Aminoglycoside blockade of Ca2+-activated K+ channel from rat brain synaptosomal membranes incorporated into planar bilayers

Summary Ca²⁺-activated K⁺ channels from rat brain synaptosomal membranes were incorporated into planar lipid bilayers, and the effects of aminoglycoside antibiotics on the single channel conductance (258±13 pS at 100mm K⁺) were investigated. Aminoglycosides reduced the single channel conductance from the cis (cytoplasmic) side in a dose- and voltage-dependent manner. Voltage dependence of the blockade indicated an interaction between positively charged amino residues of aminoglycoside antibiotics and a binding site located within the electric field of the ion-conducting pathway. The order of blocking potency was consistent with that of the number of amino residues of aminoglycosides (neomycin (6)>dibekacin (5)>ribostamycin (4)=kanamycin (4)), while the electrical distance (z=0.46–0.49) of the binding site kept almost constant for each drug. Thesezs were almost the same with those (0.46–0.51) of alkyldiamine blockers with two amino residues (total net charge of +2) and approximately twice of those (0.25–0.26) of alkylmonoamine blockers (total net charge of +1). Assuming that amino residues of aminoglycosides and alkylamines shared the same binding site located at 25% voltage drop from the cytoplasmic surface of the channel, the site would have to be at least large enough to accommodate one diamino sugar residue of the aminoglycoside in order to simultaneously interact with two positively charged amino groups. Dose- and voltage-dependent blockade of the channel by gallamine, an extremely bulky trivalent organic cation, supported the picture that the channel has a wide mouth on the cytoplasmic side and its pore region, where voltage drop occurs, may also be quite wide and nonselective, suddenly tapering to a constriction where most charged cations block the channel by occluding the K⁺-conducting pathway.     

Kinetics of Ca2+-activated K+ channels from rabbit muscle incorporated into planar bilayers. Evidence for a Ca2+ and Ba2+ blockade

The interaction of Ca2+ and Ba2+ with a Ca2+-activated K+ channel from rabbit skeletal muscle membranes is studied in planar lipid bilayers. At [Ca2+] greater than or equal to 100 microM in the cis side (the side to which the vesicles are added) and at positive voltages, the channel kinetics consisted of bursts of activity interrupted by long periods of quiescence. We found that the reciprocal of the mean burst time increases linearly with [Ca2+], whereas the mean time for the quiescent (closed) periods is independent of [Ca2+]. The number of quiescent periods is reduced by increasing [K+]. Micromolar amounts of cis Ba2+ do not activate the channel, but induce similar "slow" closings. Also, in this case, the mean burst time is inversely proportional to the [Ba2+] and the mean closed time is independent of [Ba2+]. Raising [K+] either symmetrically or only in the trans side relieved the Ba2+ effect. trans Ba2+ also induces changes in the slow kinetics, but in millimolar amounts. These results suggest that the quiescent periods correspond to a channel blocked by a Ba ion. The voltage dependence of the cis blockade indicates that the Ba2+ binding site is past the middle of the membrane field. The similarities in the slow kinetics induced by Ca2+ and Ba2+ suggest that Ca2+ blocks the channel by binding to the same site. However, binding of Ca2+ to the site is 10(5)- fold weaker.     

Gating kinetics of Ca2+-activated K+ channels from rat muscle incorporated into planar lipid bilayers. Evidence for two voltage- dependent Ca2+ binding reactions

The gating kinetics of a Ca2+-activated K+ channel from adult rat muscle plasma membrane are studied in artificial planar bilayers. Analysis of single-channel fluctuations distinguishes two Ca2+- and voltage-dependent processes: (a) short-lived channel closure (less than 1 ms) events appearing in a bursting pattern; (b) opening and closing events ranging from one to several hundred milliseconds in duration. The latter process is studied independently of the first and is denoted as the primary gating mode. At constant voltage, the mean open time of the primary gating mode is a linear function of the [Ca2+], whereas the mean closed time is a linear function of the reciprocal [Ca2+]. In the limits of zero and infinite [Ca2+], the mean open and the mean closed times are, respectively, independent of voltage. These results are predicted by a kinetic scheme consisting of the following reaction steps: (a) binding of Ca2+ to a closed state; (b) channel opening; (c) binding of a second Ca2+ ion. In this scheme, the two Ca2+ binding reactions are voltage dependent, whereas the open-closed transition is voltage independent. The kinetic constant derived for this scheme gives an accurate theoretical fit to the observed equilibrium open-state probability. The results provide evidence for a novel regulatory mechanism for the activity of an ion channel: modulation by voltage of the binding of an agonist molecule, in this case, Ca2+ ion.     

Low-conductance chloride channel from crayfish skeletal muscle incorporated into planar lipid bilayers.

Low-conductance chloride channel from skeletal muscle SR vesicles of the crayfish Astacus fluviatilis was incorporated into planar lipid bilayers and its basic characteristics were investigated. The channel has a relatively low unitary conductance of 26 pS in symmetrical 160 mmol/l choline-chloride. The dependence of the channel conductance on Cl- concentration shows saturating behavior with a maximum conductance of 37 pS and an ionic activity for half-maximum conductance Km = 75 mmol/l. The channel exhibits a complex kinetics with several modes of activity. Open state probability slightly decreases with the increasing absolute value of voltage. The channel activity does not appear to be dependent on the presence of Ca2+ ions. The channel is effectively inhibited by DIDS, a stilbene derivative. The permeability properties of the channel are similar to the specific behavior of the "double-barrelled" channel from Torpedo electroplax described by Miller and White (1984).     

Activation by divalent cations of a Ca2+-activated K+ channel from skeletal muscle membrane

Several divalent cations were studied as agonists of a Ca2+-activated K+ channel obtained from rat muscle membranes and incorporated into planar lipid bilayers. The effect of these agonists on single-channel currents was tested in the absence and in the presence of Ca2+. Among the divalent cations that activate the channel, Ca2+ is the most effective, followed by Cd2+, Sr2+, Mn2+, Fe2+, and Co2+. Mg2+, Ni2+, Ba2+, Cu2+, Zn2+, Hg2+, and Sn2+ are ineffective. The voltage dependence of channel activation is the same for all the divalent cations. The time-averaged probability of the open state is a sigmoidal function of the divalent cation concentration. The sigmoidal curves are described by a dissociation constant K and a Hill coefficient N. The values of these parameters, measured at 80 mV are: N = 2.1, K = 4 X 10(-7) mMN for Ca2+; N = 3.0, K = 0.02 mMN for Cd2+; N = 1.45, K = 0.63 mMN for Sr2+; N = 1.7, K = 0.94 mMN for Mn2+; N = 1.1, K = 3.0 mMN for Fe2+; and N = 1.1 K = 4.35 mMN for Co2+. In the presence of Ca2+, the divalent cations Cd2+, Co2+, Mn2+, Ni2+, and Mg2+ are able to increase the apparent affinity of the channel for Ca2+ and they increase the Hill coefficient in a concentration-dependent fashion. These divalent cations are only effective when added to the cytoplasmic side of the channel. We suggest that these divalent cations can bind to the channel, unmasking new Ca2+ sites.     

Competitive Mg2+ block of a large-conductance, Ca(2+)-activated K+ channel in rat skeletal muscle. Ca2+, Sr2+, and Ni2+ also block

The patch-clamp technique was used to investigate the effect of intracellular Mg2+ (Mgi2+) on the conductance of the large-conductance, Ca(2+)-activated K+ channel in cultured rat skeletal muscle. Measurements of single-channel current amplitudes indicated that Mgi2+ decreased the K+ currents in a concentration-dependent manner. Increasing Mgi2+ from 0 to 5, 10, 20, and 50 mM decreased channel currents by 34%, 44%, 56%, and 73%, respectively, at +50 mV. The magnitude of the Mgi2+ block increased with depolarization. For membrane potentials of -50, +50, and +90 mV, 20 mM Mgi2+ reduced the currents 22%, 56%, and 70%, respectively. Mgi2+ did not change the reversal potential, indicating that Mg2+ does not permeate the channel. The magnitude of the Mgi2+ block decreased as the concentration of K+ was increased. At a membrane potential of +50 mv, 20 mM Mgi2+ reduced the currents 71%, 56%, and 25% for Ki+ of 75, 150, and 500 mM. These effects of Mgi2+, voltage, and K+ were totally reversible. Although the Woodhull blocking model could approximate the voltage and concentration effects of the Mgi2+ block (Kd approximately 30 mM with 150 mM symmetrical K+; electrical distance approximately 0.22 from the inner surface), the Woodhull model could not account for the effects of K+. Double reciprocal plots of 1/single channel current vs. 1/[K+] in the presence and absence of Mgi2+, indicated that the Mgi2+ block is consistent with apparent competitive inhibition between Mgi2+ and Ki+. Cai2+, Nii2+, and Sri2+ were found to have concentration- and voltage-dependent blocking effects similar, but not identical, to those of Mgi2+. These observations suggest the blocking by Mgi2+ of the large-conductance, Ca(2+)-activated K+ channel is mainly nonspecific, competitive with K+, and at least partially electrostatic in nature.     

Ca(2+)-activated K+ channel inhibition by reactive oxygen species

We studied the effect of H(2)O(2) on the gating behavior of large-conductance Ca(2+)-sensitive voltage-dependent K(+) (K(V,Ca)) channels. We recorded potassium currents from single skeletal muscle channels incorporated into bilayers or using macropatches of Xenopus laevis oocytes membranes expressing the human Slowpoke (hSlo) alpha-subunit. Exposure of the intracellular side of K(V,Ca) channels to H(2)O(2) (4-23 mM) leads to a time-dependent decrease of the open probability (P(o)) without affecting the unitary conductance. H(2)O(2) did not affect channel activity when added to the extracellular side. These results provide evidence for an intracellular site(s) of H(2)O(2) action. Desferrioxamine (60 microM) and cysteine (1 mM) completely inhibited the effect of H(2)O(2), indicating that the decrease in P(o) was mediated by hydroxyl radicals. The reducing agent dithiothreitol (DTT) could not fully reverse the effect of H(2)O(2). However, DTT did completely reverse the decrease in P(o) induced by the oxidizing agent 5,5'-dithio-bis-(2-nitrobenzoic acid). The incomplete recovery of K(V,Ca) channel activity promoted by DTT suggests that H(2)O(2) treatment must be modifying other amino acid residues, e.g., as methionine or tryptophan, besides cysteine. Noise analysis of macroscopic currents in Xenopus oocytes expressing hSlo channels showed that H(2)O(2) induced a decrease in current mediated by a decrease both in the number of active channels and P(o).     

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.     

Leakage-channel conductance of single (Na+ + K+)-ATPase molecules incorporated into planar bilayers by fusion of liposomes

By fusing liposomes which contain in the mean only one pump unit (one intramembranous particle) to planar bilayers, and provoking the ouabain-blockable leakage conductance by the presence of n-decane, the predominant unit leakage conductance associated with one pump unit was estimated to be 40–50 pS, indicating the channel nature of the leakage pathway.     

Mechanisms of Cs+ blockade in a Ca2+-activated K+ channel from smooth muscle.

Large unitary conductance Ca2+-activated K+ channels from smooth muscle membrane were incorporated into phospholipid planar bilayers, and the blockade induced by internally and externally applied Cs+ was characterized. Internal Cs+ blockade is voltage dependent and can be explained on the basis of a Cs+ binding to a site that senses 54% of the applied voltage, with an apparent dissociation constant, Kd(0), of 70 mM. On the other hand, external Cs+ blocks the channel in micromolar amounts, and the voltage dependence of blockade is a function of Cs+ concentration. The fractional electrical distance can be as large as 1.4 at 10 mM Cs+. This last result suggests that the channel behaves as a multi-ion pore. At large negative voltages the I-V relationships in the presence of external Cs+ show an upturn, indicating relief of Cs+ block. External Cs+ blockade is relieved by increasing the internal K+ concentration, but can be enhanced by increasing the external K+. All the characteristics of external Cs+ block can be explained by a model that incorporates a "knock-on" of Cs+ by K+.     

Effect of phospholipid surface charge on the conductance and gating of a Ca2+-activated K+ channel in planar lipid bilayers

Summary A Ca-activated, K-selective channel from plasma membrane of rat skeletal muscle was studied in artificial lipid bilayers formed from either phosphatidylethanolamine (PE) or phosphatidylserine (PS). In PE, the single-channel conductance exhibited a complex dependence on symmetrical K⁺ concentration that could not be described by simple Michaelis-Menten saturation. At low K⁺ concentrations the channel conductance was higher in PS membranes, but approached the same conductance observed in PE above 0.4m KCl. At the same Ca²⁺ concentration and voltage, the probability of channel opening was significantly greater in PS than PE. The differences in the conduction and gating, observed in the two lipids, can be explained by the negative surface charge of PS compared to the neutral PE membrane. Model calculations of the expected concentrations of K⁺ and Ca²⁺ at various distances from a PS membrane surface, using Gouy-Chapman-Stern theory, suggest that the K⁺-conduction and Ca²⁺-activation sites sense a similar fraction of the surface potential, equivalent to the local electrostatic potential at a distance of 9 Å from the surface.     

Gating behaviors of a voltage-dependent and Ca2+-activated cation channel of yeast vacuolar membrane incorporated into planar lipid bilayer

Summary A voltage-dependent and Ca²⁺-activated cation channel found in the vacuolar membrane of the yeast,Saccharomyces cerevisiae, was incorporated into planar lipid bilayer and its gating characteristics were studied at the macroscopic and single-channel levels. The open-channel probability at steady state, which was estimated by the macroscopic current measurement, gave a maximum value at –10 mV and decreased in a graded fashion as the voltage became more positive or more negative. The steady-state voltage dependence was explained by assuming two independent gates, which had different rate constants and opposite voltage dependence. The fast-responding gate opened when the voltage of thecis side (the side to which the vesicles were added) was made more negative and the slow-responding gate behaved in the opposite direction. Relatively high concentrations of Ca²⁺, about 1mm, were required on thecis side for opening the slow gate in a voltage-dependent manner. DIDS increased the open-channel probability of the fast gate when added to thecis side, but was ineffective on the slow gate.     

Stretch-induced activation of Ca(2+)-activated K(+) channels in mouse skeletal muscle fibers

High-conductanceCa²⁺-activatedK⁺(K_Ca) channels werestudied in mouse skeletal muscle fibers using thepatch-clamp technique. In inside-out patches, application of negativepressure to the patch induced a dose-dependent and reversibleactivation of K_Ca channels.Stretch-induced increase in channel activity was found to be of thesame magnitude in the presence and in the absence ofCa²⁺ in the pipette. Thedose-response relationships betweenK_Ca channel activity andintracellular Ca²⁺ and betweenK_Ca channel activity and membranepotential revealed that voltage andCa²⁺ sensitivity were not alteredby membrane stretch. In cell-attached patches, in the presence of highexternal Ca²⁺ concentration,stretch-induced activation was also observed. We conclude that membranestretch is a potential mode of regulation of skeletal muscleK_Ca channel activity and could beinvolved in the regulation of muscle excitability duringcontraction-relaxation cycles.

     

Conduction and block by organic cations in a K+-selective channel from sarcoplasmic reticulum incorporated into planar phospholipid bilayers

A collection of organic cations has been used to probe the gross structural features of the ionic diffusion pathway in a K+-selective channel from sarcoplasmic reticulum (SR). Channels were incorporated into planar phospholipid bilayer membranes, and single-channel currents were measured in the presence of ammonium-derived cations in the aqueous phases. Small monovalent organic cations are able to permeate the channel: the channel conductance drops sharply for cations having molecular cross sections larger than 18-20 A2. Impermeant or poorly permeant cations such as tetraethylammonium, choline, and glucosamine, among others, block K+ conduction through the channel. This block is voltage dependent and can be described by a one-site, one-ion blocking scheme. 19 monovalent organic cations blocks primarily from the trans side of the membrane (the side defined as zero voltage), and much more weakly, if at all, from the cis side (to which SR vesicles are added). These blockers all appear to interact with a site located at 63% (average value) of the electric potential drop measured from the trans side. Furthermore, block by 1,3-bis[tris(hydroxymethyl)-methylamino] propane (BTP) shows that the presence of a blocking ion increases the duration of the apparent open state, as expected for a scheme in which the blocking site can be reached only when the channel is open. The results lead to a picture of the channel containing a wide (at least 50 A2) nonselective trans entry in series with a narrow (20 A2) constriction.     

A cation channel for K+ and Ca2+ from Tetrahymena cilia in planar lipid bilayers

The single-channel properties for monovalent and divalent cations of a voltage-independent cation channel from Tetrahymena cilia were studied in planar lipid bilayers. The single-channel conductance reached a maximum value as the K+ concentration was increased in symmetrical solutions of K+. The concentration dependence of the conductance was approximated to a simple saturation curve (a single-ion channel model) with an apparent Michaelis constant of 16.3 mM and a maximum conductance of 354 pS. Divalent cations (Ca2+, Ba2+, Sr2+, and Mg2+) also permeated this channel. The sequence of permeability determined by zero current potentials at high ionic concentrations was Ba2+ greater than or equal to K+ greater than or equal to Sr2+ greater than Mg2+ greater than Ca2+. Single-channel conductances for Ca2+ were nearly constant (13.9 pS-20.5 pS) in the concentrations between 0.5 mM and 50 mM Ca-gluconate. In the experiments with mixed solutions of K+ and Ca2+, a maximum conductance of Ca2+ (gamma Camax) and an apparent Michaelis constant of Ca2+ (K Cam) were obtained by assuming a simple competitive relation between the cations. Gamma Camax and K Cam were 14.0 pS and 0.160 mM, respectively. Single-channel conductances in mixed solutions were well-fitted to this competitive model supporting that this cation channel behaves as a single-ion channel. This channel had relatively high-affinity Ca2+-binding sites.     

Upregulation of intermediate-conductance Ca2+-activated K+ channel (IKCa1) mediates phenotypic modulation of coronary smooth muscle

A hallmark of smooth muscle cell (SMC) phenotypic modulation in atherosclerosis and restenosis is suppression of SMC differentiation marker genes, proliferation, and migration. Blockade of intermediate-conductance Ca(2+)-activated K(+) channels (IKCa1) has been shown to inhibit restenosis after carotid balloon injury in the rat; however, whether IKCa1 plays a role in SMC phenotypic modulation is unknown. Our objective was to determine the role of IKCa1 channels in regulating coronary SMC phenotypic modulation and migration. In cultured porcine coronary SMCs, platelet-derived growth factor-BB (PDGF-BB) increased TRAM-34 (a specific IKCa1 inhibitor)-sensitive K(+) current 20-fold; increased IKCa1 promoter histone acetylation and c-jun binding; increased IKCa1 mRNA approximately 4-fold; and potently decreased expression of the smooth muscle differentiation marker genes smooth muscle myosin heavy chain (SMMHC), smooth muscle alpha-actin (SMalphaA), and smoothelin-B, as well as myocardin. Importantly, TRAM-34 completely blocked PDGF-BB-induced suppression of SMMHC, SMalphaA, smoothelin-B, and myocardin and inhibited PDGF-BB-stimulated migration by approximately 50%. Similar to TRAM-34, knockdown of endogenous IKCa1 with siRNA also prevented the PDGF-BB-induced increase in IKCa1 and decrease in SMMHC mRNA. In coronary arteries from high fat/high cholesterol-fed swine demonstrating signs of early atherosclerosis, IKCa1 expression was 22-fold higher and SMMHC, smoothelin-B, and myocardin expression significantly reduced in proliferating vs. nonproliferating medial cells. Our findings demonstrate that functional upregulation of IKCa1 is required for PDGF-BB-induced coronary SMC phenotypic modulation and migration and support a similar role for IKCa1 in coronary SMC during early coronary atherosclerosis.