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.     

Comparison of mitochondrial cationic channels in wild-type and porin-deficient mutant yeast.

Bilayers were formed at the tip of microelectrodes from a suspension of proteoliposomes derived from wild-type and porin-deficient mutant yeast mitochondria. In both preparations, identical cationic channels of large conductance were recorded. This result rules out any relationship between this channel and the outer membrane voltage-dependent anion channel, the activity of which is carried by porin. The ionic selectivity and the voltage-dependence of the yeast cationic channel suggest that it is related to that recently described in mammalian mitochondria. This hypothesis is further supported by the fact that both channels are blocked by a mitochondrial addressing peptide.     

Binding of a synthetic targeting peptide to a mitochondrial channel protein

Membrane crystals of the mitochondrial outer membrane channel VDAC (porin) fromNeurospora crassa were incubated with a 20-amino-acid synthetic peptide corresponding to the N-terminal targeting region of subunit IV of cytochrome oxidase. The peptide caused disordering and contraction of the crystal lattice of the membrane arrays. Also, new stain-excluding features were observed on the peptide-treated arrays which most likely correspond to sites at which the peptide accumulates. The stain exclusion zones associated with binding of the targeting peptide (and with binding of apocytochromec in an earlier study) have been localized on a two-dimensional density map of frozen-hydrated, crystalline VDAC previously obtained by cryo-electron microscopy. The results indicate that both the peptide and cytochromec bind to protein arms which extend laterally between the channel lumens. The finding that imported polypeptides bind to a specific region of the VDAC protein implicates this channel in the process by which precursor proteins are recognized at and translocated across the mitochondrial outer membrane.     

Chemical modification of the two histidine and single cysteine residues in the channel-forming domain of colicin E1

Summary The two histidine residues of COOH-terminal channel-forming peptides of colicin E1 were modified by addition of a carbethoxy group through pretreatment with diethylpyrocarbonate. The consequences of the modification were examined by the action of the altered product on both phospholipid vesicles and planar membranes. At pH 6, where activity is low, histidine modification resulted in a decrease of the single channel conductance from 20 pS to approximately 9 pS and a decrease in the selectivity for sodium relative to chloride, showing that histidine modification affected the permeability properties of the channel. At pH 4, where activity is high, the single channel conductance and ion selectivity were not significantly altered by histidine modification. The histidine modification assayed at pH 4 resulted in a threefold increase in the rate of Cl^– efflux from asolectin vesicles, and a similar increase in conductance assayed with planar membranes. This conductance increase was inferred to arise from an increase in the fraction of bound histidine-modified colicin molecules forming channels at pH 4, since the increase in activity was not due to (i) an increase in binding of the modified peptide, (ii) a change in ion selectivity, (iii) a change of single channel conductance, or (iv) a change in the pH dependence of binding. The sole cysteine in the colicin molecule was modified in 6m urea with 5,5-dithiobis(2-nitrobenzoic acid). The activities of the colicin and its COOH-terminal tryptic peptide were found to be unaffected by cysteine modification, arguing against a role of (-SH) groups in protein insertion and/or channel formation.     

Voltage-dependent,monomeric channel activity of colicin E1 in artificial membrane vesicles

Summary The dependence of colicin channel activity on membrane potential and peptide concentration was studied in large unilamellar vesicles using colicin E1, its COOH-terminal thermolytic peptide and other channel-forming colicins. Channel activity was assayed by release of vesicle-entrapped chloride, and could be detected at a peptide: lipid molar ratio as low as 10^–7. The channel activity was dependent on the magnitude of atrans-negative potassium diffusion potential, with larger potentials yielding faster rates of solute efflux. For membrane potentials greater than –60mV (K _in ⁺ /K _out ⁺ 10), addition of valinomycin resulted in a 10-fold increase in the rate of Cl^– efflux. A delay in Cl^– efflux observed when the peptide was added to vesicles in the presence of a membrane potential implied a potential-independent binding-insertion mechanism. The initial rate of Cl^– efflux was about 1% of the single-channel conductance, implying that only a small fraction of channels were initially open, due to the delay or latency of channel formation known to occur in planar bilayers.The amount of Cl^– released as a function of added peptide increased monotonically to a concentration of 0.7 ng peptide/ml, corresponding to release of 75% of the entrapped chloride. It was estimated from this high activity and consideration of vesicle number that 50–100% of the peptide molecules were active. The dependence of the initial rate of Cl^– efflux on peptide concentration was linear to approximately the same concentration, implying that the active channel consists of a monomeric unit.     

Pores formed by Baxα5 relax to a smaller size and keep at equilibrium

Pores made by amphipathic cationic peptides (e.g., antimicrobials and fragments of pore-forming proteins) are typically studied by examining the kinetics of vesicle leakage after peptide addition or obtaining structural measurements in reconstituted peptide-lipid systems. In the first case, the pores have been considered transient phenomena that allow the relaxation of the peptide-membrane system. In the second, they correspond to equilibrium structures at minimum free energy. Here we reconcile both approaches by investigating the pore activity of the α5 fragment from the proapoptotic protein Bax (Baxα5) before and after equilibrium of peptide/vesicle complexes. Quenching assays on suspensions of large unilamellar vesicles suggest that in the presence of Baxα5, the vesicles maintain a leaky state for hours under equilibrium conditions. We proved and analyzed stable pores on single giant unilamellar vesicles (GUVs) in detail by monitoring the entrance of dyes added at different times after incubation with the peptide. When the GUVs came in contact with Baxα5, leakage started stochastically, was delayed for various periods of time, and in the majority of cases proceeded rapidly to completion. After hours in the presence of the peptide, the same individual GUVs that refilled completely at first instance maintained a porated state, which could be observed in subsequent leak-in events for serially added dyes. However, these long-term pores were smaller in size than the initial equilibration pores. Stable pores were also detected in GUVs made in the presence of Baxα5. The latter pores can be considered equilibrium states and may correspond to structures measured previously in bilayer stacks. Although pore formation may occur as a kinetic process, equilibrium pores may also be functionally relevant structures, especially in highly regulated systems such as the apoptotic mitochondrial pores induced by Bax.     

Ca2+ transport against its electrochemical gradient in cytochrome oxidase vesicles reconstituted with mitochondrial hydrophobic proteins

Cytochrome oxidase vesicles have recently been shown to accumulate Ca²⁺ in an energy-dependent manner. Energization of these vesicles with internally trapped cytochrome c and externally added ascorbate and phenazine methylsulfate generated an internally positive membrane potential and prevented Ca²⁺ influx (R. N. Rosier and T. E. Gunter, 1980, FEBS Lett.109, 99–103). In contradistinction, when cytochrome oxidase vesicles were reconstituted with complex V, a mitochondrial protein fraction containing the uncoupler binding site (Y. Hatefi, D. L. Stiggall, Y. Galante and W. G. Hanstein, 1974, Biochem. Biophys. Res. Commun.61, 313–321), both Ca²⁺ uptake and generation of an internally positive membrane potential were observed. The uptake was specifically dependent on energization of electron transport. Control experiments verified that the energization conditions used produced appropriately oriented membrane potentials. Other partially purified hydrophobic mitochondrial protein complexes were found to be less effective than complex V. The reconstituted system showed cation selectivity since Ca²⁺, Mn²⁺, and Rb⁺ were transported, while Na⁺ was not. Low levels of uncoupler, which did not affect oxidation rates, were found to partially inhibit Ca²⁺ uptake regardless of the membrane potential polarity. Uncoupling levels of uncoupler markedly inhibited Ca²⁺ uptake in internally negative cytochrome oxidase vesicles; however, inhibition in internally positive cytochrome oxidase vesicles was less relative to that at lower levels of uncoupler. The uncoupling combination of nigericin, valinomycin, and K⁺ was inhibitory to uptake regardless of membrane potential polarity. A reconstituted system of oxidative phosphorylation, which contains a hydrophobic protein fraction, energized with cytochrome oxidase similarly accumulated Ca²⁺ despite formation of an internally positive membrane potential. The results suggest that cytochrome oxidase, when coupled to appropriate hydrophobic mitochondrial proteins, can act as an electrogenic Ca²⁺ pump deriving its energy directly from electron transport.     

Characterization and function of the mitochondrial outer membrane peptide-sensitive channel

The PSC (peptide-sensitive Channel), a cationic channel of large conductance, has been characterized in yeast and mammalian mitochondria by three different methods, tip-dip, patch clamp of giant liposomes, and planar bilayers. The yeast and mammalian PSC share the common property to be blocked by basic peptides such as pCyt OX IV (1–12)Y which contains the first 12 residues of the presequence of cytochromec oxidase subunit IV. The electrophysiological data are consistent with a translocation of the peptide through the pore. Analysis of the frequency of observation of the PSC in different fractions indicates that the channel is located in the outer mitochondrial membrane. Uptake measurements of iodinated peptides by intact mitochondria from a porin-less mutant show that the peptides are translocated through the outer membrane, presumably at the level of PSC. Among the peptides active on PSC, several, such as pCyt OX IV (1–22) and the reduced form of the mast cell degranulating peptide, induce an alteration of the voltage dependence or of the inactivation rate subsisting after washing and which is eliminated only by proteolysis of the interacting peptide. These irreversible effects may account for the variability of the properties of the PSC which would interact with cytosolic or intermembrane cations, peptides, or proteins, thus modulating the channel permeability. Finally, several lines of evidence suggest the participation of the PSC in protein translocation and some interaction with the general insertion pore of the outer membrane translocation machinery.     

Three proton pumps,morphology and movements

The diameter of F₁ coupling factor and the distance it protrudes from the membrane of bovine heart submitochondrial particles were measured quantitatively using horse spleen ferritin as a standard. Employing the freeze-etch technique, particles of similar size were found on membranes of submitochondrial particles and on membranes of particles first depleted by F₁, then reconstituted by addition of F₁. The extramembranous size of F₁ is 9.7 nm and F₁ protrudes from the membrane surface by about 13.6 nm. Bacteriorhodopsin and cytochrome oxidase were incorporated into lipids derived from membranes of extremely thermoacidophilic microorganisms by the octylglucoside dilution method. The bacteriorhodopsin pump was fully functional provided high concentrations of valinomycin were added. With decanoyl-N-methylglucamide as detergent the pump was very active in the absence of valinomycin. Concentrations of gramicidin that collapsed the pH in bacteriorhodopsin liposomes prepared with soybean phospholipid had little or no effect on these rigid proteoliposomes. Very high concentrations (30 µg per ml) were partially effective, suggesting a mechanism other than formation of a gramicidin dimer channel. Cytochrome oxidase lost virtually all activity when incorporated into these rigid liposomes but was fully reactivated on addition of suitable detergents.Abbreviations SMP submitochondrial vesicles prepared from bovine heart mitochondria exposed to sonic oscillation in the presence of pyrophosphate - F₁ the water-soluble coupling factor of the mitochondrial ATPase complex - CF₁ the water-soluble coupling factor of the chloroplast ATPase complex - ASU vesicles submitochondrial vesicles prepared from bovine heart mitochondria disrupted by sonic oscillation in ammonia, then passed through Sephadex and treated with urea - OSCP oligomycin sensitivity-conferring protein - Mega 8, 9, and 10 for octoylnanoyl, and decanoyl-N-methylglucamide - 1799 bis-(hexafluoroacetonyl)acetone - PMS N-methylphenazonium methosulfate     

Ionic mitochondrial channels: characteristics and possible role in protein translocation

Most of the mitochondrial proteins are synthesized in the cytoplasm as precursors which are then translocated into the organelle. These precursors have a NH2-terminal extension which functions as a mitochondrial targeting signal. The import process through mitochondrial membranes is voltage-dependent; its mechanism is still unknown. Translocation has been proposed to occur through specific channels, thus, indicating the interest of the study of mitochondrial ionic channels. Two anion channels with different electrical characteristics have been described in the outer and the inner membranes. Using the technique of "Tip-Dip", we have shown the existence of a cation channel of large conductance in mitochondria. The characteristics of this channel differ from that of the other mitochondrial anion channels. A positively charged 13-residue synthetic peptide, with the sequence of the amino terminal extremity of the nuclear-coded subunit IV of yeast cytochrome C oxidase, induces a blockade of the cationic channel. From the characteristics of the blockade, it is likely that the channel could be permeable to the peptide. The specificity of this effect suggests that this channel might be involved in protein translocation.     

Reversible and irreversible effects of basic peptides on the mitochondrial cationic channel.

We have previously shown that a 13-residue basic peptide, derived from the presequence of a mitochondrial precursor, blocked the cationic channel of the outer mitochondrial membrane. The properties of the blockade suggested that the peptide could go through the pore in the presence of a sufficient driving force. In an attempt to evaluate more precisely the relevance of such an interpretation, we have examined the effect on the same channel of basic peptides from 16 to 34 residues, most of which are parts of or derive from mitochondrial presequences. Two peptides were found to induce a reversible voltage-dependent blockade, the properties of which were the same as those of the blockade induced by the 13-residue peptide. The others had a similar effect, but triggered in addition a modification of the voltage gating that persisted after washing the peptide out. The modification was in turn abolished by trypsin added to the side of the channel previously exposed to the peptide. The protease acted on the bound peptide and not on the channel itself. The irreversible modification of the voltage gating, the mechanism of which remains obscure, was not specific for mitochondrial-addressing sequences.     

Cytochemical studies on the localization and functional properties of calcium in anterior pituitary cells

Summary The localization of calcium and its functional properties in anterior pituitary cells were studied using a potassium pyroantimonate technique. In all kinds of secretory cells, the precipitates of the calcium-pyroantimonate complex were distributed on the limiting membrane of the secretory granule. They were present also in the cytoplasmic matrix, the mitochondrial matrix, small smooth vesicles, coated vesicles, and in the nuclear euchromatin area. The precipitates were usually seen at the contact region between the limiting membranes of two adjacent secretory granules, or between the granule limiting membrane and the plasma membrane. When the tissues were incubated in the medium containing A23187 (10 M) for 5 min, the deposits on the granule limiting membrane were increased in number and those on the mitochondrial matrix were decreased; the reaction products almost disappeared on the limiting membranes of the secretory granules after membrane fusion following single or multigranular exocytosis induced by A23187-treatment. In addition, small vesicles in the capillary endothelium contained reaction precipitates. Based on these results we propose a hypothetical model for the relationship between the localization of calcium and secretory activity.This study was supported by grants from the Japan Ministry of Education     

Escherichia coli Membranes Depleted of SecYEG Elicit SecA-Dependent Ion-Channel Activity but Lose Signal Peptide Specificity

We have developed a sensitive method to detect the opening of SecA-dependent, protein-conducting channels in Xenopus oocytes. In this study, we determined the ionic current activities of the SecA-dependent channel from membrane vesicles depleted of SecYEG. We found that these SecYEG-depleted membranes produced SecA-dependent ionic currents in the oocytes, as did membranes containing SecYEG. However, reconstituted membranes depleted of SecYEG required higher concentrations of SecA to elicit ionic currents like those in membranes containing SecYEG. In contrast to membranes containing SecYEG, the proofreading capacity of signal peptides was lost for those membranes lacking SecYEG. These findings are consistent with loss of signal peptide specificity in channel activity from membranes of SecY suppressor or SecY plug domain mutants. The signal peptide specificity of the reconstituted membranes, like SecA-liposomes, can be restored by the addition of SecYEG proteoliposomes. On the other hand, the channel activity efficiency of reconstituted membranes was fully restored, while SecA-liposomes could only be partially enhanced by the addition of SecYEG, indicating that, in addition to SecYEG, other membrane proteins contribute to the efficiency of channel activity. The SecA-dependent channels in membranes that lacked SecYEG also lost ion selectivity to monovalent cations but retained selective permeability to large anions. Thus, the electrophysiological evidence presented here indicates that SecYEG is not obligatory for the channel activity of Escherichia coli membranes, as previously shown for protein translocation, and that SecYEG is important for maintenance of the efficiency and specificity of SecA-dependent channels.     

Protein rotational diffusion measurements on the interaction of bee venom melittin with bacteriorhodopsin in lipid vesicles

The rotational diffusion of bacteriorhodopsin reconstituted into dimyristoylphosphatidylcholine vesicles was measured by the technique of flash-induced transient dichroism. In the presence of melittin, a cell lysing peptide from honey bee (Apis mellifera) venom, dose-dependent loss of rotational mobility was observed. Chemically modified melittin derivatives, in which free amine groups were either acetylated or succinylated, were impaired in their ability to induce immobilisation of bacteriorhodopsin. Bacteriorhodopsin reconstitutions of differing lipid/protein ratio were tested and it was found that the bacteriorhodopsin immobilisation phenomena depended on the melittin/protein ratio, not the melittin/lipid ratio. This suggests that melittin produces its effect via direct interaction with bacteriorhodopsin. A mechanism is proposed in which the aggregation of bacteriorhodopsin is induced by electrostatic attraction between its anionic surface moieties and the highly cationic C-terminal segment of melittin.     

Activation of the ADP/ATP carrier from mitochondria by cationic effectors

The ADP/ATP carrier from the mitochondrial inner membrane was found to be influenced by cationic substances from the hydrophilic surroundings. Under low-ionic-strength conditions, addition of these cationic effectors fully activated the reconstituted adenine nucleotide translocator. The list of activators included divalent cations, polyamines, peptides and cationic proteins. The minimum requirement for an activator to be effective was the presence of at least two positive net charges, regardless of the size of the molecule. Cationic molecules were not activating when an intramolecular charge compensation was possible or when the two charges were too far apart from one another. The affinity of these activators varied from several hundred microM (diaminoalkanes, divalent cations) to 1 microM (cytochrome c, spermine) and even down to a few nM (polylysine). The activation by cations was fully reversible and was not due to fusion processes. It was not mediated by an interaction with the anionic substrates ADP and ATP, nor by interaction with the liposomes. The stimulation could directly and functionally be correlated to the reconstituted carrier protein. Activation was not observed in intact mitochondria, but could be demonstrated when the outer mitochondrial membrane had been removed by treatment with digitonin. These mitoplasts were stimulated by polycations similar to the ADP/ATP carrier in the reconstituted system.     

Tubulin-blocked state of VDAC studied by polymer and ATP partitioning

Recently reported functional interaction between voltage-dependent anion channel of the outer mitochondrial membrane, VDAC, and dimeric tubulin is observed as a reversible channel blockage. Using partitioning of poly-(ethylene glycol)s of different molecular weights and reversal potential measurements, we probe the size and ion selectivity of the fully open and tubulin-blocked states of VDAC reconstituted into planar lipid bilayers. While the effective radius of the channel decreases by only a factor of 1.34±0.15, the selectivity reverses from initially anionic to cationic. Directly measuring ATP partitioning we demonstrate that these changes prohibit ATP from entering the channel in its tubulin-blocked state.     

Reconstitution of adenosine 3'-phosphate 5'-phosphosulfate transporter from rat brain

Adenosine 3'-phosphate 5'-phosphosulfate (PAPS), the "active" sulfate donor for sulfated macromolecules, is synthesized in the cytosolic fraction of rat brains. This molecule is then translocated into the lumen of the Golgi apparatus so that it is available to the sulfotransferase enzymes. The protein responsible for the PAPS translocating activity has been solubilized from vesicles enriched in enzyme markers for the Golgi apparatus and reconstituted into liposomes. In reconstituted liposomes translocating activity has a pH optimum of 7.0 and activity was increased 3-fold by divalent cations, although EDTA produced no inhibition. The affinity of the reconstituted translocator for PAPS showed a Km of 1.2 mM with a Vmax of 14 pmol of PAPS translocated/min/mg of protein. Specificity of the translocator activity was tested with a number of nucleotide analogues and only 3',5'-adenosine diphosphate was a competitive inhibitor. Inhibitors of the mitochondrial ADP/ATP transporter and the red cell anion channel blocked transport of PAPS only at very high concentrations.     

Membrane components can modulate the substrate specificity of protein kinase C

The cationic amphiphile, cholesteryl-3-carboxyamidoethylene-trimethylammonium iodide, can alter the substrate specificity of protein kinase C (PKC). The phosphorylation of histone catalyzed by PKC requires the binding of the enzyme to phospholipid vesicles. This cationic amphiphile reduces both the binding of PKC to lipid and as a consequence its rate of phosphorylation of histone. In contrast, PKC bound to large unilamellar vesicles (LUVs) composed of 50 mol % POPS, 20 mol % POPC, and 30 mol % of this amphiphile catalyzes protamine sulfate phosphorylation by an almost 4 fold greater rate. This activation requires phosphatidylserine (PS) and is inhibited by Ca²⁺. The extent of activation is affected by the time of incubation of PKC with LUVs. This data suggests a novel mechanism by which PKC-dependent signal transduction pathways may be altered by altering the protein targets of this enzyme.     

Isolation and functional reconstitution of a 38-kDa chloride channel protein from bovine tracheal membranes.

Secretion of chloride ions via apically located anion-selective channels in epithelia regulates fluid formation and cytosolic Cl- homeostasis. In order to understand the biochemical basis of Cl- channel function, we attempted to isolate this transporter from bovine tracheal apical membranes. Initially, peripheral polypeptides were removed from apically enriched vesicles by washing with alkaline buffer (pH 10.8) containing 2 mM CHAPS. The resulting pellet contained 50-60% of the original protein and displayed 2-fold enhanced Cl- channel activity compared to untreated vesicles. The pellet was treated with Triton X-100, and the solubilized proteins were separated on the cationic exchanger CM-cellufine. Washing the resin with a pH 8.0-8.3 buffer eluted a fraction with enriched Cl- channel activity. This fraction contained less than 5% of the total solubilized protein. A subsequent separation was performed using the anionic exchanger AM-cellufine. The highest activity was found in the fractions eluted by 80-120 mM KCl. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis showed a major 38,000-Da protein band. This band was electroeluted from the gel under nondenaturing and nonreducing conditions and reconstituted into phosphatidylcholine liposomes. KCl-loaded vesicles containing the purified 38-kDa protein transported up to 5 nmol of 125I-/mg of protein/5 min. This value was 15-fold higher than the uptake measured in vesicles reconstituted with total solubilized membrane proteins and 4-fold higher compared to the CM-cellufine-enriched fraction. The observed 125I- uptake was 90% inhibited by 100 microM 4,4-bis(isothiocyano)-2,2'-stilbenedisulfonate or 10 microM valinomycin. In summary, we have developed a biochemical protocol for the isolation of a 38 kDa protein mediating potential-dependent and 4,4-bis(isothiocyano)-2,2'-stilbenedisulfonate-sensitive Cl- channel activity.     

Interaction between ion channel-inactivating peptides and anionic phospholipid vesicles as model targets.

Studies of rapid (N-type) inactivation induced by different synthetic inactivating peptides in several voltage-dependent cation channels have concluded that the channel inactivation "entrance" (or "receptor" site for the inactivating peptide) consists of a hydrophobic vestibule within the internal mouth of the channel, separated from the cytoplasm by a region with a negative surface potential. These protein domains are conformed from alternative sequences in the different channels and thus are relatively unrestricted in terms of primary structure. We are reporting here on the interaction between the inactivating peptide of the Shaker B K+ channel (ShB peptide) or the noninactivating ShB-L7E mutant with anionic phospholipid vesicles, a model target that, as the channel's inactivation "entrance," contains a hydrophobic domain (the vesicle bilayer) separated from the aqueous media by a negatively charged vesicle surface. When challenged by the anionic phospholipid vesicles, the inactivating ShB peptide 1) binds to the vesicle surface with a relatively high affinity, 2) readily adopts a strongly hydrogen-bonded beta-structure, likely an intramolecular beta "hairpin," and 3) becomes inserted into the hydrophobic bilayer by its folded N-terminal portion, leaving its positively charged C-terminal end exposed to the extravesicular aqueous medium. Similar experiments carried out with the noninactivating, L7E-ShB mutant peptide show that this peptide 1) binds also to the anionic vesicles, although with a lower affinity than does the ShB peptide, 2) adopts only occasionally the characteristic beta-structure, and 3) has completely lost the ability to traverse the anionic interphase at the vesicle surface and to insert into the hydrophobic vesicle bilayer. Because the negatively charged surface and the hydrophobic domains in the model target may partly imitate those conformed at the inactivation "entrance" of the channel proteins, we propose that channel inactivation likely includes molecular events similar to those observed in the interaction of the ShB peptide with the phospholipid vesicles, i.e., binding of the peptide to the region of negative surface potential, folding of the bound peptide as a beta-structure, and its insertion into the channel's hydrophobic vestibule. Likewise, we relate the lack of channel inactivation seen with the mutant ShB-L7E peptide to the lack of ability shown by this peptide to cross through the anionic interphase and insert into the hydrophobic domains of the model vesicle target.