutations alter gating currents of Shaker K channels.

Activation of voltage-dependent channels involves charge-moving conformational changes of the voltage sensor that can be detected as gating currents. In Shaker K channels, the S4 sequence comprises at least part of the voltage sensor. We have measured gating currents in three S4 mutants: R368Q, R377K, and R371Q. R368Q enhances the separation of two components of charge movement and greatly reduces the valence of one component. R377K partially uncouples charge movement from channel opening. In contrast, the gating currents of R371Q resemble those of the control. Two other S4 mutations, R377Q and K374Q, make proteins that are not properly processed and transported to the cell surface and thereby eliminate the gating current. To explain the effects of R368Q, we hypothesize that R368 is part of a salt bridge that is broken early in activation. Subsequently, the S4 segment undergoes a conformational change, and, after a final, relatively voltage-independent step, the channel opens.

Purification and reconstitution of functional Shaker K+ channels assayed with a light-driven voltage-control system.

Voltage-dependent potassium channels are integral membrane proteins that control the excitability of nerve and muscle. The cloning of genes for K+ channels has led to structure/function analysis using a combination of site-directed mutagenesis and electrophysiology. As a result, much has been learned about how these proteins work. A deeper understanding of their function will require detailed structural characterization, however. We now report the purification of Shaker K+ channels from an insect expression system using immunoaffinity methods. The purified channels have been reconstituted, assayed using a novel, light-driven, vesicular voltage-control system, and shown to be functional. This approach will enable us to compare and optimize methods for protein production and purification. Purification of active protein is a prerequisite for detailed structural analysis, since activity is the key indication that the structural integrity of the channel has been preserved during biochemical procedures. Thus, this work represents a first step toward the determination of the structure of Shaker K+ channels.

Voltage activation of reconstituted sodium channels: use of bacteriorhodopsin as a light-driven current source.

We present a general method for the development and control of transmembrane potentials (delta psi) in reconstituted vesicles. The light-driven proton pump bacteriorhodopsin (bR) from Halobacterium halobium is the current source in the system, and the intensity of light controls the magnitude of delta psi at any given time. Transmembrane potentials were determined from the equilibrium distribution of hydrophobic ions, which was monitored by using either electron paramagnetic resonance spectroscopy and spin-labeled phosphonium ions or a tetraphenylphosphonium-selective electrode. A bias or holding potential was generated by using gradients of anions of limited permeability in the presence of an impermeable cation. Using n-methylglucamine or the polymer poly(ethylene imide) as the impermeable cation, the anions NO3- and SCN- were most effective in producing large (> -60 mV) negative diffusion potentials in egg phosphatidylcholine vesicles. In the presence of a NO(3-)-based negative holding potential (approx. -65 mV), bR is capable of depolarizing the membrane to at least the 0-mV level within a few seconds. More rapid depolarizations can be achieved by the application of a brief intense illumination preceding the preset illumination level (supercharging). The technique was successfully used to activate for the first time a population of unmodified voltage-dependent sodium channels purified from eel electroplax.

Gating currents from a nonconducting mutant reveal open-closed conformations in Shaker K+ channels.

In voltage-dependent ion channels, a voltage sensor region is responsible for channel activation and an aqueous pore is responsible for ion conduction. These two processes have been traditionally considered to be independent. We describe here a mutation in the putative pore region (W434F) that completely abolishes ion conduction without affecting the gating charge of the channel. Gating currents in the nonconductive mutant were found to be identical in their kinetic and steady-state properties to those in conductive channels. Gating current measurements could be performed without subtracting pulses and in the presence of normal physiological solutions. Application of internal tetraethylammonium (an open channel blocker) induced Off charge immobilization for large depolarizations, suggesting that the internal tetraethylammonium-binding site becomes available upon depolarization. We concluded that for this mutant, although the conduction pathway is not functional, the channel can still undergo the closed-open conformation in response to voltage changes.