Bcl-2 is the prototypical member of a large family of apoptosis-regulating proteins, consisting of blockers and promoters of cell death. The three-dimensional structure of a Bcl-2 homologue, Bcl-X L , suggests striking similarity to the pore-forming domains of diphtheria toxin and the bacterial colicins, prompting exploration of whether Bcl-2 is capable of forming pores in lipid membranes. Using chloride efflux from KCl-loaded unilamellar lipid vesicles as an assay, purified recombinant Bcl-2 protein exhibited pore-forming activity with properties similar to those of the bacterial toxins, diphtheria toxin, and colicins, i.e., dependence on low pH and acidic lipid membranes. In contrast, a mutant of Bcl-2 lacking the two core hydrophobic ␣-helices (helices 5 and 6), predicted to be required for membrane insertion and channel formation, produced only nonspecific effects. In planar lipid bilayers, where detection of single channels is possible, Bcl-2 formed discrete ion-conducting, cation-selective channels, whereas the Bcl-2 (⌬h5, 6) mutant did not. The most frequent conductance observed (18 ؎ 2 pS in 0.5 M KCl at pH 7.4) is consistent with a four-helix bundle structure arising from Bcl-2 dimers. However, larger channel conductances (41 ؎ 2 pS and 90 ؎ 10 pS) also were detected with progressively lower occurrence, implying the step-wise formation of larger oligomers of Bcl-2 in membranes. These findings thus provide biophysical evidence that Bcl-2 forms channels in lipid membranes, suggesting a novel function for this antiapoptotic protein.

Identification of the minimum number of ways in which open and closed states communicate is a crucial step in defining the gating kinetics of multistate channels. We used certain correlation functions to extract information about the pathways connecting the open and closed states of the cation channel of the purified nicotinic acetylcholine receptor and of the chloride channel of Torpedo californica electroplax membranes. Single channel currents were recorded from planar lipid bilayers containing the membrane channel proteins under investigation. The correlation functions are conveniently computed from single channel current records and yield information on E, the minimum number of entry/exit states into the open or closed aggregates. E gives a lower limit on the numbers of transition pathways between open and closed states. For the acetylcholine receptor, the autocorrelation analysis shows that there are at least two entry/exit states through which the open and closed aggregates communicate. The chloride channel fluctuates between three conductance substates, here indentified as C, M, and H for closed, intermediate, and high conductance, respectively. Correlation analysis shows that E is greater than or equal to 2 for the M aggregate, indicating that there are at least two distinct entry/exit states in the M aggregate. In contrast, there is no evidence for the existence of more than one entry/exit state in the C or H aggregates. Thus, these correlation functions provide a simple and general strategy to extract information on channel gating kinetics.

A class of proteins that mimic the fundamental pore structure of authentic ionic channels has been designed, synthesized, and characterized. The design is based on our earlier result that a 23-mer peptide with the sequence of the M2 segment of the Torpedo californica acetylcholine receptor 8 subunit-Glu-Lys-Met-Ser-Thr-Ala-Ile-Ser-Val-Leu-Leu-AlaGln-Ala-Val-Phe-Leu-Leu-Leu-Thr-Ser-Gln-Arg-forms cation-selective channels in lipid bilayers, presumably by selfassembly of conductive oligomers. Accordingly, a tethered parallel tetramer was synthesized with four M28 peptides attached to a carrier template-a 9-amino acid backbone with four attachment sites. As In one approach to this problem, a hypothesis is first formulated about the existence of such functional segments. The design of a functional peptide that mimics the predicted structural element is followed by its chemical synthesis using solid-phase methods, and the peptide's ability to form ion channels is tested by incorporating it into a synthetic lipid bilayer. The channel activity of the peptide is characterized in terms of ion conduction and channel gating. This characterization is compared with the specific features of the authentic channel. The comparison, in turn, leads to a redesign of the peptide to match the anticipated characteristics of the authentic channel. Finally, the identification of a specific residue thought to be critical for the function under study can then be tested by substitution (2, 3).A test case using this approach has been the nicotinic acetylcholine receptor (AcChoR (2). This molecular-engineering approach has the great virtue of simplicity. The design, however, dictates that these peptides self-assemble in the membrane to generate discrete conductive oligomers, expressed as channel units. We show here that this lack of control over the final product (oligomeric number) can be circumvented by synthesizing a tethered parallel tetramer with four Torpedo AcChoR M28 peptides attached to a carrier template-a 9-amino acid backbone with four attachment sites (6). When incorporated in lipid bilayers, the complete 101-residue protein forms channels that reproduce several features that are characteristic of authentic AcChoR channels: single-channel conductance (y), cation selectivity, transitions between closed and open states in the millisecond time range, and sensitivity to a local anesthetic channel blocker. Thus, synthetic pore proteinssynporins-provide a strategy to investigate structurefunction relationships in channel proteins. A preliminary account of this research was presented elsewhere (7).MATERIALS AND METHODS Materials. 1,2-Diphytanoyl-sn-glycero-3-phosphocholine (PC), 1-palmitoyl-2-oleyl-sn-glycero-3-phosphocholine (POPC), and 1-palmitoyl-2-oleyl-sn-glycero-3-phosphoethanolamine (POPE) were obtained from Avanti. All other chemicals were of the highest purity available commercially.Synthesis of Proteins. Synthesis of the four-helix bundles was accomplished by a two-step procedure. A common 9-amino acid template was ...

We recorded single channel currents activated by agonist binding to purified acetylcholine receptors from the electric organ of Torpedo californica reconstituted in planar lipid bilayers. Analysis of single channel records indicate that the acetylcholine receptor channel displays two kinetically distinct open states which differ in their mean open times but have similar channel conductances.

To establish the relationship between cholinergic ligand occupancy and channel open states, we recorded single-channel currents activated by different acetylcholine (ACh) concentrations from Torpedo californica ACh receptors reconstituted in lipid bilayers. Inspection of single-channel records shows that the frequency of occurrence of long openings increases with ACh concentration. Analysis of the probability distribution of open dwell times indicates that the ACh receptor channel has two distinct channel open states, short- and long-lived. The frequency of occurrence of the long openings over the short increased with ACh concentration, whereas the corresponding time constants were virtually unaltered. The extent of agonist occupancy at the ACh-binding sites in the purified cholinergic receptor appears, therefore, to correlate with an increased probability of the long-lived open state. These results are consistent with the notion that the two open-channel states arise from different extents of ligand occupancy at the receptor molecule.