Blocking Pore-open Mutants of CLC-0 by Amphiphilic Blockers

The blockade of CLC-0 chloride channels by p-chlorophenoxy acetate (CPA) has been thought to be state dependent; the conformational change of the channel pore during the “fast gating” alters the CPA binding affinity. Here, we examine the mechanism of CPA blocking in pore-open mutants of CLC-0 in which the residue E166 was replaced by various amino acids. We find that the CPA-blocking affinities depend upon the volume and the hydrophobicity of the side chain of the introduced residue; CPA affinity can vary by three orders of magnitude in these mutants. On the other hand, mutations at the intracellular pore entrance, although affecting the association and dissociation rates of the CPA block, generate only a modest effect on the steady-state blocking affinity. In addition, various amphiphilic compounds, including fatty acids and alkyl sulfonates, can also block the pore-open mutants of CLC-0 through a similar mechanism. The blocking affinity of fatty acids and alkyl sulfonates increases with the length of these amphiphilic blockers, a phenomenon similar to the block of the Shaker K⁺ channel by long-chain quaternary ammonium (QA) ions. These observations lead us to propose that the CPA block of the open pore of CLC-0 is similar to the blockade of voltage-gated K⁺ channels by long-chain QAs or by the inactivation ball peptide: the blocker first uses the hydrophilic end to “dock” at the pore entrance, and the hydrophobic part of the blocker then enters the pore to interact with a more hydrophobic region of the pore. This blocking mechanism appears to be very general because the block does not require a precise structural fit between the blocker and the pore, and the blocking mechanism applies to the cation and anion channels with unrelated pore architectures.     

Molecular mechanisms of centipede toxin SsTx-4 inhibition of inwardly rectifying potassium channels

Inwardly rectifying potassium channels (Kirs) are important drug targets, with antagonists for the Kir1.1, Kir4.1, and pancreatic Kir6.2/SUR1 channels being potential drug candidates for treating hypertension, depression, and diabetes, respectively. However, few peptide toxins acting on Kirs are identified and their interacting mechanisms remain largely elusive yet. Herein, we showed that the centipede toxin SsTx-4 potently inhibited the Kir1.1, Kir4.1, and Kir6.2/SUR1 channels with nanomolar to submicromolar affinities and intensively studied the molecular bases for toxin–channel interactions using patch-clamp analysis and site-directed mutations. Other Kirs including Kir2.1 to 2.4, Kir4.2, and Kir7.1 were resistant to SsTx-4 treatment. Moreover, SsTx-4 inhibited the inward and outward currents of Kirs with different potencies, possibly caused by a K⁺ “knock-off” effect, suggesting the toxin functions as an out pore blocker physically occluding the K⁺-conducting pathway. This conclusion was further supported by a mutation analysis showing that M137 located in the outer vestibule of the Kir6.2/ΔC26 channel was the key residue mediating interaction with SsTx-4. On the other hand, the molecular determinants within SsTx-4 for binding these Kir channels only partially overlapped, with K13 and F44 being the common key residues. Most importantly, K11A, P15A, and Y16A mutant toxins showed improved affinity and/or selectivity toward Kir6.2, while R12A mutant toxin had increased affinity for Kir4.1. To our knowledge, SsTx-4 is the first characterized peptide toxin with Kir4.1 inhibitory activity. This study provides useful insights for engineering a Kir6.2/SUR1 channel–specific antagonist based on the SsTx-4 template molecule and may be useful in developing new antidiabetic drugs.     

Nonindependent K+ Movement through the Pore in IRK1 Potassium Channels

We measured unidirectional K⁺ in- and efflux through an inward rectifier K channel (IRK1) expressed in Xenopus oocytes. The ratio of these unidirectional fluxes differed significantly from expectations based on independent ion movement. In an extracellular solution with a K⁺ concentration of 25 mM, the data were described by a Ussing flux-ratio exponent, n′, of ∼2.2 and was constant over a voltage range from −50 to −25 mV. This result indicates that the pore of IRK1 channels may be simultaneously occupied by at least three ions. The IRK1 n′ value of 2.2 is significantly smaller than the value of 3.5 obtained for Shaker K channels under identical conditions. To determine if other permeation properties that reflect multi-ion behavior differed between these two channel types, we measured the conductance (at 0 mV) of single IRK1 channels as a function of symmetrical K⁺ concentration. The conductance could be fit by a saturating hyperbola with a half-saturation K⁺ activity of 40 mM, substantially less than the reported value of 300 mM for Shaker K channels. We investigated the ability of simple permeation models based on absolute reaction rate theory to simulate IRK1 current–voltage, conductance, and flux-ratio data. Certain classes of four-barrier, three-site permeation models are inconsistent with the data, but models with high lateral barriers and a deep central well were able to account for the flux-ratio and single channel data. We conclude that while the pore in IRK1 and Shaker channels share important similarities, including K⁺ selectivity and multi-ion occupancy, they differ in other properties, including the sensitivity of pore conductance to K⁺ concentration, and may differ in the number of K⁺ ions that can simultaneously occupy the pore: IRK1 channels may contain three ions, but the pore in Shaker channels can accommodate four or more ions.     

Ion Conduction through C-Type Inactivated Shaker Channels

C-type inactivation of Shaker potassium channels involves entry into a state (or states) in which the inactivated channels appear nonconducting in physiological solutions. However, when Shaker channels, from which fast N-type inactivation has been removed by NH₂-terminal deletions, are expressed in Xenopus oocytes and evaluated in inside-out patches, complete removal of K⁺ ions from the internal solution exposes conduction of Na⁺ and Li⁺ in C-type inactivated conformational states. The present paper uses this observation to investigate the properties of ion conduction through C-type inactivated channel states, and demonstrates that both activation and deactivation can occur in C-type states, although with slower than normal kinetics. Channels in the C-type states appear “inactivated” (i.e., nonconducting) in physiological solutions due to the summation of two separate effects: first, internal K⁺ ions prevent Na⁺ ions from permeating through the channel; second, C-type inactivation greatly reduces the permeability of K⁺ relative to the permeability of Na⁺, thus altering the ion selectivity of the channel.     

Control of voltage-gated K+ channel permeability to NMDG+ by a residue at the outer pore

Crystal structures of potassium (K⁺) channels reveal that the selectivity filter, the narrow portion of the pore, is only ∼3-Å wide and buttressed from behind, so that its ability to expand is highly constrained, and the permeation of molecules larger than Rb⁺ (2.96 Å in diameter) is prevented. N-methyl-d-glucamine (NMDG⁺), an organic monovalent cation, is thought to be a blocker of Kv channels, as it is much larger (∼7.3 Å in mean diameter) than K⁺ (2.66 Å in diameter). However, in the absence of K⁺, significant NMDG⁺ currents could be recorded from human embryonic kidney cells expressing Kv3.1 or Kv3.2b channels and Kv1.5 R487Y/V, but not wild-type channels. Inward currents were much larger than outward currents due to the presence of intracellular Mg²⁺ (1 mM), which blocked the outward NMDG⁺ current, resulting in a strong inward rectification. The NMDG⁺ current was inhibited by extracellular 4-aminopyridine (5 mM) or tetraethylammonium (10 mM), and largely eliminated in Kv3.2b by an S6 mutation that prevents the channel from opening (P468W) and by a pore helix mutation in Kv1.5 R487Y (W472F) that inactivates the channel at rest. These data indicate that NMDG⁺ passes through the open ion-conducting pore and suggest a very flexible nature of the selectivity filter itself. 0.3 or 1 mM K⁺ added to the external NMDG⁺ solution positively shifted the reversal potential by ∼16 or 31 mV, respectively, giving a permeability ratio for K⁺ over NMDG⁺ (P_K⁺/P_NMDG⁺) of ∼240. Reversal potential shifts in mixtures of K⁺ and NMDG⁺ are in accordance with P_K⁺/P_NMDG⁺, indicating that the ions compete for permeation and suggesting that NMDG⁺ passes through the open state. Comparison of the outer pore regions of Kv3 and Kv1.5 channels identified an Arg residue in Kv1.5 that is replaced by a Tyr in Kv3 channels. Substituting R with Y or V allowed Kv1.5 channels to conduct NMDG⁺, suggesting a regulation by this outer pore residue of Kv channel flexibility and, as a result, permeability.     

Lack of Negatively Charged Residues at the External Mouth of Kir2.2 Channels Enable the Voltage-Dependent Block by External Mg2+

Kir channels display voltage-dependent block by cytosolic cations such as Mg²⁺ and polyamines that causes inward rectification. In fact, cations can regulate K channel activity from both the extracellular and intracellular sides. Previous studies have provided insight into the up-regulation of Kir channel activity by extracellular K⁺ concentration. In contrast, extracellular Mg²⁺ has been found to reduce the amplitude of the single-channel current at milimolar concentrations. However, little is known about the molecular mechanism of Kir channel blockade by external Mg²⁺ and the relationship between the Mg²⁺ blockade and activity potentiation by permeant K⁺ ions. In this study, we applied an interactive approach between theory and experiment. Electrophysiological recordings on Kir2.2 and its mutants were performed by heterologous expression in Xenopus laevis oocytes. Our results confirmed that extracellular Mg²⁺ could reduce heterologously expressed WT Kir2.2 currents in a voltage dependent manner. The kinetics of inhibition and recovery of Mg²⁺ exhibit a 3∼4s time constant. Molecular dynamics simulation results revealed a Mg²⁺ binding site located at the extracellular mouth of Kir2.2 that showed voltage-dependent Mg²⁺ binding. The mutants, G119D, Q126E and H128D, increased the number of permeant K⁺ ions and reduced the voltage-dependent blockade of Kir2.2 by extracellular Mg²⁺.     

Multi-ion distributions in the cytoplasmic domain of inward rectifier potassium channels

Inward rectifier potassium (Kir) channels act as cellular diodes, allowing unrestricted flow of potassium (K⁺) into the cell while preventing currents of large magnitude in the outward direction. The rectification mechanism by which this occurs involves a coupling between K⁺ and intracellular blockers—magnesium (Mg²⁺) or polyamines—that simultaneously occupy the permeation pathway. In addition to the transmembrane pore, Kirs possess a large cytoplasmic domain (CD) that provides a favorable electronegative environment for cations. Electrophysiological experiments have shown that the CD is a key regulator of both conductance and rectification. In this study, we calculate and compare averaged equilibrium probability densities of K⁺ and Cl⁻ in open-pore models of the CDs of a weak (Kir1.1-ROMK) and a strong (Kir2.1-IRK) rectifier through explicit-solvent molecular-dynamics simulations in ∼1 M KCl. The CD of both channels concentrates K⁺ ions greater than threefold inside the cytoplasmic pore while IRK shows an additional K⁺ accumulation region near the cytoplasmic entrance. Simulations carried out with Mg²⁺ or spermine (SPM⁴⁺) show that these ions interact with pore-lining residues, shielding the surface charge and reducing K⁺ in both channels. The results also show that SPM⁴⁺ behaves differently inside these two channels. Although SPM⁴⁺ remains inside the CD of ROMK, it diffuses around the entire volume of the pore. In contrast, this polyatomic cation finds long-lived conformational states inside the IRK pore, interacting with residues E224, D259, and E299. The strong rectifier CD is also capable of sequestering an additional SPM⁴⁺ at the cytoplasmic entrance near a cluster of negative residues D249, D274, E275, and D276. Although understanding the actual mechanism of rectification blockade will require high-resolution structural information of the blocked state, these simulations provide insight into how sequence variation in the CD can affect the multi-ion distributions that underlie the mechanisms of conduction, rectification affinity, and kinetics.     

Cloning and functional characterization of inward-rectifying potassium (Kir) channels from Malpighian tubules of the mosquito Aedes aegypti

Inward-rectifying K⁺ (Kir) channels play critical physiological roles in a variety of vertebrate cells/tissues, including the regulation of membrane potential in nerve and muscle, and the transepithelial transport of ions in osmoregulatory epithelia, such as kidneys and gills. It remains to be determined whether Kir channels play similar physiological roles in insects. In the present study, we sought to 1) clone the cDNAs of Kir channel subunits expressed in the renal (Malpighian) tubules of the mosquito Aedes aegypti, and 2) characterize the electrophysiological properties of the cloned Kir subunits when expressed heterologously in oocytes of Xenopus laevis. Here, we reveal that three Kir subunits are expressed abundantly in Aedes Malpighian tubules (AeKir1, AeKir2B, and AeKir3); each of their full-length cDNAs was cloned. Heterologous expression of the AeKir1 or the AeKir2B subunits in Xenopus oocytes elicits inward-rectifying K⁺ currents that are blocked by barium. Relative to the AeKir2B-expressing oocytes, the AeKir1-expressing oocytes 1) produce larger macroscopic currents, and 2) exhibit a modulation of their conductive properties by extracellular Na⁺. Attempts to functionally characterize the AeKir3 subunit in Xenopus oocytes were unsuccessful. Lastly, we show that in isolated Aedes Malpighian tubules, the cation permeability sequence of the basolateral membrane of principal cells (Tl⁺ > K⁺ > Rb⁺ > NH₄⁺) is consistent with the presence of functional Kir channels. We conclude that in Aedes Malpighian tubules, Kir channels contribute to the majority of the barium-sensitive transepithelial transport of K⁺.     

Kir4.1/Kir5.1 channels possess strong intrinsic inward rectification determined by a voltage-dependent K+-flux gating mechanism

Inwardly rectifying potassium (Kir) channels are broadly expressed in both excitable and nonexcitable tissues, where they contribute to a wide variety of cellular functions. Numerous studies have established that rectification of Kir channels is not an inherent property of the channel protein itself, but rather reflects strong voltage dependence of channel block by intracellular cations, such as polyamines and Mg²⁺. Here, we identify a previously unknown mechanism of inward rectification in Kir4.1/Kir5.1 channels in the absence of these endogenous blockers. This novel intrinsic rectification originates from the voltage-dependent behavior of Kir4.1/Kir5.1, which is generated by the flux of potassium ions through the channel pore; the inward K⁺-flux induces the opening of the gate, whereas the outward flux is unable to maintain the gate open. This gating mechanism powered by the K⁺-flux is convergent with the gating of PIP₂ because, at a saturating concentration, PIP₂ greatly reduces the inward rectification. Our findings provide evidence of the coexistence of two rectification mechanisms in Kir4.1/Kir5.1 channels: the classical inward rectification induced by blocking cations and an intrinsic voltage-dependent mechanism generated by the K⁺-flux gating.     

Potassium Channels in Motor Cells of Samanea saman: A Patch-Clamp Study

Leaflet movements in Samanea saman are driven by the shrinking and swelling of cells in opposing (extensor and flexor) regions of the motor organ (pulvinus). Changes in cell volume, in turn, depend upon large changes in motor cell content of K⁺, Cl⁻ and other ions. We performed patch-clamp experiments on extensor and flexor protoplasts, to determine whether their plasma membranes contain channels capable of carrying the large K⁺ currents that flow during leaflet movement. Recordings in the “whole-cell” mode reveal depolarization-activated K⁺ currents in extensor and flexor cells that increase slowly (t_½ = ca. 2 seconds) and remain active for minutes. Recordings from excised patches reveal a single channel conductance of ca. 20 picosiemens in both cell types. The magnitude of the K⁺ currents is adequate to account quantitatively for K⁺ loss, previously measured in vivo during cell shrinkage. The K⁺ channel blockers tetraethylammonium (5 millimolar) or quinine (1 millimolar) blocked channel opening and decreased light- and dark-promoted movements of excised leaflets. These results provide evidence for the role of potassium channels in leaflet movement.     

The ligand-sensitive gate of a potassium channel lies close to the selectivity filter

Potassium channels selectively conduct K⁺ ions across cell membranes and have key roles in cell excitability. Their opening and closing can be spontaneous or controlled by membrane voltage or ligand binding. We used Ba²⁺ as a probe to determine the location of the ligand-sensitive gate in an inwardly rectifying K⁺ channel (Kir6.2). To a K⁺ channel, Ba²⁺ and K⁺ are of similar sizes, but Ba²⁺ blocks the pore by binding within the selectivity filter. We found that internal Ba²⁺ could still access its binding site when the channel was shut, which indicates that the ligand-sensitive gate lies above the Ba²⁺-block site, and thus within or above the selectivity filter. This is in marked contrast to the voltage-dependent gate of K_V channels, which is located at the intracellular mouth of the pore.     

Single-Channel Characteristics of Wild-Type IKs Channels and Channels formed with Two MinK Mutants that Cause Long QT Syndrome

I_Ks channels are voltage dependent and K⁺ selective. They influence cardiac action potential duration through their contribution to myocyte repolarization. Assembled from minK and KvLQT1 subunits, I_Ks channels are notable for a heteromeric ion conduction pathway in which both subunit types contribute to pore formation. This study was undertaken to assess the effects of minK on pore function. We first characterized the properties of wild-type human I_Ks channels and channels formed only of KvLQT1 subunits. Channels were expressed in Xenopus laevis oocytes or Chinese hamster ovary cells and currents recorded in excised membrane patches or whole-cell mode. Unitary conductance estimates were dependent on bandwidth due to rapid channel “flicker.” At 25 kHz in symmetrical 100-mM KCl, the single-channel conductance of I_Ks channels was ∼16 pS (corresponding to ∼0.8 pA at 50 mV) as judged by noise-variance analysis; this was fourfold greater than the estimated conductance of homomeric KvLQT1 channels. Mutant I_Ks channels formed with D76N and S74L minK subunits are associated with long QT syndrome. When compared with wild type, mutant channels showed lower unitary currents and diminished open probabilities with only minor changes in ion permeabilities. Apparently, the mutations altered single-channel currents at a site in the pore distinct from the ion selectivity apparatus. Patients carrying these mutant minK genes are expected to manifest decreased K⁺ flux through I_Ks channels due to lowered single-channel conductance and altered gating.     

Non-Equilibrium Dynamics Contribute to Ion Selectivity in the KcsA Channel

The ability of biological ion channels to conduct selected ions across cell membranes is critical for the survival of both animal and bacterial cells. Numerous investigations of ion selectivity have been conducted over more than 50 years, yet the mechanisms whereby the channels select certain ions and reject others are not well understood. Here we report a new application of Jarzynski’s Equality to investigate the mechanism of ion selectivity using non-equilibrium molecular dynamics simulations of Na⁺ and K⁺ ions moving through the KcsA channel. The simulations show that the selectivity filter of KcsA adapts and responds to the presence of the ions with structural rearrangements that are different for Na⁺ and K⁺. These structural rearrangements facilitate entry of K⁺ ions into the selectivity filter and permeation through the channel, and rejection of Na⁺ ions. A mechanistic model of ion selectivity by this channel based on the results of the simulations relates the structural rearrangement of the selectivity filter to the differential dehydration of ions and multiple-ion occupancy and describes a mechanism to efficiently select and conduct K⁺. Estimates of the K⁺/Na⁺ selectivity ratio and steady state ion conductance for KcsA from the simulations are in good quantitative agreement with experimental measurements. This model also accurately describes experimental observations of channel block by cytoplasmic Na⁺ ions, the “punch through” relief of channel block by cytoplasmic positive voltages, and is consistent with the knock-on mechanism of ion permeation.     

Coupled Ion Movement Underlies Rectification in an Inward-Rectifier K+ Channel

We studied block of the internal pore of the ROMK1 inward-rectifier K⁺ channel by Mg²⁺ and five quaternary ammoniums (tetramethylammonium, tetraethylammonium, tetrapropylammonium, tetrabutylammonium, and tetrapentylammonium). The apparent affinity of these blockers varied as a function of membrane voltage. As a consequence, the channel conducted K⁺ current more efficiently in the inward than the outward direction; i.e., inward rectification. Although the size of some monovalent quaternary ammoniums is rather large, the zδ values (which measure voltage dependence of their binding to the pore) were near unity in symmetric 100 mM K⁺. Furthermore, we observed that not only the apparent affinities of the blockers themselves, but also their dependence on membrane voltage (or zδ), varied as a function of the concentration of extracellular K⁺. These results suggest that there is energetic coupling between the binding of blocking and permeating (K⁺) ions, and that the voltage dependence of channel blockade results, at least in part, from the movement of K⁺ ions in the electrical field. A further quantitative analysis of the results explains why the complex phenomenon of inward rectification depends on both membrane voltage and the equilibrium potential for K⁺.     

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.     

Do voltage-gated Kv1.1 and inward rectifier Kir2.1 potassium channels form heteromultimers?

Possible heteromultimer formation between Kv- and Kir-type K⁺ channels was investigated, in connection with the known functional diversity of K⁺ channels in vivo. Voltage-clamp experiments were performed on Xenopus oocytes, either injected with concatenated Kir2.1-Kv1.1 mRNA, or co-injected with Kv1.1 and Kir2.1 mRNA. K⁺ currents could be approximated by the algebraic sum of the 2 K⁺ current types alone. The tandem construct did not show functional expression, although it could be detected by Western blotting. We conclude that Kv1.1 and Kir2.1 α-subunit proteins fail to assemble and do not contribute functional diversity to K⁺ channels.     

K+ Block Is the Mechanism of Functional Asymmetry in Bacterial Nav Channels

Crystal structures of several bacterial Na_v channels have been recently published and molecular dynamics simulations of ion permeation through these channels are consistent with many electrophysiological properties of eukaryotic channels. Bacterial Na_v channels have been characterized as functionally asymmetric, and the mechanism of this asymmetry has not been clearly understood. To address this question, we combined non-equilibrium simulation data with two-dimensional equilibrium unperturbed landscapes generated by umbrella sampling and Weighted Histogram Analysis Methods for multiple ions traversing the selectivity filter of bacterial Na_vAb channel. This approach provided new insight into the mechanism of selective ion permeation in bacterial Na_v channels. The non-equilibrium simulations indicate that two or three extracellular K⁺ ions can block the entrance to the selectivity filter of Na_vAb in the presence of applied forces in the inward direction, but not in the outward direction. The block state occurs in an unstable local minimum of the equilibrium unperturbed free-energy landscape of two K⁺ ions that can be ‘locked’ in place by modest applied forces. In contrast to K⁺, three Na⁺ ions move favorably through the selectivity filter together as a unit in a loose “knock-on” mechanism of permeation in both inward and outward directions, and there is no similar local minimum in the two-dimensional free-energy landscape of two Na⁺ ions for a block state. The useful work predicted by the non-equilibrium simulations that is required to break the K⁺ block is equivalent to large applied potentials experimentally measured for two bacterial Na_v channels to induce inward currents of K⁺ ions. These results illustrate how inclusion of non-equilibrium factors in the simulations can provide detailed information about mechanisms of ion selectivity that is missing from mechanisms derived from either crystal structures or equilibrium unperturbed free-energy landscapes.     

Long-pore Electrostatics in Inward-rectifier Potassium Channels

Inward-rectifier potassium (Kir) channels differ from the canonical K⁺ channel structure in that they possess a long extended pore (~85 Å) for ion conduction that reaches deeply into the cytoplasm. This unique structural feature is presumably involved in regulating functional properties specific to Kir channels, such as conductance, rectification block, and ligand-dependent gating. To elucidate the underpinnings of these functional roles, we examine the electrostatics of an ion along this extended pore. Homology models are constructed based on the open-state model of KirBac1.1 for four mammalian Kir channels: Kir1.1/ROMK, Kir2.1/IRK, Kir3.1/GIRK, and Kir6.2/KATP. By solving the Poisson-Boltzmann equation, the electrostatic free energy of a K⁺ ion is determined along each pore, revealing that mammalian Kir channels provide a favorable environment for cations and suggesting the existence of high-density regions in the cytoplasmic domain and cavity. The contribution from the reaction field (the self-energy arising from the dielectric polarization induced by the ion's charge in the complex geometry of the pore) is unfavorable inside the long pore. However, this is well compensated by the electrostatic interaction with the static field arising from the protein charges and shielded by the dielectric surrounding. Decomposition of the static field provides a list of residues that display remarkable correspondence with existing mutagenesis data identifying amino acids that affect conduction and rectification. Many of these residues demonstrate interactions with the ion over long distances, up to 40 Å, suggesting that mutations potentially affect ion or blocker energetics over the entire pore. These results provide a foundation for understanding ion interactions in Kir channels and extend to the study of ion permeation, block, and gating in long, cation-specific pores.     

Permeation Redux: Thermodynamics and Kinetics of Ion Movement through Potassium Channels

The fundamental biophysics underlying the selective movement of ions through ion channels was launched by George Eisenman in the 1960s, using glass electrodes. This minireview examines the insights from these early studies and the explosive progress made since then.The recent passing of George Eisenman (December 18, 2013) inspired us to revisit the topic most associated with his passionate input, namely how the membrane proteins known as ion channels control passive movements of ions across biological membranes. Ion permeation has captivated biophysicists for more than half a century, and only now, with the combined advent of atomic-level structures and sophisticated computational wizardry, are the secrets of this amazing process beginning to be revealed. Why “amazing”? For example, because K⁺-selective ion channels can discriminate between K⁺ and Na⁺ ions, which differ in radius by a mere 0.38 Ångstrom, and do so with 1000:1 reliability and at lightning speed near the diffusion limit, the dwell time of an ion in the pore of a channel is as fleeting as ∼10⁻⁸ s. Understanding this remarkably-tuned process in K⁺ channels requires attention to two perspectives: the ability of specific channels to discriminate between the ions they might encounter (i.e., selectivity); and the kinetics of ion movement across the channel pore (i.e., conduction).The classical thermodynamic explanation of ion selectivity is that the relative free energy difference of ions in the pore relative to the bulk solution is the critical quantity to consider (1–4). Some of the earliest insights into thermodynamic selectivity derive from studies of ion binding to aluminosilicate glass electrodes (5,6). Depending on the composition of the glass, these electrodes, originally developed for their proton sensitivity, can exhibit a dramatic range of selectivities among the five alkali metal cations. In rank order, one might expect as many as 5 × 4 × 3 × 2 × 1 = 120 different sequences of selectivities among these five cations. Remarkably, however, in the vast literature of selectivity in biological membranes, typically only 11 sequences are observed (with some exceptions). These became known as the “Eisenman sequences”. The exact same selectivity sequences are observed in glass electrodes of various compositions.Why are the free energy differences the way they are for a given system? To answer this question, one needs a physical mechanism. For Eisenman, numerical calculations stood as a critical component of the process of better understanding Nature. In other words, proposing a physical mechanism that is qualitatively reasonable is not enough—one must also test it by constructing atomic models leading to actual quantitative predictions (Fig. 1). In the early days, the concept of the anionic field strength of a binding site was formulated and tested with direct calculations based on exceedingly simple atomic hard-sphere models of ions, water molecules, and coordinating ligands such as shown in Fig. 1 A (2,5). Remarkably, these simple calculations led to the Eisenman selectivity sequences. Eisenman was able to account for the limited class of sequences by considering the equilibrium binding of cations to the glass, and the energetic competition between water and glass for the ions. The critical factor that determines the selectivity sequence of a given glass is the anionic field strength of the binding site on the glass. Briefly, the smallest group Ia cation, Li⁺, holds water most tenaciously, so it will only dehydrate and bind in the presence of a strongly negative electrostatic potential.Open in a separate windowFigure 1Structural models used in theoretical studies of ion selectivity. (A) Simple model used to introduce the concept of field strength leading to 11 cationic selectivity sequences (2,5,6). Ions, water, and ligands are represented by simple hard-spheres with embedded point charges. Selectivity arises from the difference in the interaction energy of the cation with a water molecule (top) and an anionic coordinating ligand (bottom). (B) Ion-selective transfer process is depicted with atomic models incorporating all molecular details in the case of solvation in liquid water (top) and binding to the K⁺-selective ionophore valinomycin (bottom). Such atomic models were used to carry out some of the earliest MD free energy simulations on ion binding selectivity (12,13,15).By contrast, the largest cation, Cs⁺, holds water least tenaciously. It cannot bind readily to a strongly negative site because the site itself greedily clings to water molecules, and thus prevents Cs⁺ binding. However, Cs⁺ is more willing, relative to the smaller cations, to dehydrate and bind in the presence of a weakly negative electrostatic potential. At the extremes, the highest anionic field strength glass shows a selectivity sequence ofLi⁺ > Na⁺ > K⁺ > Rb⁺ > Cs⁺(sequence XI), and the lowest anionic field strength glass shows a selectivity sequence ofCs⁺ > Rb⁺ > K⁺ > Na⁺ > Li⁺(sequence I).A very simple model, based on the relative Gibbs’ free energies of binding and hydration, explains why there are only 11 sequences (5–7). The critical factor underlying the pattern of these selectivity sequences is that the “ion-site interaction energies fall off as a function of cation size as a lower power of the cation radius than do ion-water interaction energies” (5,6). The icing on the cake is that ion selectivity of channels in membranes appears to follow similar principles (7). The thermodynamic principles are evidently analogous. Moreover, Eisenman’s contributions went far beyond the monovalent cation selectivity of potassium channels. His theoretical approach was seminal in understanding both cation and anion selectivity in a diverse range of physical and biological systems (8,9).The advent of molecular dynamics (MD) simulations around this period was of critical importance to the field. This made it possible to construct increasingly realistic models of proteins (10), including ion channels (11), and examine the ion selectivity of carriers using the alchemical free energy perturbation (FEP) technique (12,13). With no experimental structures yet available for the ion-selective regions of biological K⁺ channels, an important step forward was Eisenman’s realization that other ion-selective systems could be used to computationally test the structural basis of his selectivity theory. Both peptidelike small ionophores, such as valinomycin and nonactin, and the ion-coordinating fivefold symmetry sites in icosahedral virus structures, thus caught his attention (13). As it turned out, these types of structures were indeed very relevant for the selectivity problem, because K⁺-channel filters were eventually shown to be lined likewise by carbonyl groups (14). With the crystallographically determined valinomycin structure at hand, its selectivity could be energetically analyzed by atomistic computer simulations, as illustrated in Fig. 1 B (12,15). The anionic field strength (represented by the carbonyl ligand dipole moment) could then be varied artificially, and the successive progression through the different selectivity sequences, as a function of field strength, directly observed. Likewise, Eisenman and Alvarez (13) made computational predictions for the binding energetics and selectivity of the Ca²⁺ binding site at the fivefold symmetry axis of satellite tobacco necrosis virus, and they subsequently showed experimentally that this binding site had a marked rare-earth ion size selectivity (16). To this day, the general computational FEP/MD framework based on equilibrium thermodynamics used in these studies continues to be a critical tool to understand ion channels (17), transporters (18), and pumps (19).Despite these early insights, it was always clear to Eisenman that explanations of selectivity solely based on thermodynamic equilibrium were too simple to account for the detailed properties observed in biological systems. Since the halcyon days of equilibrium binding studies on glass electrodes, the permeation landscape presented by the pores of ion channels has emerged as richer than anticipated. One important realization is that binding and conduction of ions through a channel may act as contradictory processes, because although an ion has to leave the comfort of its hydration shells to selectively enter the mouth of a channel pore, if it binds the channel too tightly, it cannot move rapidly through it. This mini-conundrum is most apparent, perhaps, for K⁺-channels, which attract K⁺ ions much more forcefully than Na⁺ ions, yet conduct K⁺ ions much faster than Na⁺ ions.Another factor evident in early studies of permeation is that ions encounter a series of obstacles (i.e., energy barriers) and binding sites (i.e., energy wells) as they wend their way through the pore. One approach to understanding permeation is to consider that ions hopscotch from one well to the next over a series of barriers. When the number of barriers is rather limited, say <5, one can use so-called “rate theory” (20) to analyze and formulate the free energy profile experienced by an ion crossing the membrane. Hille (21) proposed that selectivity derives largely from the selectivities of the barriers, not the wells. Eisenman and Horn (7) later considered the possibility that binding sites and barriers within a particular channel might have different selectivity sequences. For example, if a channel presents two barriers, one of which has selectivity sequence I and the other has selectivity sequence XI, the channel as a whole will have an intermediate selectivity sequence that is not an Eisenman sequence at all. Rather, it is a so-called “polarizability sequence” (7). Interestingly, contemporary studies indicate that successive binding sites along K⁺-selective channels display different selectivities (22). Another concept based on Eyring barrier models is that the energy levels for wells and barriers may not be static, and may therefore fluctuate on a timescale relevant to ion permeation (23). Finally, the biophysics of ion permeation and later structural studies show that multiple ions may cohabit the same channel simultaneously, and the interactions among these ions have profound consequences for ion conduction and selectivity.Fast forward to the 21st century: atomic-level structures and all-atom simulations seem to have blown the permeation field wide open, as suggested by recent reviews (24–27). Once the KcsA channel structure was solved (14), the structural origin of K⁺-ion permeation could finally be addressed by computer simulations of the “real structure” and a number MD simulation studies provided novel insight (22,28–31). Needless to say, George Eisenman took great interest in these simulations even though he had by then retired. Also, in the case of KcsA, the initial work largely revolved around calculations of equilibrium ion binding and selectivity, barrier heights, and energy landscape mapping (22,31), because direct all-atom simulations of spontaneous permeation were not possible. However, the general type of knock-on mechanism with multiion occupancy of the channel selectivity filter, involving key distinct states (22,31), and a surprisingly flat energy landscape (22), appear to be robust features of these channels.Even with the advent of MD simulations, the concept of field strength has kept its relevance. For example, the selectivity filter in MD simulations of the KcsA channel displayed a range of atomic flexibility that seemed somewhat shocking at the time because a traditional host-guest mechanism of selectivity would require a fairly rigid cavity-size. Yet, free energy computations indicated that this was not strictly necessary to establish the thermodynamic free energy differences needed to support ion selectivity (32). The resilience of Eisenman’s ideas is not entirely surprising because, as foreseen early on by Bertil Hille (21), the concept of field strength remains “useful if the dipoles of the channel are free to move and can be pulled in by small ions and pushed back by large ones”.Nevertheless, despite the exciting progress, the chapter on ion selectivity in K⁺ channels is far from closed. Very recently, a number of studies have revealed some extremely intriguing multiion aspects of selectivity in K⁺ channels that appear to stand squarely outside the realm of equilibrium thermodynamics. By examining the properties of MthK (33) and NaK (34) mutants, Liu and Lockless (35) and Sauer et al. (36) showed that the channel becomes K⁺-selective only if there are four consecutive binding sites along the filter. This has culminated more recently with studies of two engineered mutants of the NaK channel, referred to as “NaK2K” and “NaK2CNG”. According to reversal potential measurements from single-channel electrophysiology, the NaK2K construct is K⁺-selective and the NaK2CNG construct is nonselective. Remarkably, despite being nonselective in ion permeation, the NaK2CNG filter displays an equilibrium preference for binding K⁺ over Na⁺, as indicated by measurements with isothermal titration calorimetry and concentration-dependent ion replacement within the filter observed through crystallographic titration experiments.K⁺-selective channels bind two or more K⁺ ions in the narrow filter, whereas the nonselective channels bind fewer ions. Based on the crystallographic titration experiments, the NaK2K construct has two high-affinity K⁺ sites whereas the NaK2CNG construct has only one K⁺-selective site. These experiments show that both K⁺-selective and nonselective channels select K⁺ over Na⁺ ions at equilibrium, implying that equilibrium selectivity is insufficient to determine the selectivity of ion permeation (35,36). The data indicate that having multiple K⁺ ions bound simultaneously is required for selective K⁺ conduction, and that a reduction in the number of bound K⁺ ions destroys the multiion selectivity mechanism utilized by K⁺ channels. Although these experimental results are intriguing, the underlying microscopic mechanisms remain unclear. The implication is that the multiion character of the permeation process must, somehow, be a critical element for establishing selective ion conduction through K⁺ channels.The progress made, and the challenges that remain, are perhaps best illustrated by returning to computational studies of the simplest membrane spanning structure known, namely the gramicidin A channel. Before detailed studies of selectivity and conductance of K⁺-channels were launched, computational work on ion conduction through membrane channels was largely focused on this simple channel (37–41). In this case the permeation selectivity was monotonically size-dependent (Eisenman sequence I) and, in this respect, less interesting than K⁺-selective channels. However, from an energetic point of view it was puzzling how this single helical structure could yield free energy barriers low enough to permit high conductivity (7,42). Computer simulations of increasing complexity in this case established that the combined effect of several contributions to ion stabilization along the pore (from the protein, membrane, single-file waters, and bulk solution) indeed results in low barriers to permeation (11,39,40). Furthermore, the most realistic model comes in close agreement with experimental measurements (11,43), although it is clear that work is still needed.     

Pregnenolone Sulfate Potentiates the Inwardly Rectifying K+ Channel Kir2.3

Background

Neurosteroids have various physiological and neuropsychopharmacological effects. In addition to the genomic effects of steroids, some neurosteroids modulate several neurotransmitter receptors and channels, such as N-methyl-D-aspartate receptors, γ-aminobutyric acid type A (GABA_A) receptors, and σ₁ receptors, and voltage-gated Ca²⁺ and K⁺ channels. However, the molecular mechanisms underlying the various effects of neurosteroids have not yet been sufficiently clarified. In the nervous system, inwardly rectifying K⁺ (Kir) channels also play important roles in the control of resting membrane potential, cellular excitability and K⁺ homeostasis. Among constitutively active Kir2 channels in a major Kir subfamily, Kir2.3 channels are expressed predominantly in the forebrain, a brain area related to cognition, memory, emotion, and neuropsychiatric disorders.

Methodology/Principal Findings

The present study examined the effects of various neurosteroids on Kir2.3 channels using the Xenopus oocyte expression assay. In oocytes injected with Kir2.3 mRNA, only pregnenolone sulfate (PREGS), among nine neurosteroids tested, reversibly potentiated Kir2.3 currents. The potentiation effect was concentration-dependent in the micromolar range, and the current-voltage relationship showed inward rectification. However, the potentiation effect of PREGS was not observed when PREGS was applied intracellularly and was not affected by extracellular pH conditions. Furthermore, although Kir1.1, Kir2.1, Kir2.2, and Kir3 channels were insensitive to PREGS, in oocytes injected with Kir2.1/Kir2.3 or Kir2.2/Kir2.3 mRNA, but not Kir2.1/Kir2.2 mRNA, PREGS potentiated Kir currents. These potentiation properties in the concentration-response relationships were less potent than for Kir2.3 channels, suggesting action of PREGS on Kir2.3-containing Kir2 heteromeric channels.

Conclusions/Significance

The present results suggest that PREGS acts as a positive modulator of Kir2.3 channels. Kir2.3 channel potentiation may provide novel insights into the various effects of PREGS.