Ion selectivity of colicin E1: II. Permeability to organic cations

Channels formed by colicin E1 in planar lipid bilayers have large diameters and conduct both cations and anions. The rates at which ions are transported, however, are relatively slow, and the relative anion-to-cation selectivity is modulated over a wide range by the pH of the bathing solutions. We have examined the permeability of these channels to cationic probes having a variety of sizes, shapes, and charge distributions. All of the monovalent probes were found to be permeant, establishing a minimum diameter at the narrowest part of the pore of approximately 9 A. In contrast to this behavior, all of the polyvalent organic cations were shown to be impermeant. This simple exclusionary rule is interpreted as evidence that, when steric restrictions require partial dehydration of an ion, the structure of the channel is able to provide a substitute electrostatic environment for only one charged group at time.     

The permeation of organic cations through cAMP-gated channels in mammalian olfactory receptor neurons

The permeation of monovalent organic cations through adenosine 3,5-cyclic monophosphate-(cAMP) activated channels was studied by recording macroscopic currents in excised inside-out membrane patches from the dendritic knobs of isolated mammalian olfactory receptor neurons (ORNs). Current-voltage relations were measured when bathing solution Na⁺ was replaced by monovalent organic cations. Permeability ratios relative to Na⁺ ions were calculated from changes in reversal potentials. Some of the small organic cations tested included ammonium (NH ₄ ⁺ ), hydroxylammonium and formamidinium, with relative permeability ratios of 1.41, 2.3 and 1.01 respectively. The larger methylated and ethylated ammonium ions studied included: DMA (dimethylammonium), TMA (tetramethylammonium) and TEA (tetraethylammonium) and they all had permeability ratios larger than 0.09. Even large cations such as choline, arginine and tris(hydroxymethyl)aminomethane (Tris) were appreciably permeant through the cAMP-activated channel with permeability ratios ranging from 0.19 to 0.7. The size of the permeating cations, as assessed by molecular weight, was a good predictor of the permeability. The permeability sequence of the cAMP-activated channel in our study was P_NH4 > P_Na > p_DMA > p_TMA > P_Choline > P_TEA. Higher permeability ratios of hydroxylammonium, arginine and tris(hydroxymethyl)aminomethane cannot be explained by ionic size alone. Our results indicate that: (i) cAMP-activated channels poorly select between monovalent cations; (ii) the pore dimension must be at least 6.5 × 6.5 Å, in order to allow TEA and Tris to permeate and (iii) molecular sieving must be an important mechanism for the permeation of large organic ions through the channels with specific ion binding playing a smaller role than in other structurally similar channels. In addition, the results clearly indicate that cyclic nucleotide-gated (CNG) channels in different cells are not the same, the olfactory CNG channel being different from that of the photoreceptors, particularly with respect to the permeation of large organic cations, which the ORN channels allow to permeate readily.This work was supported by the Australian Research Council of Australia.     

Channels formed by colicin E1 in planar lipid bilayers are large and exhibit pH-dependent ion selectivity

Summary The E1 subgroup (E1, A, Ib, etc.) of antibacterial toxins called colicins are known to form voltage-dependent channels in planar lipid bilayers. The genes for colicins E1, A and Ib have been cloned and sequenced, making these channels interesting models for the widespread phenomenon of voltage dependence in cellular channels. In this paper we investigate ion selectivity and channel size—properties relevant to model building. Our major finding is that the colicin E1 channel is large, having a diameter ofat least 8 Å at its narrowest point. We established this from measurements of reversal potentials for gradients formed by salts of large cations or large anions. In so doing, we exploited the fact that the colicin channel is permeable to both cations and anions, and its relative selectivity to them is a functions and anions, and its relative selectivity to them is a function of pH. The channel is anion selective (Cl^– over K⁺) in neutral membranes, and the degree of selectivity is highly dependent on pH. In negatively charged membranes, it becomes cation selective at pH's higher than about 5. Experiments with pH gradients cross the membrane suggest that titratable groups both within the channel lumen and near the channel ends affect the selectivity. Individual E1 channels have more than one open conductance state, all displaying comparable ion selectivity. Colicins A and Ib also exhibit pH-dependent ion selectivity, and appear to have even larger lumens than E1.     

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.     

Anion-cation permeability correlates with hydrated counterion size in glycine receptor channels

The functional role of ligand-gated ion channels depends critically on whether they are predominantly permeable to cations or anions. However, these, and other ion channels, are not perfectly selective, allowing some counterions to also permeate. To address the mechanisms by which such counterion permeation occurs, we measured the anion-cation permeabilities of different alkali cations, Li⁺ Na⁺, and Cs⁺, relative to either Cl⁻ or anions in both a wild-type glycine receptor channel (GlyR) and a mutant GlyR with a wider pore diameter. We hypothesized and showed that counterion permeation in anionic channels correlated inversely with an equivalent or effective hydrated size of the cation relative to the channel pore radius, with larger counterion permeabilities being observed in the wider pore channel. We also showed that the anion component of conductance was independent of the nature of the cation. We suggest that anions and counterion cations can permeate through the pore as neutral ion pairs, to allow the cations to overcome the large energy barriers resulting from the positively charged selectivity filter in small GlyR channels, with the permeability of such ion pairs being dependent on the effective hydrated diameter of the ion pair relative to the pore diameter.     

Properties of the kainate channel in rat brain mRNA injected Xenopus oocytes: ionic selectivity and blockage

The properties of kainate receptor/channels were studied in Xenopus oocytes injected with mRNA that was isolated from adult rat striatum and cerebellum and partially purified by sucrose gradient fractionation. Kainate (3-1000 microM) induced a smooth inward current that was competitively inhibited by gamma-D-glutamyl-aminomethanesulfonate (GAMS, 300 microM). In striatal mRNA-injected oocytes, the kainate current displayed nearly linear voltage-dependence and mean reversal potential (Er) of -6.1 +/- 0.5 mV. In cerebellar mRNA-injected oocytes; Er was nearly identical (-5.1 +/- 1.2 mV) but there was marked inward rectification of the kainate current. Ion replacement studies reveal that the kainate channel is selective for cations over anions, but relatively non-selective among small monovalent cations. Large monovalent cations such as tetrabutylammonium are impermeant and induce a non-competitive block of kainate current that is strongly voltage-dependent. Divalent cations are relatively impermeant in the kainate channel and Cd++ and other polyvalent metals were shown to block kainate current by a mechanism that is only weakly voltage-dependent. A model of the kainate channel is proposed based upon these observations.     

Properties of the kainate channel in rat brain mRNA injected Xenopus oocytes: ionic selectivity and blockage

The properties of kainate receptor/channels were studied in Xenopus oocytes injected with mRNA that was isolated from adult rat striatum and cerebellum and partially purified by sucrose gradient fractionation. Kainate (3–1000 µ.M) induced a smooth inward current that was competitively inhibted by gamma-D-glutamyl-aminomethanesulfonate (GAMS, 300 µM). In striatal mRNA-injected oocytes, the kainate current displayed nearly linear voltage-dependence and mean reversal potential (E_r) of -6.1 ± 0.5 mV In cerebellar mRNA-injected oocytes; E_r was nearly identical (-5.1 ± 1.2 mV) but there was marked inward rectification of the kainate current. Ion replacement studies reveal that the kainate channel is selective for cations over anions, but relatively non-selective among small monovalent cations. Large monovalent cations such as tetrabutylammonium are impermeant and induce a non-competitive block of kainate current that is strongly voltage-dependent. Divalent cations are relatively impermeant in the kainate channel and Cd⁺⁺ and other polyvalent metals were shown to block kainate current by a mechanism that is only weakly voltage-dependent. A model of the kainate channel is proposed based upon these observations.     

Structural and functional properties of colicin B

Colicin B was isolated in pure form from cells of Escherichia coli that contained the colicin activity and immunity genes cloned on a multi-copy plasmid. Active colicin B consisted of a single polypeptide with Mr of about 60,000. The sequence of 44 amino acids from the amino-terminal portion is presented. The isoelectric point of the protein was at 4.5. Colicin B inhibited the membrane potential-dependent transport of proline and enhanced the uptake of alpha-methylglucoside via the phosphoenolpyruvate-dependent phosphotransferase system. Colicin B formed small, ion permeable channels with an average single-channel conductance of 13.7 pS (1 pS = 10(-12) siemens) in 1 M KCl. Channel formation was voltage-dependent in the pH range between 4.5 and 6. At pH 7 the channels were voltage independent. Voltage-dependent channels were only formed when the trans compartment (the protein was added to the cis compartment) was negative by at least 70 mV. Evidence for an asymmetric single channel conductance was obtained. With KCl a hyperbolic conductance-concentration relationship was observed. The conductance for monovalent cations was minimal for Li+ and was maximal for NH+4. The single channel conductance of colicin B was larger than that of colicin A as judged from lipid bilayer experiments under otherwise identical conditions.     

Anomalous proton selectivity in a large channel: colicin A

Some of the bactericidal proteins known as colicins exert their toxic action by forming a large, nonselective channel in the inner membrane of target bacteria. The structure of this channel is unknown. It conducts large ions but has a much smaller conductance than would be expected for a channel of its deduced size. Here we report that the colicin channel, particularly the colicin A channel, is selective for protons over other cations (and anions) by many orders of magnitude. This was deduced from measurements of reversal potentials in pH gradients across planar lipid bilayers containing these channels. For example, in symmetric 0.1 M KCl with a pH 5/pH 8 gradient across the membrane, the reversal potential of colicin A is -21 mV, rather than 0. Such a result would be unremarkable for a narrow channel but is beyond explanation by current understanding of permeation for a channel of its diameter. For this reason, we re-examined the issue of the diameter of the channel lumen and confirmed that the lumen is indeed "too large" ( approximately 10 A) to select for protons by the amount that we measure. We are thus compelled to propose that an unorthodox mechanism is at work in this protein.     

The characterization of a monovalent cation-selective channel of mammalian cardiac muscle sarcoplasmic reticulum

Summary Rabbit cardiac muscle sarcoplasmic reticulum (SR) was isolated and separated into ryanodine-sensitive and-insensitive fractions (L.R. Jones and S.E. Cala,J. Biol. Chem. 256:11809–11818, 1981). Vesicles of cardiac SR were incorporated into planar phospholipid bilayers by fusion and the channel activity of the membrane studied under voltage-clamp conditions (C. Miller,J. Membrane Biol. 40: 1–23, 1978). Both fractions contain a monovalent cation-selective three-state channel. In the presence of 75mm K₂SO₄, the fully open state () conductance of this channel is 157.2±30 pS and the sub-state () conductance is 100.7±21 pS. Both open states display the same selectivity sequence for monovalent cations, i.e. K⁺>NH ₄ ⁺ >Rb⁺>Na⁺>Li⁺ and may be blocked by the skeletal muscle relaxants decamethonium and hexamethonium. Block occurs when the compounds are added to either side of the membrane. The properties of the cardiac SR cation channel are compared with those of the previously reported monovalent cation-selective channels of mammalian and amphibian skeletal muscle SR.     

Binding of organic solvent molecules influences the P1′−P2′ stereo-and sequence-specificity of α-chymotrypsin in kinetically controlled peptide synthesis

In kinetically controlled peptide synthesis catalyzed by -chymotrypsin (EC 3.4.21.1, CT) the P₁-specificity and stereospecificity was found to increase for hydrophobic and decrease for positively charged nucleophiles in systems with hydrophobic organic solvent molecules. This was observed in the following systems: (i) buffer saturated with organic solvent molecules; (ii) the enzyme adsorbed on a hydrophobic adsorbent (Phenylbutylamine-Eupergit). These results show that organic molecules bound to the enzyme surface perturb the P₁-S₁ interactions and change the catalytic properties of the enzyme.     

Gating processes of channels induced by colicin A,its C-terminal fragment and colicin E1 in planar lipid bilayers

The dependence on pH and membrane potential of the pore formed by colicin A and its C-terminal 20 kDa fragment has been measured using planar lipid bilayers. The single channel conductance of the pore formed by both colicin A and the fragment increases with pH with an apparent pK of 6.0. At pH 5.0 the gating by membrane potential of the channels formed by either colicin A or its fragment is identical. At the same pH, quite similar pore properties were found when using the related bacteriocin, colicin E₁. In agreement with previous studies, these data indicate that the protein structure containing the lumen of the pore resides in the 20 kDa C-terminal part of the colicin A and favours the recently proposed model, based on protein sequence analysis, which proposes that colicin A, E₁ and I_B C-terminal domains are folded in the same three-dimensional structure. However, it is also shown that colicin A and not its C-terminal fragment undergoes a pH dependent transition between an acidic and a basic form of the pore with an apparent pK of 5.3. The two forms of the pore differ by their gating charge but not by the channel size. These results suggest that there is a pH dependent association between the C-terminal domain carrying the lumen of the pore and another domain of the molecule which affect the pore sensitivity to membrane potential.     

Studies on the cation permeability of human red cell ghosts

Summary Net K movements in reconstituted human red cell ghosts and the resealing of ghosts to cations after osmotic hemolysis of red cells have been studied as functions of the free Ca ion concentration. The Ca-dependent specific increase in K permeability was shown to be mediated by a site close to the internal surface of the membrane with an apparent dissociation constant at pH 7.2 for Ca (K _D1) of 3–5×10^–7 m, for Sr of 7×10^–6 m. Ba and Mg did not increase the K-permeability of the membrane but inhibited the Ca-mediated permeability changes.K _D1 decreased in a nonlinear fashion when the pH was increased from 6.0 to 8.5. Two different pK values of this membrane site were found at pH 8.3 and 6.3. The Ca-activated net K efflux into a K-free medium was almost completely inhibited by an increase in intracellular Na from 4 to 70mm. Extracellular K antagonized this Na effect. Changes in the extracellular Na (0.1–140mm) or K(0.1–6mm) concentrations had little effect and did not changeK _D1. The Ca-stimulated recovery of a low cation permeability in ghost cells appeared to be mediated by a second membrane site which was accessible to divalent cations only during the process of hemolysis in media of low ionic strength. The apparent dissociation constant for Ca at this site (K _D2) varied between 6×10^–7 and 4×10^–6 m at pH 7.2. Mg, Sr, and Ba could replace Ca functionally. The selectivity sequence was Ca>Sr>Ba>Mg.K _D2 was independt on the pH value in the range between 6.0 and 8.0. Hill coefficients of 2 were observed for the interaction of Ca with both membrane sites suggesting that more than one Ca ion is bound per site. The Hill coefficients were affected neither by the ion composition nor by the pH values of the intra- and extracellular media. It is concluded that two different pathways for the permeation of cations across the membrane are controlled by membrane sites with high affinities for Ca: One specific for K, one unspecific with respect to cations. The K-specific channel has properties similar to the K channel in excitable tissues.     

Kinetic characteristics of the sulfate self-exchange in human red blood cells and red blood cell ghosts

Summary The sulfate and the chloride self-exchange fluxes were determined by measuring the rate of the tracer efflux from radioactively labeled human red blood cells and red blood cell ghosts. The concentration dependence and the pH-dependence of the sulfate self-exchange flux were studied. In addition, the effects of some monovalent and divalent anions on the sulfate and the chloride self-exchange fluxes were investigated.The sulfate self-exchange fluxes saturate, exhibiting a concentration maximum at sulfate concentrations between 100 and 300mm (25°C). The position of the concentration maximum depends upon pH. At high sulfate concentrations a self-inhibition of the flux becomes apparent. The apparent half-saturation constant and the apparent self-inhibition constant at pH 7.2 were 30mm and 400mm respectively. Within the pH range of 6.3–8.5, both constants decreased with increasing pH. No saturation of the sulfate self-exchange flux was observed if the sulfate concentration was raised by substituting sulfate for isoosmotic amounts of a second salt (NaCl, NaNO₃, Na-acetate, Na-lactate, Na-succinate or Na₂HPO₄). Red blood cells and red blood cell ghosts display the same pattern of concentration responsiveness.The sulfate self-exchange flux exhibits a pH-maximum at about pH 6.2 (37°C). The location of the pH-maximum is little affected by variations of the sulfate concentration. The logarithmic plots (log vs. pH) revealed that the flux/pH relation can be approximated by two straight lines. The slopes of the alkaline branches of the flux/pH curves range from –0.55 to –0.86, the slopes of the branches of the curves range from 0.08 to 1.14 and were strongly affected by changes of the sulfate concentrations. The apparent pK's obtained from the alkaline and from the acidic branches of the flux/pH curves were about 7.0 and 6.0, respectively. Intact red blood cells and red blood cell ghosts display the same type of pH-dependency of the sulfate self-exchange flux.The sulfate self-exchange flux is competitively inhibited by nitrate, chloride, acetate, oxalate and phosphate. The chloride self-exchange flux is competitively inhibited by thiocyanate, nitrate, sulfate and phosphate. The inhibition constants for the various anion species increase in the given sequence.The results of our studies indicate that the sulfate self-exchange flux is mediated by a two-site transport mechanism consisting either of a mobile carrier or a two-site pore. The experiments reported in this paper do not permit distinguishing between both transport mechanisms. The similarities of the sulfate and the chloride self-exchange flux and the mutual competition between sulfate and chloride point to a common transport system for both anion species.     

External monovalent cations that impede the closing of K channels

We have examined the effects of a variety of monovalent cations on K channel gating in squid giant axons. The addition of the permeant cations K, Rb, or Cs to the external medium decreases the channel closing rate and causes a negative shift of the conductance-voltage relationship. Both of these effects are larger in Rb than in K. The opening kinetics of the K channel are, on the other hand, unaffected by these monovalent cations. Other permeant species, like NH4 and Tl, slightly increase the closing rate, whereas the relatively impermeant cations Na, Li, and Tris have little or no effect on K channel gating. The permeant cations have different effects on the reversal potential and the shape of the instantaneous current-voltage relationship. These effects give information about entry and binding of the cations in K channels. Rb, for example, enters the pore readily (large shift of the reversal potential), but binds tightly to the channel interior, inhibiting current flow. We find a correlation between the occupancy of the channel by a monovalent cation and the closing rate, and conclude that the presence of a monovalent cation in the pore inhibits channel closing, and thereby causes a leftward shift in the activation-voltage curve. In causing these effects, the cations appear to bind near the inner surface of the membrane.     

Diltiazem at high concentration increases the ionic permeability of biological membranes

Summary The effects of diltiazem, a drug which inhibits the calcium channels in cardiac muscle as well as the light-sensitive channels in photoreceptor cells, were studied on ionic fluxes in both membrane and intact cell preparations. Diltiazem nonselectively increased the ionic permeability to both anions and cations in photoreceptor rod outer segment and synaptic membrane vesicles as well as in intact erythrocytes. Under our conditions, the estimated threshold for the diltiazem effect varied between 12.5 and 200 m. In each case the concentration dependence exhibited the sigmoidal shape characteristic of positive cooperativity. The effect of diltiazem on ionic fluxes from phospholipid vesicles were strongly influenced by phospholipid composition and membrane charge. By contrast, diltiazem inhibited the efflux of⁸⁶Rb from photoreceptor cells of intact aspartate-isolated retina, an effect opposite to that of diltiazem on ionic permeabilities in photoreceptor membrane vesicle preparations.These data raise serious doubts on the specificity of diltiazem as a calcium channel blocker or as a cGMP channel blocker when used at concentrations higher than 10 m.     

The Ca2+-induced leak current in Xenopus oocytes is indeed mediated through a Cl− channel

Defolliculated oocytes of Xenopus laevis responded to removal of external divalent cations with large depolarizations and, when voltage clamped, with huge currents. Single channel analysis revealed a Cl^– channel with a slope conductance of about 90 pS at positive membrane potentials with at least four substates. Single channel amplitudes and mean channel currents had a reversal potential of approximately –15 mV as predicted by the Nernst equation for a channel perfectly selective for Cl^–. Readdition of Ca²⁺ immediately inactivated the channel and restored the former membrane potential or clamp current. The inward currents were mediated by a Ca²⁺ inactivated Cl^– channel (CaIC). The inhibitory potency of Ca²⁺ was a function of the external Ca²⁺ concentration with a half maximal blocker concentration of about 20 m.These channels were inhibited by the Cl^– channel blockers flufenamic acid, niflumic acid and diphenylamine-2-carboxylate (DPC). In contrast, 4,4-acetamido-4-isothiocyanatostilbene-2,2-disulfonicacid (SITS), another Cl^– channel blocker, led to activation of this Cl^– channel. Like other Cl^– channels, the CaIC was activated by cytosolic cAMP. Extracellular ATP inhibited the channel while ADP was without any effect. Injection of phorbol 12-myristate 13-acetate (PMA), a protein kinase C activating phorbol ester, stimulated the Cl^– current. Cytochalasin D, an actin filament disrupting compound, reversibly decreased the clamp current demonstrating an influence of the cytoskeleton.The results indicate that removal of divalent cations activates Cl^– channels in Xenopus oocytes which share several features with Cl^– channels of the CLC family. The former so-called leak current of oocytes under divalent cation-free conditions is nothing else than an activation of Cl^– channels.The microelectrode measurements are part of the PhD thesis of K. Liebold; the patch clamp contributions are part of the PhD thesis of F.W. Reifarth. This study was supported by the Deutsche Forschungsgemeinschaft (We1858/2-l) and by Sonderforschungsbereich 249.     

Single chloride channels in endosomal vesicle preparations from rat kidney cortex

Summary Endocytotic vesicles from rat kidney cortex, isolated by differential centrifugation and enriched on a Percoll gradient, contain both an electrogenic H⁺ translocation system and a conductive chloride pathway. Using the dehydration/rehydration method, we fused vesicles of enriched endosomal vesicle preparations and thereby made them accessible to the patch-clamp technique. In the fused vesicles, we observed Cl^– channels with a single-channel conductance of 73±2 pS in symmetrical 140mm KCl solution (n=25). The current-voltage relationship was linear in the range of –60 to +80 mV, but channel kinetic properties dependended on the clamp potential. At positive potentials, two sublevels of conductance were discernible and the mean open time of the channel was 10–15 msec. At negative voltages, only one substate could be resolved and the mean open time decreased to 2–6 msec. Clamp voltages more negative than –50 mV caused reversible channel inactivation. The channel was selective for anions over cations. Ion substitution experiments revealed an anion permeability sequence of Cl^–=Br^–=I^–>SO ₄ ^2– F^–. Gluconate, methanesulfonate and cyclamate were impermeable. The anion channel blockers 4,4-diisothiocyanatostilbene-2,2-disulfonic acid (DIDS, 1.0mm) and 5-nitro-2-(3-phenylpropylamino)-benzoic acid (NPPB, 0.1mm) totally inhibited channel activity. Comparisons with data obtained from radiolabeled Cl^–-flux measurements and studies on the H⁺ pump activity in endocytotic vesicle suspensions suggest that the channel described here is involved in maintenance of electroneutrality during ATP-driven H⁺ uptake into the endosomes.     

Gating of retinal rod cation channel by different nucleotides: Comparative study of unitary currents

Summary Single channels are observed after incorporation of native vesicles from bovine rod outer segment membranes into planar lipid bilayers. The activity of a single channel in the presence of cGMP is compared to that induced by the analog 8-bromo-cGMP and by cAMP. At +80 mV, K _0.5 is about 3 m for 8Br-cGMP, 18 m for cGMP and 740 m for cAMP. In cAMP, the amplitude of the current is smaller than in cGMP or 8Br-cGMP and depends on the filter cut-off frequency. The open/closed transition rates of the channel are slightly slower with 8Br-cGMP than with cGMP while they are 5 to 10 times faster with cAMP. Addition of Ni²⁺ ions to either cGMP or cAMP increases the open probability: the open/closed transition rates and amplitude of the current in cAMP are then comparable to those in cGMP. A dual effect of the addition of cAMP on the cGMPor 8Br-cGMP dependent activity previously reported (Furman, R.E., Tanaka, J.C. 1989. Biochemistry 28:2785–2788) is observed with a single channel: addition of subthreshold cAMP concentrations to cGMP (or to 8Br-cGMP) markedly increases P _o; addition of cAMP concentrations higher than about 70 m progressively accelerates the kinetics and reduces the amplitude to values observed in cAMP alone. The results are discussed in relation with the model previously proposed to account for the existence of four current levels (Ildefonse, M., Bennett, N. 1991. J. Membrane Biol. 123:133–147).     

Comparison of the macroscopic and single channel conductance properties of colicin E1 and its COOH-terminal tryptic peptide

A COOH-terminal tryptic fragment (Mr approximately equal to 20,000) of colicin E1 has been proposed to contain the membrane channel-forming domain of the colicin molecule. A comparison is made of the conductance properties of colicin E1 and its COOH-terminal fragment in planar bilayer membranes. The macroscopic and single channel properties of colicin E1 and its COOH-terminal tryptic fragment are very similar, if not indistinguishable, implying that the NH2-terminal, two-thirds of the colicin E1 molecule, does not significantly influence its channel properties. The channel-forming activity of both polypeptides is dependent upon the presence of a membrane potential, negative on the trans side of the membrane. The average single channel conductance of colicin E1 and the COOH-terminal fragment is 20.9 +/- 3.9 and 19.1 +/- 2.9 picosiemens, respectively. The rate at which both proteins form conducting channels increases as the pH is lowered from 7 to 5. Both molecules require negatively charged lipids for activity to be expressed, exhibit the same ion selectivity, and rectify the current to the same extent. Both polypeptides associate irreversibly with the membrane in the absence of voltage, but subsequent formation of conducting channels requires a negative membrane potential.