Potassium permeation through the KcsA channel: a density functional study

We present a theoretical study on structural and electronic aspects of K+ permeation through the binding sites of the KcsA channel's selectivity filter. Density functional calculations are carried out on models taken from selected snapshots of a molecular dynamics simulation recently reported [FEBS Lett. 477 (2000) 37]. During the translocation process from one binding site to the other, the coordination number of the permeating K+ ion turns out to decrease and K+ ion polarizes significantly its ligands, backbone carbonyl groups and a water molecule. K+-induced polarization increases significantly at the transition state (TS) between the two binding sites. These findings suggest that polarization effects play a significant role in the microscopic mechanisms regulating potassium permeation.     

Characterization of a calcium-activated potassium channel in human fibroblasts

The cell-attached and inside-out patch clamp techniques were used to record single-channel currents from human epidermal fibroblasts. A large-conductance channel (320 pS in symmetric 140 mM KCl) with high potassium selectivity was observed in many patches, particularly those located at the borders of the cells. The channel exhibited both voltage and calcium sensitivity and, therefore, was regarded as a variety of the large-conductance calcium-activated potassium channels reported in many preparations. Probability density functions, fitted to histograms of open and closed time durations at 35 degrees C, usually displayed a minimum of one open state and two closed states. However, kinetic analysis by the fractal method suggested more complicated behavior, particularly for the closed condition. It was not uncommon to observe several channels in one patch. This was distinguishable from the presence of subconductances, which were also observed. Although this channel could have many roles, it seems likely to mediate the calcium-activated conductance that underlies the hyperpolarizing response of fibroblasts to mechanical, electrical, or chemical stimuli.     

Sodium permeability of a cloned small-conductance calcium-activated potassium channel

Small-conductance Ca2+-activated potassium channels (SK(Ca) channels) are heteromeric complexes of pore-forming main subunits and constitutively bound calmodulin. SK(Ca) channels in neuronal cells are activated by intracellular Ca2+ that increases during action potentials, and their ionic currents have been considered to underlie neuronal afterhyperpolarization. However, the ion selectivity of neuronal SK(Ca) channels has not been rigorously investigated. In this study, we determined the monovalent cation selectivity of a cloned rat SK(Ca) channel, rSK2, using heterologous expression and electrophysiological measurements. When extracellular K+ was replaced isotonically with Na+, ionic currents through rSK2 reversed at significantly more depolarized membrane potentials than the value expected for a Nernstian relationship for K+. We then determined the relative permeability of rSK2 for monovalent cations and compared them with those of the intermediate- and large-conductance Ca2+-activated K+ channels, IK(Ca) and BK(Ca) channels. The relative permeability of the rSK2 channel was determined as K+(1.0)>Rb+(0.80)>NH(4)+(0.19) approximately Cs+(0.19)>Li+(0.14)>Na+(0.12), indicating substantial permeability of small ions through the channel. Although a mutation near the selectivity filter mimicking other K+-selective channels influenced the size-selectivity for permeant ions, Na+ permeability of rSK2 channels was still retained. Since the reversal potential of endogenous SK(Ca) current is determined by Na+ permeability in a physiological ionic environment, the ion selectivity of native SK(Ca) channels should be reinvestigated and their in vivo roles may need to be restated.     

Neuroleptics antagonize a calcium-activated potassium channel in airway smooth muscle

We examined the effect of neuroleptics on Ca-activated K channels from dog airway smooth muscle cells. Because these agents inhibit a variety of other Ca-mediated processes, it seemed possible that they might also inhibit Ca-activated K channels. In excised, inside-out patches, several neuroleptics potently and reversibly inhibited the K channel from the internal but not the external surface of the patch. Measurements of the effect on open probability and open- and closed-state durations support a simple kinetic model in which neuroleptics bind to and block the open channel. Inhibition by neuroleptics was moderately voltage dependent, with blockers less potent at hyperpolarizing voltages. The relationship between voltage and the dissociation constant for the blocker suggests that the binding site is one-third of the way across the channel's electrical field. Equilibrium dissociation constants for the drug-channel complex were: haloperidol, 1.0 +/- 0.1 microM; trifluoperazine, 1.4 +/- 0.1 microM; thioridazine, 2.4 +/- 0.1 microM; and chlorpromazine, 2.0 microM. This rank-order potency is different from their potency as calmodulin inhibitors, which suggests that neuroleptics bind to the channel rather than a calmodulin-channel complex.     

Simulations of ion permeation through a potassium channel: molecular dynamics of KcsA in a phospholipid bilayer

Potassium channels enable K(+) ions to move passively across biological membranes. Multiple nanosecond-duration molecular dynamics simulations (total simulation time 5 ns) of a bacterial potassium channel (KcsA) embedded in a phospholipid bilayer reveal motions of ions, water, and protein. Comparison of simulations with and without K(+) ions indicate that the absence of ions destabilizes the structure of the selectivity filter. Within the selectivity filter, K(+) ions interact with the backbone (carbonyl) oxygens, and with the side-chain oxygen of T75. Concerted single-file motions of water molecules and K(+) ions within the selectivity filter of the channel occur on a 100-ps time scale. In a simulation with three K(+) ions (initially two in the filter and one in the cavity), the ion within the central cavity leaves the channel via its intracellular mouth after approximately 900 ps; within the cavity this ion interacts with the Ogamma atoms of two T107 side chains, revealing a favorable site within the otherwise hydrophobically lined cavity. Exit of this ion from the channel is enabled by a transient increase in the diameter of the intracellular mouth. Such "breathing" motions may form the molecular basis of channel gating.     

The barium site in a potassium channel by x-ray crystallography

X-ray diffraction data were collected from frozen crystals (100 degrees K) of the KcsA K(+) channel equilibrated with solutions containing barium chloride. Difference electron density maps (F(barium) - F(native), 5.0 A resolution) show that Ba(2+) resides at a single location within the selectivity filter. The Ba(2+) blocking site corresponds to the internal aspect (adjacent to the central cavity) of the "inner ion" position where an alkali metal cation is found in the absence of the blocking Ba(2+) ion. The location of Ba(2+) with respect to Rb(+) ions in the pore is in good agreement with the findings on the functional interaction of Ba(2+) with K(+) (and Rb(+)) in Ca(2+)-activated K(+) channels (Neyton, J., and C. Miller. 1988. J. Gen. Physiol. 92:549-567). Taken together, these structural and functional data imply that at physiological ion concentrations a third ion may interact with two ions in the selectivity filter, perhaps by entering from one side and displacing an ion on the opposite side.     

Modeling permeation energetics in the KcsA potassium channel

The thermodynamics of cation permeation through the KcsA K(+) channel selectivity filter is studied from the perspective of a physically transparent semimicroscopic model using Monte Carlo free energy integration. The computational approach chosen permits dissection of the separate contributions to ionic stabilization arising from different parts of the channel (selectivity filter carbonyls, single-file water, cavity water, reaction field of bulk water, inner helices, ionizable residues). All features play important roles; their relative significance varies with the ion's position in the filter. The cavity appears to act as an electrostatic buffer, shielding filter ions from structural changes in the inner pore. The model exhibits K(+) vs. Na(+) selectivity, and roughly isoenergetic profiles for K(+) and Rb(+), and discriminates against Cs(+), all in agreement with experimental data. It also indicates that Ba(2+) and Na(+) compete effectively with permeant ions at a site near the boundary between the filter and the cavity, in the vicinity of the barium blocker site.     

大电导钙激活钾通道（BKCa）及其开放剂研究进展

大电导钙激活钾通道（BKCa）广泛分布在哺乳动物各种组织（不含心肌细胞）中,并参与细胞内信号转导、细胞的兴奋及代谢调节等生理过程。BKCa功能异常牵涉到特发性癫痫、高血压等疾病的发生。BKCa通道是治疗高血压、尿失禁、哮喘、冠心病及缺血性脑中风等疾病的潜在药物靶点。探索高活性、高选择性、细胞通透性优良、类药性好的BKCa通道开放剂,不仅有助于阐明BKCa通道在生理病理条件下的作用机制,而且为治疗心脑血管疾病的药物研发奠定基础。对各类BKCa通道开放剂做一概述。     

Potassium-selective block of barium permeation through single KcsA channels

Ba(2+), a doubly charged analogue of K(+), specifically blocks K(+) channels by virtue of electrostatic stabilization in the permeation pathway. Ba(2+) block is used here as a tool to determine the equilibrium binding affinity for various monovalent cations at specific sites in the selectivity filter of a noninactivating mutant of KcsA. At high concentrations of external K(+), the block-time distribution is double exponential, marking at least two Ba(2+) sites in the selectivity filter, in accord with a Ba(2+)-containing crystal structure of KcsA. By analyzing block as a function of extracellular K(+), we determined the equilibrium dissociation constant of K(+) and of other monovalent cations at an extracellular site, presumably S1, to arrive at a selectivity sequence for binding at this site: Rb(+) (3 μM) > Cs(+) (23 μM) > K(+) (29 μM) > NH(4)(+) (440 μM) > Na(+) and Li(+) (>1 M). This represents an unusually high selectivity for K(+) over Na(+), with |ΔΔG(0)| of at least 7 kcal mol(-1). These results fit well with other kinetic measurements of selectivity as well as with the many crystal structures of KcsA in various ionic conditions.     

Cation permeation through the voltage-dependent potassium channel in the squid axon. Characteristics and mechanisms

Characteristics of cation permeation through voltage-dependent delayed rectifier K channels in squid giant axons were examined. Axial wire voltage-clamp measurements and internal perfusion were used to determine conductance and permeability properties. These K channels exhibit conductance saturation and decline with increases in symmetrical K+ concentrations to 3 M. They also produce ion- and concentration-dependent current-voltage shapes. K channel permeability ratios obtained with substitutions of internal Rb+ or NH+4 for K+ are higher than for external substitution of these ions. Furthermore, conductance and permeability ratios of NH+4 or Rb+ to K+ are functions of ion concentration. Conductance measurements also reveal the presence of an anomalous mole fraction effect for NH+4, Rb+, or Tl+ to K+. Finally, internal Cs+ blocks these K channels in a voltage-dependent manner, with relief of block by elevations in external K+ but not external NH+4 or Cs+. Energy profiles for K+, NH+4, Rb+, Tl+, and Cs+ incorporating three barriers and two ion-binding sites are fitted to the data. The profiles are asymmetric with respect to the center of the electric field, have different binding energies and electrical positions for each ion, and (for K+) exhibit concentration-dependent barrier positions.     

Potassium excretion through ROMK potassium channel expressed in gill mitochondrion-rich cells of Mozambique tilapia

Despite recent progress in physiology of fish ion homeostasis, the mechanism of plasma K+ regulation has remained unclear. Using Mozambique tilapia, a euryhaline teleost, we demonstrated that gill mitochondrion-rich (MR) cells were responsible for K+ excretion, using a newly invented technique that insolubilized and visualized K+ excreted from the gills. For a better understanding of the molecular mechanism of K+ excretion in the gills, cDNA sequences of renal outer medullary K+ channel (ROMK), potassium large conductance Ca(2+)-activated channel, subfamily M (Maxi-K), K(+)-Cl(-) cotransporters (KCC1, KCC2, and KCC4) were identified in tilapia as the candidate molecules that are involved in K+ handling. Among the cloned candidate molecules, only ROMK showed marked upregulation of mRNA levels in response to high external K+ concentration. In addition, immunofluorescence microscopy revealed that ROMK was localized in the apical opening of gill MR cells, and that the immunosignals were most intense in the fish acclimated to the environment with high K+ concentration. To confirm K+ excretion via ROMK, K+ insolubilization-visualization technique was applied again in combination with K+ channel blockers. The K+ precipitation was prevented in the presence of Ba2+, indicating that ROMK has a pivotal role in K+ excretion. The present study is the first to demonstrate that the fish excrete K+ from the gill MR cells, and that ROMK expressed in the apical opening of the MR cells is a main molecular pathway responsible for K+ excretion.     

Single channel recordings from calcium-activated potassium channels in cultured rat muscle

Single channel recordings from cultured rat skeletal muscle have revealed a large conductance (230 pS) channel with a high selectivity for K⁺ over Na⁺. In excised patches of membrane, the probability of channel opening is sensitive to micromolar concentrations of calcium ions at the intracellular surface of the patch. Channel openings appear grouped together into bursts whose duration increases with Ca²⁺ and membrane depolarization. Statistical analysis of the individual open times during each burst showed that there are two distinct open states of similar conductance but dissimilar average lifetimes. These channels might contribute to a macroscopic calcium-activated potassium conductance in rat skeletal muscle and other preparations.     

Temperature dependence of proton permeation through a voltage-gated proton channel

Voltage-gated proton channels are found in many different types of cells, where they facilitate proton movement through the membrane. The mechanism of proton permeation through the channel is an issue of long-term interest, but it remains an open question. To address this issue, we examined the temperature dependence of proton permeation. Under whole cell recordings, rapid temperature changes within a few milliseconds were imposed. This method allowed for the measurement of current amplitudes immediately before and after a temperature jump, from which the ratios of these currents (I_ratio) were determined. The use of I_ratio for evaluating the temperature dependence minimized the contributions of factors other than permeation. Temperature jumps of various degrees (ΔT, −15 to 15°C) were applied over a wide temperature range (4–49°C), and the Q₁₀s for the proton currents were evaluated from the I_ratios. Q₁₀ exhibited a high temperature dependence, varying from 2.2 at 10°C to 1.3 at 40°C. This implies that processes with different temperature dependencies underlie the observed Q₁₀. A novel resistivity pulse method revealed that the access resistance with its low temperature dependence predominated in high temperature ranges. The measured temperature dependence of Q₁₀ was decomposed into Q₁₀ of the channel and of the access resistances. Finally, the Q₁₀ for proton permeation through the voltage-gated proton channel itself was calculated and found to vary from 2.8 at 5°C to 2.2 at 45°C, as expected for an activation enthalpy of 64 kJ/mol. The thermodynamic features for proton permeation through proton-selective channels were discussed for the underlying mechanism.     

Role of the C-terminus of the high-conductance calcium-activated potassium channel in channel structure and function

The role of ion channels in cell physiology is regulated by processes occurring after protein biosynthesis, which are critical for both channel function and targeting of channels to appropriate cell compartments. Here we apply biochemical and electrophysiological methods to investigate the role of the high-conductance, calcium-activated potassium (Maxi-K) channel C-terminal domain in channel tetramerization, association with the beta1 subunit, trafficking of the channel complex to the cell surface, and channel function. No evidence for channel tetramerization, cell surface expression, or function was observed with Maxi-K(1)(-)(323), a construct truncated three residues after the S(6) transmembrane domain. However, Maxi-K(1)(-)(343) and Maxi-K(1)(-)(441) are able to form tetramers and to associate with the beta1 subunit. Maxi-K(1)(-)(343)-beta1 and Maxi-K(1)(-)(441)-beta1 complexes are efficiently targeted to the cell surface and cannot be pharmacologically distinguished from full-length channels in binding experiments but do not form functional channels. Maxi-K(1)(-)(651) forms tetramers and associates with beta1; however, the complex is not present at the cell surface, but is retained intracellularly. Maxi-K(1)(-)(651) surface expression and channel function can be fully rescued after coexpression with its C-terminal complement, Maxi-K(652)(-)(1113). However coexpression of Maxi-K(1)(-)(343) and Maxi-K(1)(-)(441) with their respective C-terminal complements did not rescue channel function. Together, these data demonstrate that the domain(s) in the Maxi-K channel necessary for formation of tetramers, coassembly with the beta1 subunit, and cell surface expression resides within the S(0)-S(6) linker domain of the protein, and that structural constraints within the gating ring in the C-terminal region can regulate trafficking and function of constructs truncated in this region.     

Oleic acid blocks the calcium-activated chloride channel TMEM16A/ANO1

The calcium-activated chloride channel TMEM16A (ANO1) supports the passive movement of chloride ions across membranes and controls critical cell functions. Here we study the block of wild-type and mutant TMEM16A channels expressed in HEK293 cells by oleic acid, a monounsaturated omega-9 fatty acid beneficial for cardiovascular health. We found that oleic acid irreversibly blocks TMEM16A in a dose- and voltage-dependent manner at low intracellular Ca²⁺. We tested whether oleic acid interacted with the TMEM16A pore, varying the permeant anion concentration and mutating pore residues. Lowering the permeating anion concentration in the intracellular side did nothing but the blockade was intensified by increasing the anion concentration in the extracellular side. However, the blockade of the pore mutants E633A and I641A was voltage-independent, and the I641A IC₅₀, a mutant with the inner hydrophobic gate in disarray, increased 16-fold. Furthermore, the uncharged methyl-oleate blocked 20–24% of the wild-type and I641A channels regardless of voltage. Our findings suggest that oleic acid inhibits TMEM16A by an allosteric mechanism after the electric field drives oleic acid's charged moiety inside the pore. Block of TMEM16A might be why oleic acid has a beneficial impact on the cardiovascular system.     

Characterization of a calcium-activated potassium channel from rabbit intestinal smooth muscle incorporated into planar bilayers

Summary Interaction of vesicles from a microsomal fraction of rabbit intestinal smooth muscle with planar bilayers promotes the incorporation of a large conductance potassium-selective channel. The channel conductance fluctuates between two states: closed and open and the fraction of time the channel dwells in the open state is a function of the electric potential difference and the calcium concentrations. This channel seems to correspond to a Ca-activated K channel described by other authors in smooth muscle cells with the patch-clamp technique. Single-channel conductance is a saturating function of the potassium concentration. The relationship between conductance and concentration cannot be described by a hyperbolic function, suggesting multiple occupancy of the channel. The single-channel conductance is 230 pS in symmetrical 0.1m KCl. Current is a linear function of the applied voltage in the range between –100 and +100 mV, at concentrations of 0.1m KCl or higher. At lower concentrations, current-to-voltage curves bend symmetrically to the voltage axis. Sodium, lithium and cesium ions do not pass through the channel and the permeability for Rb is 66% that of potassium. All these alkali cations and Ca²⁺ block the channel in a voltage-dependent manner. A two-site three-barrier model on Eyring absolute reaction rate theory can account for the conduction and blocking characteristics.     

Induction of calcium-activated potassium channel activity by hemin in human erythroleukemia cells

The agent hemin has been demonstrated to be able to initiate a coordinated differentiation program in several cell types. In the present study, we examined the ability of hemin on inducing cell differentiation and Ca(2+)-activated K(+) channel activity in erythroleukemic K562 cells. Treating undifferentiated K562 cells with hemin (0.1 mM) for five days caused these cells to display differentiation-like characteristics including chromatin aggregation, nuclear degradation, pseudopod extension of the membrane and increased hemoglobin production. However, overall cell viability was not significantly changed by the presence of hemin. After hemin treatment for different periods, the Ca(2+)-activated K(+) channel was activated by the addition of ionomycin (1 microM), and was inhibited by either clotrimazole, charybdotoxin, or EGTA. Before hemin treatment there was no significant Ca(2+)-activated K(+) channel activity present in undifferentiated K562 cells. After hemin treatment for 5 days, a significant Ca(2+)-activated K(+) channel activity was detected. This increasing Ca(2+)-activated K(+) channel activity may be contributed from a subtype of Ca(2+)-activated K(+) channel, KCNN4. These results suggest that the ability of hemin to induce increasing Ca(2+)-activated K(+) channel activity may contribute to the mechanism of hemin-induced K562 cell differentiation.     

Reconstitution in phospholipid vesicles of calcium-activated potassium channel from outer renal medulla

Summary A barium-sensitive Ca-activated K⁺ channel in the luminal membrane of the tubule cells in thick ascending limb of Henle's loop is required for maintenance of the lumen positive transepithelial potential and may be important for regulation of NaCl reabsorption. In this paper we examine if the K⁺ channel can be solubilized and reconstituted into phospholipid vesicles with preservation of its native properties. The K⁺ channel in luminal plasma membrane vesicles can be quantitatively solubilized in CHAPS at a detergent/protein ratio of 3. For reconstitution, detergent is removed by passage over a column of Sephadex G 50 (coarse). K⁺-channel activity is assayed by measurement of⁸⁶Rb⁺ uptake against a large opposing K⁺ gradient. The reconstituted K⁺ channel is activated by Ca²⁺ in the physiological range of concentration (K_1/22×10^–7 m at pH 7.2) as found for the K⁺ channel in native plasma membrane vesicles and shows the same sensitivity to inhibitors (Ba²⁺, trifluoperazine, calmidazolium, quinidine) and to protons. Reconstitution of the K⁺ channel into phospholipid vesicles with full preservation of its native properties is an essential step towards isolation and purification of the K⁺-channel protein.Titration with Ca²⁺ shows that most of the active K⁺ channels in reconstituted vesicles have their cytoplasmic aspect facing outward in contrast to the orientation in plasma membrane vesicles, which requires also addition of Ca²⁺ ionophore in order to observe Ca²⁺ stimulation. The reconstituted K⁺ channel is highly sensitive to tryptic digestion. Brief digestion leads to activation of the K⁺ channel in absence of Ca²⁺, to the level of activity seen with saturating concentrations of Ca²⁺. This tryptic split is located in a cytoplasmic aspect of the K⁺ channel that appears to be involved in opening and closing the K⁺ channel in response to Ca²⁺ binding.     

Testosterone relaxes coronary arteries by opening the large-conductance, calcium-activated potassium channel

Cardiovascular diseases are often considered to be a predominantly male health problem, and it has been suggested that testosterone exerts deleterious effects on cardiovascular function; however, few experimental studies support this suggestion. Moreover, the cellular and molecular mechanism(s) underlying vascular responses to testosterone is unknown. The present study has investigated the acute effects of testosterone on porcine coronary artery smooth muscle at the tissue and cellular levels. Contractile studies demonstrated that testosterone or dihydrotestosterone (a nonaromatizable metabolite) relaxed these arteries by an endothelium-independent mechanism involving potassium efflux. Direct evidence from patch-clamp studies confirmed that testosterone opened K(+) channels in single coronary myocytes, and further analysis identified this protein as the large-conductance, calcium- and voltage-activated potassium (BK(Ca)) channel. Moreover, inhibiting BK(Ca) channel activity significantly attenuated testosterone-induced coronary relaxation. These findings indicate that testosterone relaxes porcine coronary arteries predominantly by opening BK(Ca) channels in coronary myocytes, and this response may be associated with accumulation of cGMP. This novel mechanism may provide a better understanding of testosterone-induced vasorelaxation reported in recent experimental and early clinical studies.