Isoflurane activates rat mitochondrial ATP-sensitive K+ channels reconstituted in lipid bilayers

Activation of mitochondrial ATP-sensitive K(+) (mitoK(ATP)) channels is critical in myocardial protection induced by preconditioning with volatile anesthetics or brief periods of ischemia. In this study, we characterized rat mitoK(ATP) channels reconstituted in lipid bilayers and examined their direct regulation by isoflurane. Mitochondria and the inner membrane fraction were isolated from rat ventricles and fused into lipid bilayers. On the basis of their inhibition by 5-hydroxydecanoate (5-HD)/ATP or activation by diazoxide, mitoK(ATP) channels of several conductance states were observed in symmetrical (150 mM) potassium glutamate (26, 47, 66, 83, and 105 pS). Isoflurane (0.8 mM) increased the cumulative open probability from 0.09 +/- 0.02 at baseline to 0.50 +/- 0.09 (P < 0.05, n = 5), which was inhibited by 5-HD. Isoflurane caused a dose-dependent rightward shift in ATP inhibition of mitoK(ATP) channels, which increased the IC(50) for ATP from 335 +/- 4 to 940 +/- 34 microM at 0.8 mM (P < 0.05, n = 5 approximately 8). We conclude that direct activation of the mitoK(ATP) channel by isoflurane is likely to contribute to volatile anesthetic-induced myocardial preconditioning.     

Inward rectification of the minK potassium channel

The minK protein induces a slowly activating voltage-dependent potassium current when expressed in Xenopus oocytes. We have used macroscopic minK currents to determine the open channel current-voltage relationship for the channel, and have found that the minK current is inwardly rectifying. The channel passes inward current at least 20fold more readily than outward current. Both rat and human minK exhibit this property. The rectification of minK is similar to that reported for a slow component of the cardiac delayed rectifier, strengthening the hypothesis that minK is responsible for that current.We would like to thank Drs. Steve Goldstein and Chris Miller for the artificial rat minK gene, and Dr. Rick Swanson for the human minK construct. This work was supported by NIH grant GM-48851 to L.K.K.     

IRK1 inward rectifier K(+) channels exhibit no intrinsic rectification

In intact cells the depolarization-induced outward IRK1 currents undergo profound relaxation so that the steady-state macroscopic I-V curve exhibits strong inward rectification. A modest degree of rectification persists after the membrane patches were perfused with artificial solutions devoid of Mg(2+) and polyamines, which has been interpreted as a reflection of intrinsic channel gating and led to the view that inward rectification results from enhancement of the intrinsic gating by intracellular cations rather than simple pore block. Furthermore, IRK1 exhibits significant extracellular K(+)-sensitive relaxation of its inward current, a feature that has been likened to the C-type inactivation observed in the voltage-activated Shaker K(+) channels. We found that both these current relaxations can be accounted for by impurities in some common constituents of recording solutions, such as residual hydroxyethylpiperazine in HEPES and ethylenediamine in EDTA. Therefore, inherently, IRK1 channels are essentially ohmic at the macroscopic level, and the voltage jump-induced current relaxations do not reflect IRK1 gating but the unusually high affinity of its pore for cations. Furthermore, our study helps define the optimal experimental conditions for studying IRK1.     

C-terminus determinants for Mg2+ and polyamine block of the inward rectifier K+ channel IRK1.

Critical loci for ion conduction in inward rectifier K+ channels are only now being discovered. The C-terminal region of IRK1 plays a crucial role in Mg2+i blockade and single-channel K+ conductance. A negatively charged aspartate in the putative second transmembrane domain (position 172) is essential for time-dependent block by the cytoplasmic polyamines spermine and spermidine. We have now localized the C-terminus effect in IRK1 to a single, negatively charged residue (E224). Mutation of E224 to G, Q and S drastically reduced rectification. Furthermore, the IRK1 E224G mutation decreased block by Mg2+i and spermidine and, like the E224Q mutation, caused a dramatic reduction in the apparent single-channel K+ conductance. The double mutation IRK1 D172N+ E224G was markedly insensitive to spermidine block, displaying an affinity similar to ROMK1. The results are compatible with a model in which the negatively charged residue at position 224, E224, is a major determinant of pore properties in IRK1. By means of a specific interaction with the negatively charged residue at position 172, D172, E224 contributes to the formation of the binding pocket for Mg2+ and polyamines, a characteristic of strong inward rectifiers.     

Mechanism of the voltage sensitivity of IRK1 inward-rectifier K+ channel block by the polyamine spermine

IRK1 (Kir2.1) inward-rectifier K+ channels exhibit exceedingly steep rectification, which reflects strong voltage dependence of channel block by intracellular cations such as the polyamine spermine. On the basis of studies of IRK1 block by various amine blockers, it was proposed that the observed voltage dependence (valence approximately 5) of IRK1 block by spermine results primarily from K+ ions, not spermine itself, traversing the transmembrane electrical field that drops mostly across the narrow ion selectivity filter, as spermine and K+ ions displace one another during channel block and unblock. If indeed spermine itself only rarely penetrates deep into the ion selectivity filter, then a long blocker with head groups much wider than the selectivity filter should exhibit comparably strong voltage dependence. We confirm here that channel block by two molecules of comparable length, decane-bis-trimethylammonium (bis-QA(C10)) and spermine, exhibit practically identical overall voltage dependence even though the head groups of the former are much wider ( approximately 6 A) than the ion selectivity filter ( approximately 3 A). For both blockers, the overall equilibrium dissociation constant differs from the ratio of apparent rate constants of channel unblock and block. Also, although steady-state IRK1 block by both cations is strongly voltage dependent, their apparent channel-blocking rate constant exhibits minimal voltage dependence, which suggests that the pore becomes blocked as soon as the blocker encounters the innermost K+ ion. These findings strongly suggest the existence of at least two (potentially identifiable) sequentially related blocked states with increasing numbers of K+ ions displaced. Consequently, the steady-state voltage dependence of IRK1 block by spermine or bis-QA(C10) should increase with membrane depolarization, a prediction indeed observed. Further kinetic analysis identifies two blocked states, and shows that most of the observed steady-state voltage dependence is associated with the transition between blocked states, consistent with the view that the mutual displacement of blocker and K+ ions must occur mainly as the blocker travels along the long inner pore.     

Acetylcholine receptor in planar lipid bilayers. Characterization of the channel properties of the purified nicotinic acetylcholine receptor from Torpedo californica reconstituted in planar lipid bilayers

The properties of the channel of the purified acetylcholine receptor (AChR) were investigated after reconstitution in planar lipid bilayers. The time course of the agonist-induced conductance exhibits a transient peak that relaxes to a steady state value. The macroscopic steady state membrane conductance increases with agonist concentration, reaching saturation at 10(-5) M for carbamylcholine (CCh). The agonist-induced membrane conductance was inhibited by d-tubocurarine (50% inhibition, IC50, at approximately 10(-6) M) and hexamethonium (IC50 approximately 10(-5) M). The single channel conductance, gamma, is ohmic and independent of the agonist. At 0.3 M monovalent salt concentrations, gamma = 28 pS for Na+, 30 pS for Rb+, 38 pS for Cs+, and 50 pS for NH+4. The distribution of channel open times was fit by a sum of two exponentials, reflecting the existence of two distinct open states. tau o1 and tau o2, the fast and slow components of the distribution of open times, are independent of the agonist concentration: for CCh this was verified in the range of 10(-6) M less than C less than 10(-3)M. tau 01 and tau o2 are approximately three times longer for suberyldicholine ( SubCh ) than for CCh. tau o1 and tau o2 are moderately voltage dependent, increasing as the applied voltage in the compartment containing agonist is made more positive with respect to the other. At desensitizing concentrations of agonist, the AChR channel openings occurred in a characteristic pattern of sudden paroxysms of channel activity followed by quiescent periods. A local anesthetic derivative of lidocaine ( QX -222) reduced both tau o1 and tau o2. This effect was dependent on both the concentration of QX -222 and the applied voltage. Thus, the AChR purified from Torpedo electric organ and reconstituted in planar lipid bilayers exhibits ion conduction and kinetic and pharmacological properties similar to AChR in intact muscle postsynaptic membranes.     

Evidence for sequential ion-binding loci along the inner pore of the IRK1 inward-rectifier K+ channel

Steep rectification in IRK1 (Kir2.1) inward-rectifier K(+) channels reflects strong voltage dependence (valence of approximately 5) of channel block by intracellular cationic blockers such as the polyamine spermine. The observed voltage dependence primarily results from displacement, by spermine, of up to five K(+) ions across the narrow K(+) selectivity filter, along which the transmembrane voltage drops steeply. Spermine first binds, with modest voltage dependence, at a shallow site where it encounters the innermost K(+) ion and impedes conduction. From there, spermine can proceed to a deeper site, displacing several more K(+) ions and thereby producing most of the observed voltage dependence. Since in the deeper blocked state the leading amine group of spermine reaches into the cavity region (internal to the selectivity filter) and interacts with residue D172, its trailing end is expected to be near M183. Here, we found that mutation M183A indeed affected the deeper blocked state, which supports the idea that spermine is located in the region lined by the M2 and not deep in the narrow K(+) selectivity filter. As to the shallower site whose location has been unknown, we note that in the crystal structure of homologous GIRK1 (Kir3.1), four aromatic side chains of F255, one from each of the four subunits, constrict the intracellular end of the pore to approximately 10 A. For technical simplicity, we used tetraethylammonium (TEA) as an initial probe to test whether the corresponding residue in IRK1, F254, forms the shallower site. We found that replacing the aromatic side chain with an aliphatic one not only lowered TEA affinity of the shallower site approximately 100-fold but also eliminated the associated voltage dependence and, furthermore, confirmed that similar effects occurred also for spermine. These results establish the evidence for physically separate, sequential ion-binding loci along the long inner pore of IRK1, and strongly suggest that the aromatic side chains of F254 underlie the likely innermost binding locus for both blocker and K(+) ions in the cytoplasmic pore.     

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.     

Contributions of a negatively charged residue in the hydrophobic domain of the IRK1 inwardly rectifying K+ channel to K(+)-selective permeation.

Inwardly rectifying K+ channels are highly selective for K+ ions and show strong interaction with ions in the pore. Both features are important for the physiological functions of these channels and pose intriguing mechanistic questions of ion permeation. The aspartate residue in the second putative transmembrane segment of the IRK1 inwardly rectifying K+ channel, previously implicated in inward rectification gating due to cytoplasmic Mg2+ and polyamine block, is found in this study to be crucial for the channel's ability to distinguish between K+ and Rb+ ions. Mutation of this residue also perturbs the interaction between the channel pore and the Sr2+ blocking ion. Our studies suggest that this aspartate residue contributes to a selectivity filter near the cytoplasmic end of the pore.     

Fragments from alpha-actinin insert into reconstituted lipid bilayers.

Recent experiments have indicated that alpha-actinin interacts with phospholipid membranes. Using computer analysis methods we determined two possible lipid binding sites capable of membrane attachment/insertion, residues 281-300 and 720-739 of the primary amino acid sequence on smooth muscle alpha-actinin. Having expressed these regions as fusion proteins with schistosomal GST (glutathione S-transferase), we used differential scanning calorimetry (DSC) to investigate their interaction with mixtures of zwitterionic (dimyristoyl-l-alpha-phosphatidylcholine, DMPC) and anionic (dimyristoyl-l-alpha-phosphatidylglycerol, DMPG) phospholipids in reconstituted lipid bilayers. Calorimetric measurements showed that as fusion protein concentration increased, the main chain transition enthalpy decreased and chain melting temperatures shifted, which is indicative of partial protein insertion into the hydrophobic region of the lipid membranes. Centrifugation assay and subsequent SDS/Page chromatography confirmed this finding.     

Single channel behavior of recombinant beta 2 gap junction connexons reconstituted into planar lipid bilayers.

The beta 2 gap junction protein (Cx26) was expressed in an insect cell line by infection with a baculovirus vector containing the rat beta 2 cDNA. Isolated beta 2 gap junction connexons were reconstituted into planar lipid bilayers. Single channel activity was observed with a unitary conductance of 35-45 pS in 200 mM KCl. Channels with conductance values of 60 pS and 90-110 pS also coexisted with the lower conducting channel suggesting that there are channels with different conductance properties within a population of connexons. Channel activity was observed at voltages of up to 150 mV. Furthermore, the characterization of these channel properties from the beta 2 connexons that were generated by this heterologous expression system has provided the basis for identifying an endogenous beta 2 connexon channel in material reconstituted from native rat liver gap junctions.     

Characterization of the channel properties of a neuronal acetylcholine receptor reconstituted into planar lipid bilayers

An alpha-toxin-binding membrane protein, isolated from the head and thoracic ganglia of the locus (Locusta migratoria), was reconstituted into planar lipid bilayers. Cholinergic agonists such as acetylcholine, carbamylcholine, and suberyldicholine induced fluctuations of single channels, which suggests that the protein represents a functional cholinergic receptor channel. The antagonist d-tubocurarine blocked the activation of the channels, whereas hexamethonium had only a weak effect; similar properties have been described for nicotinic insect receptors in situ. The channel was selectively permeable to monovalent cations but was impermeable to anions. The conductance of the channel (75 pS in 100 mM NaCl) was independent of the type of agonist used to activate the receptor. Kinetic analysis of the channel gating revealed that, at high agonist concentrations (50 microM carbamylcholine), more than one closed state exists and that multiple gating events, bursting as well as fast flickering, appeared. At very high agonist concentrations (500 microM carbamylcholine), desensitization was observed. Channel kinetics were dependent on the transmembrane potential. Comparing the conductance, the kinetics, and the pharmacology of nicotinic acetylcholine receptor from insect ganglia and fish electroplax reconstituted into bilayers revealed obvious similarities but also significant differences.     

Large-conductance cholesterol-amphotericin B channels in reconstituted lipid bilayers

The antimycotic activity of amphotericin B (AmB) depends on its ability to make complexes sterols to form ion channels that cause membrane leakage. To study this phenomenon, surface pressure (pi) as a function of surface area (A) and pi-A hysteresis were measured in monolayers of AmB-cholesterol mixtures on the water-air interface. The most stable monolayers were produced from molecules of AmB and cholesterol with 2:1 stoichiometry. At this ratio, AmB and cholesterol interact to form ion channels in lipid bilayers with millisecond dwell times and conductances of 4-400 pS. The AmB-cholesterol complexes assemble in three, four, etc., subunit aggregates to form ion channels of diverse and large-conductances. Their I-V characteristics were linear over a range of +/-200 mV. The channel currents were inhibited by the addition of tetraethylammonium (TEA), potassium channel blocker, to the cis-side of the membrane. Likewise, AmB-cholesterol complexes reconstituted in membrane-coated nanoporous silicon dioxide surfaces showed single channel behavior with large amplitudes at various voltages. Large-conductance ion channels show great promise for use in biosensors on solid supports.     

Inward rectification of the AKT2 channel abolished by voltage-dependent phosphorylation

The Arabidopsis K(+) channel AKT2 possesses the remarkable property that its voltage threshold for activation can be either within the physiological range (gating mode 1), or shifted towards considerably more positive voltages (gating mode 2). Gating mode 1 AKT2 channels behave as delayed K(+)-selective inward rectifiers; while gating mode 2 AKT2 channels are K(+)-selective 'open leaks' in the physiological range of membrane potential. In the present study we have investigated modulation of AKT2 current by effectors of phosphatases/kinases in COS cells and Xenopus oocytes. These experiments show that (i) dephosphorylation can result in AKT2 channel silencing; and (ii) phosphorylation by protein kinase A (PKA) favors both recruitment of silenced AKT2 channels and transition from gating mode 1 to gating mode 2. Interestingly, phosphorylation of AKT2 by PKA in COS cells and Xenopus oocytes is favored by hyperpolarization. Two PKA phosphorylation sites (S210 and S329) were pinpointed in the region of the pore inner mouth. The role of these phosphorylation sites in the switch between the two gating modes was assessed by electrophysiological characterization of mutant channels. The molecular aspects of AKT2 regulation by phosphorylation, and the possible physiological meaning of such regulation in the plant context, are discussed.     

Transport kinetics of uncoupling proteins. Analysis of UCP1 reconstituted in planar lipid bilayers

According to alternative hypotheses, mitochondrial uncoupling protein 1 (UCP1) is either a proton channel ("buffering model") or a fatty acid anion carrier ("fatty acid cycling"). Transport across the proton channel along a chain of hydrogen bonds (Grotthus mechanism) may include fatty acid carboxyl groups or occur in the absence of fatty acids. In this work, we demonstrate that planar bilayers reconstituted with UCP1 exhibit an increase in membrane conductivity exclusively in the presence of fatty acids. Hence, we can exclude the hypothesis considering a preexisting H+ channel in UCP1, which does not require fatty acid for function. The augmented conductivity is nearly completely blocked by ATP. Direct application of transmembrane voltage and precise current measurements allowed determination of ATP-sensitive conductances at 0 and 150 mV as 11.5 and 54.3 pS, respectively, by reconstituting nearly 3 x 10(5) copies of UCP1. The proton conductivity measurements carried out in presence of a pH gradient (0.4 units) allowed estimation of proton turnover numbers per UCP1 molecule. The observed transport rate of 14 s-1 is compatible both with carrier and channel nature of UCP1.     

Kinetic analysis of bacterial reaction centers reconstituted in lipid bilayers

(1) Reaction center-lipid complexes were extracted into octane solutions. Different methods for generating an assymetric membrane distribution of reaction centers are discussed, which allow the measurement of electrical signals upon illumination. (2) The dichroism of the chromophoric groups in the reaction centers was investigated in planar lipid bilayers and the angle β between each transition moment and the normal to the membrane could be determined to be β(757 nm) = 29.5 ± 1.2, β(801 nm) = 34 ± 1.0 and β(860 nm) = 41.3 ± 0.9°. (3) The kinetics of the reaction centers from Rhodopseudomonas sphaeroides were analysed by electrical measurements and the relevant rate constants could be determined. In addition, the interaction between reaction centers and the intramembrane, ubiquinone-containing pool was investigated and described in a kinetic model. (4) The interaction between the electron-donating ferrocytochromes exhibited two distinguishable sources, a fast accessible, membrane-bound pool, which is limited by diffusion, and a pool consisting of an aqueous solution of ferrocytochrome c, which is accessible with a slower rate constant.     

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.     

Protons decrease the single channel conductance of the sarcoplasmic reticulum K+ channel in neutral and negatively charged bilayers.

The conductance of rabbit sarcoplasmic reticulum K+ channels incorporated into artificial bilayers of varying lipid composition was measured at different K+ and proton concentrations. Protons competitively inhibit the K+ conductance with a Ki of 0.5 microM. In negatively charged membranes, the conductance is well described by Gouy-Chapman-Stern theory modified to include the inhibitory effect of protons of the conductance and assuming that the channel mouth is isolated by 5-10 A from bilayer surface.