Colicin Ia is a 69 kDa protein that kills susceptible Escherichia coli cells by binding to a specific receptor in the outer membrane, colicin I receptor (70 kDa), and subsequently translocating its channel forming domain across the periplasmic space, where it inserts into the inner membrane and forms a voltage-dependent ion channel. We determined crystal structures of colicin I receptor alone and in complex with the receptor binding domain of colicin Ia. The receptor undergoes large and unusual conformational changes upon colicin binding, opening at the cell surface and positioning the receptor binding domain of colicin Ia directly above it. We modelled the interaction with full-length colicin Ia to show that the channel forming domain is initially positioned 150 Å above the cell surface. Functional data using full-length colicin Ia show that colicin I receptor is necessary for cell surface binding, and suggest that the receptor participates in translocation of colicin Ia across the outer membrane.

The translocation (T) domain plays a key role in the action of diphtheria toxin and is responsible for transferring the N-terminus-attached catalytic domain across the endosomal membrane into the cytosol in response to acidification. The T-domain undergoes a series of pH-triggered conformational changes that take place in solution and on the membrane interface, and ultimately result in transbilayer insertion and N-terminus translocation. Structure-function studies along this pathway have been hindered because the protein population occupies multiple conformations at the same time. Here we report that replacement of the three C-terminal histidine residues, H322, H323, and H372, in triple-R or triple-Q mutants prevents effective translocation of the N-terminus. Introduction of these mutations in the full-length toxin results in decrease of its potency. In the context of isolated T-domain, these mutations cause loss of characteristic conductance in planar bilayers. Surprisingly, these mutations do not affect general folding in solution, protein interaction with the membranes, insertion of the consensus transmembrane helical hairpin TH8-9, or the ability of the T-domain to destabilize vesicles to cause leakage of fluorescent markers. Thus, the C-terminal histidine residues are critical for the transition from the inserted intermediate state to the open-channel state in the insertion/translocation pathway of the T-domain.

Colicin Ia, a bacterial protein toxin of 626 amino acid residues, forms voltagedependent channels in planar lipid bilayer membranes. We have exploited the high affinity binding of streptavidin to biotin to map the topology of the channel-forming domain (roughly 175 residues of the COOH-terminal end) with respect to the membrane. That is, we have determined, for the channel's open and closed states, which parts of this domain are exposed to the aqueous solutions on either side of the membrane and which are inserted into the bilayer. This was done by biotinylating cysteine resid:~es introduced by site-directed mutagenesis, and monitoring by electrophysiological methods the effect of streptavidin addition on channel behavior. We have identified a region of at least 68 residues that flips back and forth across the membrane in association with channel opening and closing. This identification was based on our observations that for mutants biotinylated in this region, streptavidin added to the cis (colicin-containing) compartment interfered with channel opening, and trans streptavidin interfered with channel closing. (If biotin was linked to the colicin by a disulfide bond, the effects of streptavidin on channel closing could be reversed by detaching the streptavidin-biotin complex from the colicin, using a water-soluble reducing agent. This showed that the cysteine sulfur, not just the biotin, is exposed to the trans solution.) The upstream and downstream segments flanking the translocated region move into and out of the bilayer during channel opening and closing, forming two transmembrane segments. Surprisingly, if any of several residues near the upstream end of the translocated region is held on the cis side by streptavidin, the colicin still forms voltage-dependent channels, indicating that a part of the protein that normally is fully translocated across the membrane can become the upstream transmembrane segment. Evidently, the identity of the upstream transmembrane segment is not crucial to channel formation, and several open channel structures can exist.

Colicin Ia is a bactericidal protein that forms voltage-dependent, ion-conducting channels, both in the inner membrane of target bacteria and in planar bilayer membranes. Its amino acid sequence is rich in charged residues, except for a hydrophobic segment of 40 residues near the carboxyl terminus. In the crystal structure of colicin Ia and related colicins, this segment forms an alpha-helical hairpin. The hydrophobic segment is thought to be involved in the initial association of the colicin with the membrane and in the formation of the channel, but various orientations of the hairpin with respect to the membrane have been proposed. To address this issue, we attached biotin to a residue at the tip of the hydrophobic hairpin, and then probed its location with the biotin-binding protein streptavidin, added to one side or the other of a planar bilayer. Streptavidin added to the same side as the colicin prevented channel opening. Prior addition of streptavidin to the opposite side protected channels from this effect, and also increased the rate of channel opening; it produced these effects even before the first opening of the channels. These results suggest a model of membrane association in which the colicin first binds with the hydrophobic hairpin parallel to the membrane; next the hairpin inserts in a transmembrane orientation; and finally the channel opens. We also used streptavidin binding to obtain a stable population of colicin molecules in the membrane, suitable for the quantitative study of voltage-dependent gating. The effective gating charge thus determined is pH-independent and relatively small, compared with previous results for wild-type colicin Ia.

One cannot always distinguish different Markov models of ion-channel kinetics solely on the basis of steady-state kinetic data. If two generator (or transition) matrices are related by a similarity transformation that does not combine states with different conductances, then the models described by these generator matrices have the same observable steady-state statistics. This result suggests a procedure for expressing the model in a unique form, and sometimes reducing the number of parameters in a model. I apply the similarity transformation procedure to a number of simple models. When a model specifies the dependence of the rates of transition on an experimentally variable parameter such as the concentration of a ligand or the membrane potential, the class of equivalent models may be further restricted, but a model is not always uniquely determined even under these conditions. Voltage-step experiments produce non-stationary data that can also be used to distinguish models.

The translocation (T) domain plays a key role in the entry of diphtheria toxin into the cell. Upon endosomal acidification, the T-domain undergoes a series of conformational changes that lead to its membrane insertion and formation of a channel. Recently, we have reported that the triple replacement of the C-terminal histidines H322, H323 and H372 with glutamines prevents the formation of open channels in planar lipid bilayers. Here, we report that this effect is primarily due to the mutation of H322. We further examine the relationship between the loss of functionality and membrane folding in a series of mutants with C-terminal histidine substitutions using spectroscopic assays. The membrane insertion pathway for the mutants differs from that of the wild type as revealed by membrane-induced red-shift of tryptophan fluorescence at pH 6.0–6.5. T-domain mutants with replacements at H323 and H372, but not at H322, regain wild type-like spectroscopic signature upon further acidification. Circular dichroism measurements confirm that affected mutants misfold during insertion into vesicles. Conductance measurements reveal that substituting H322 dramatically reduces the numbers of properly folded channels in a planar bilayer, but the properties of the active channels appear to be unaltered. We propose that H322 plays an important role in the formation of open channels and is involved in guiding the proper insertion of the N-terminal region of the T-domain into the membrane.

We are deigning simple peptide ion channels as model sstems for the study of the physical principles controlling conduction through Ion-channel proteins. Here we report on an uncharg peptide, Ac-(Leu-Ser-Ser-Leu-LeuSer-Leu)3-CONH2, de to form an aggregate of parale, amphiphilic, membrane-snuing a-helices around a central water-filed pore. This peptide in planar lipid bilayers forms ion channels that show singl-hannel current rectification in smmetrnc 1 M KCI. The current at a given holding membrane potential is larger than the current measured through the same channel when the potential is reversed. Based on our hypothesized gating mechm, the larger currents flow from the peptide carboxyl terminus-toward the amino terminus. We present an Ionic electrodiffusion model based on the helicaldipole potential and the dielectric interfacial polarization energy, which with reasonable values for dipole magnitude and dielecic constants, accurately replicates the current-voltage data.Many aspects of signal transduction and transmission in living cells depend on ionic currents, which flow into or out of the cell through ion-channel proteins. The currents of several types of channel have an asymmetric dependence on the transmembrane potential; if the larger current is directed toward the cell interior, the channel is said to be inwardly rectifying. Whole-cell rectification, arising from the summed currents of many individual channels, can arise from a difference in the channel opening-closing equilibrium at positive and negative membrane potentials (asymmetric voltage gating). However, a single open channel can also show rectification. This rectification can be caused by asymmetries in Mg2+ blocking (1-3), permeant ion concentration (4, 5), and fixed electrical charges or dipoles at the membrane surface or in the channel (6).Both fixed charges and dipoles are likely to affect conduction in ion-channel proteins. Charge effects have been demonstrated in the nicotinic acetylcholine receptor, where single-channel conductance and rectification properties depend on the charge of certain amino acid side chains thought to be near the conducting pore (7). However, the effect of dipoles on conduction has not been as clearly demonstrated, even though they could be important in pores lined by a-helices. The dipole moments of the peptide bonds in an a-helix add together to form an electrical macrodipole (8,9). Transmembrane a-helices are major structural features of membrane proteins such as bacteriorhodopsin (10) and the photosynthetic reaction center (11) and are thought to line the pores of ion channels such as the gapjunction (12, 13) and the nicotinic receptor (14). [Dipoles might also influence rectification in channels without a-helices, such as certain forms of the model channel gramicidin (15,16).]We are using minimalist-designed peptide ion channels to study the physical principles underlying conduction through protein ion channels. To study purely dipolar effects on rectification, we have designed and synthesized an unc...

To help determine how amino acid sequence can influence ionic conduction properties in α-helical structures, we have synthesized and studied three closely related, channel-forming peptides. The sequences are based on a 21-residue amphiphilic Leu-Ser-Ser-Leu-Leu-Ser-Leu heptad repeat motif and differ in having either neutral, negatively, or positively charged N-termini. The channels formed by the neutral peptide are modestly cation selective and exhibit asymmetric current-voltage curves arising from the partial charges at the ends of the α-helix. Addition of a negatively charged Glu residue converted the channel to a completely cation-selective structure and essentially eliminated its rectification. Addition of a positively charged Arg residue near the N-terminus of the peptide reduced this channel's cation selectivity and increased the extent of rectification. These effects on channel ionic conductance can be explained by a theoretical electrostatic model and provide insights into the workings of more complex channel proteins.