Saturation and Microsecond Gating of Current Indicate Depletion-induced Instability of the MaxiK Selectivity Filter

Patch clamp experiments on single MaxiK channels expressed in HEK293 cells were performed with a high temporal resolution (50-kHz filter) in symmetrical solutions with 50, 150, or 400 mM KCl and 2.5 mM CaCl2 and 2.5 mM MgCl2. At membrane potentials >+100 mV, the single-channel current showed a negative slope resistance, concomitantly with a flickery block, which was not influenced by Ca2+ or Mg2+. The analysis of the amplitude histograms by beta distributions revealed that current in this voltage range was reduced by two effects: rate limitation at the cytosolic side of the pore and gating with rate constants 10–20-fold higher than the cutoff frequency of the filter (i.e., dwell times in the microsecond range). The data were analyzed in terms of a model that postulates a coupling between both effects; if the voltage over the selectivity filter withdraws ions from the cavity at a higher rate than that of refilling from the cytosol, the selectivity filter becomes instable because of ion depletion, and current is interrupted by the resulting flickering. The fit of the IV curves revealed a characteristic voltage of 35 mV. In contrast, the voltage dependence of the gating factor R, i.e., the ratio between true and apparent single-channel current, could be fitted by exponentials with a characteristic voltage of 60 mV, suggesting that only part of the transmembrane potential is felt by the flux through the selectivity filter

Asymmetric Interplay Between K+ and Blocker and Atomistic Parameters From Physiological Experiments Quantify K+ Channel Blocker Release

Modulating the activity of ion channels by blockers yields information on both the mode of drug action and on the biophysics of ion transport. Here we investigate the interplay between ions in the selectivity filter (SF) of K+ channels and the release kinetics of the blocker tetrapropylammonium in the model channel KcvNTS. A quantitative expression calculates blocker release rate constants directly from voltage-dependent ion occupation probabilities in the SF. The latter are obtained by a kinetic model of single-channel currents recorded in the absence of the blocker. The resulting model contains only two adjustable parameters of ion-blocker interaction and holds for both symmetric and asymmetric ionic conditions. This data-derived model is corroborated by 3D reference interaction site model (3D RISM) calculations on several model systems, which show that the K+ occupation probability is unaffected by the blocker, a direct consequence of the strength of the ion-carbonyl attraction in the SF, independent of the specific protein background. Hence, KcvNTS channel blocker release kinetics can be reduced to a small number of system-specific parameters. The pore-independent asymmetric interplay between K+ and blocker ions potentially allows for generalizing these results to similar potassium channels

Minimal Viral Potassium Channels for Studying Protein/Lipid Interaction

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The Wooster Voice (Wooster, OH), 1997-10-16

This edition of the College of Wooster student run newspaper was published on October 16 of 1997 and it is twelve pages long. Jilted surgeon general calls for more health education, Dr. Jocelyn Elders presents during the Wooster Forum series about health care and education. Ebert to be dedicated this weekend, the Ebert Art Center is going to be formally dedicated. Renowned musical trio combines classical with new, the Cleveland Duo and saxophonist James Umble perform at the Gault Recital Hall. Athletic updates for the past week are highlighted on pages ten to twelve.https://openworks.wooster.edu/voice1991-2000/1177/thumbnail.jp

A small viral potassium ion channel with an inherent inward rectification

Some algal viruses have coding sequences for proteins with structural and functional characteristics of pore modules of complex K+ channels. Here we exploit the structural diversity among these channel orthologs to discover new basic principles of structure/function correlates in K+ channels. The analysis of three similar K+ channels with ≤ 86 amino acids (AA) shows that one channel (Kmpv1) generates an ohmic conductance in HEK293 cells while the other two (KmpvSP1, KmpvPL1) exhibit typical features of canonical Kir channels. Like Kir channels, the rectification of the viral channels is a function of the K+ driving force. Reconstitution of KmpvSP1 and KmpvPL1 in planar lipid bilayers showed rapid channel fluctuations only at voltages negative of the K+ reversal voltage. This rectification was maintained in KCl buffer with 1 mM EDTA, which excludes blocking cations as the source of rectification. This means that rectification of the viral channels must be an inherent property of the channel. The structural basis for rectification was investigated by a chimera between rectifying and non-rectifying channels as well as point mutations making the rectifier similar to the ohmic conducting channel. The results of these experiments exclude the pore with pore helix and selectivity filter as playing a role in rectification. The insensitivity of the rectifier to point mutations suggests that tertiary or quaternary structural interactions between the transmembrane domains are responsible for this type of gating

Identification of Intrahelical Bifurcated H‑Bonds as a New Type of Gate in K+ Channels

Gating of ion channels is based on structural transitions between open and closed states. To uncover the chemical basis of individual gates, we performed a comparative experimental and computational analysis between two K+ channels, KcvS and KcvNTS. These small viral encoded K+ channel proteins, with a monomer size of only 82 amino acids, resemble the pore module of all complex K+ channels in terms of structure and function. Even though both proteins share about 90% amino acid sequence identity, they exhibit different open probabilities with ca. 90% in KcvNTS and 40% in KcvS. Single channel analysis, mutational studies and molecular dynamics simulations show that the difference in open probability is caused by one long closed state in KcvS. This state is structurally created in the tetrameric channel by a transient, Ser mediated, intrahelical hydrogen bond. The resulting kink in the inner transmembrane domain swings the aromatic rings from downstream Phes in the cavity of the channel, which blocks ion flux. The frequent occurrence of Ser or Thr based helical kinks in membrane proteins suggests that a similar mechanism could also occur in the gating of other ion channels. Includes Supporting Informatio

Identification of Intrahelical Bifurcated H‑Bonds as a New Type of Gate in K+ Channels

Gating of ion channels is based on structural transitions between open and closed states. To uncover the chemical basis of individual gates, we performed a comparative experimental and computational analysis between two K+ channels, KcvS and KcvNTS. These small viral encoded K+ channel proteins, with a monomer size of only 82 amino acids, resemble the pore module of all complex K+ channels in terms of structure and function. Even though both proteins share about 90% amino acid sequence identity, they exhibit different open probabilities with ca. 90% in KcvNTS and 40% in KcvS. Single channel analysis, mutational studies and molecular dynamics simulations show that the difference in open probability is caused by one long closed state in KcvS. This state is structurally created in the tetrameric channel by a transient, Ser mediated, intrahelical hydrogen bond. The resulting kink in the inner transmembrane domain swings the aromatic rings from downstream Phes in the cavity of the channel, which blocks ion flux. The frequent occurrence of Ser or Thr based helical kinks in membrane proteins suggests that a similar mechanism could also occur in the gating of other ion channels. Includes Supporting Informatio

Fast and slow gating are inherent properties of the pore module of the K+ channel Kcv

Kcv from the chlorella virus PBCV-1 is a viral protein that forms a tetrameric, functional K+ channel in heterologous systems. Kcv can serve as a model system to study and manipulate basic properties of the K+ channel pore because its minimalistic structure (94 amino acids) produces basic features of ion channels, such as selectivity, gating, and sensitivity to blockers. We present a characterization of Kcv properties at the single-channel level. In symmetric 100 mM K+, single-channel conductance is 114 ± 11 pS. Two different voltage-dependent mechanisms are responsible for the gating of Kcv. “Fast” gating, analyzed by β distributions, is responsible for the negative slope conductance in the single-channel current–voltage curve at extreme potentials, like in MaxiK potassium channels, and can be explained by depletion-aggravated instability of the filter region. The presence of a “slow” gating is revealed by the very low (in the order of 1–4%) mean open probability that is voltage dependent and underlies the time-dependent component of the macroscopic current

Reconstitution and functional characterization of ion channels from nanodiscs in lipid bilayers

Recent studies have shown that membrane proteins can be efficiently synthesized in vitro before spontaneously inserting into soluble nanoscale lipid bilayers called nanodiscs (NDs). In this paper, we present experimental details that allow a combination of in vitro translation of ion channels into commercially available NDs followed by their direct reconstitution from these nanobilayers into standard bilayer setups for electrophysiological characterization. We present data showing that two model K+ channels, Kcv and KcsA, as well as a recently discovered dual-topology F− channel, Fluc, can be reliably reconstituted from different types of NDs into bilayers without contamination from the in vitro translation cocktail. The functional properties of Kcv and KcsA were characterized electrophysiologically and exhibited sensitivity to the lipid composition of the target DPhPC bilayer, suggesting that the channel proteins were fully exposed to the target membrane and were no longer surrounded by the lipid/protein scaffold. The single-channel properties of the three tested channels are compatible with studies from recordings of the same proteins in other expression systems. Altogether, the data show that synthesis of ion channels into NDs and their subsequent reconstitution into conventional bilayers provide a fast and reliable method for functional analysis of ion channels

Modulation of enrofloxacin binding in OmpF by Mg2+ as revealed by the analysis of fast flickering single-porin current

One major determinant of the efficacy of antibiotics on Gram-negative bacteria is the passage through the outer membrane. During transport of the fluoroquinolone enrofloxacin through the trimeric outer membrane protein OmpF of Escherichia coli, the antibiotic interacts with two binding sites within the pore, thus partially blocking the ionic current. The modulation of one affinity site by Mg2+ reveals further details of binding sites and binding kinetics. At positive membrane potentials, the slow blocking events induced by enrofloxacin in Mg2+-free media are converted to flickery sojourns at the highest apparent current level (all three pores flickering). This indicates weaker binding in the presence of Mg2+. Analysis of the resulting amplitude histograms with beta distributions revealed the rate constants of blocking (k(OB)) and unblocking (k(BO)) in the range of 1,000 to 120,000 s(-1). As expected for a bimolecular reaction, k(OB) was proportional to blocker concentration and k(BO) independent of it. k(OB) was approximately three times lower for enrofloxacin coming from the cis side than from the trans side. The block was not complete, leading to a residual conductivity of the blocked state being similar to 25% of that of the open state. Interpretation of the results has led to the following model: fast flickering as caused by interaction of Mg2+ and enrofloxacin is related to the binding site at the trans side, whereas the cis site mediates slow blocking events which are also found without Mg2+. The difference in the accessibility of the binding sites also explains the dependency of k(OB) on the side of enrofloxacin addition and yields a means of determining the most plausible orientation of OmpF in the bilayer. The voltage dependence suggests that the dipole of the antibiotic has to be adequately oriented to facilitate binding