Ion selectivity of colicin E1: III. Anion permeability.

The antibiotic protein colicin E1 forms ion channels in planar lipid bilayers that are capable of conducting monovalent organic cations having mean diameters of at least 9 A. Polyvalent organic cations appear to be completely impermeant, regardless of size. All permeant ions, whether large or small, positively or negatively charged, are conducted by this channel at very slow rates. We have examined the permeability of colicin E1 channels to anionic probes having a variety of sizes, shapes, and charge distributions. In contrast to the behavior of cations, polyvalent as well as monovalent organic anions were found to permeate the colicin E1 channel. Inorganic sulfate was able to permeate the channel only when the pH was 4 or less, conditions under which the colicin E1 protein is predominantly in an anion-preferring conformational state. The less selective state(s) of the colicin E1 channel, observed when the pH was 5 or greater, was not permeable to inorganic sulfate. The sulfate salt of the impermeant cation Bis-T6 (N,N,N',N'-tetramethyl-1,6-hexanediamine) had no effect on the single channel conductance of colicin E1 channels exposed to solutions containing 1 M NaCl at pH 5. The complete lack of blocking activity by either of these two impermeant ions indicates that both are excluded from the channel lumen. These results are consistent with our hypothesis that there is but a single location in the lumen of the colicin E1 channel where positively charged groups can be effectively hydrated. This site may coincide with the location of the energetic barrier which impedes the movement of anions.

First principles: physical science concepts as a foundation for advanced studies in physiology.

The importance of mathematics and physical science to physiology is amply evident in both the classic and current literature of this field. Students who have completed typical medical school preparatory programs, however, have been poorly equipped to embark on serious graduate study in physiology. Because undergraduate education in the sciences is typically structured as narrowly defined, disjointed degree programs in separate disciplines, students of biology have had only limited access to the resources of physical science departments. Attempts by physiology faculty to remedy specific deficiencies on a piecemeal basis have been found to be both time consuming and ineffective. We describe an intensive one-semester course as an alternative. Maintaining high levels of generality and concentrating on the development of physical insight were possible only when the mathematical foundation attained by the students was adequate. Although physiological applications were stressed throughout, extended examination of complex problems was limited to a few examples of particular interest. Emphasis was placed on rigor and sophistication rather than proficiency and detail.

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.

Ion selectivity of colicin E1: modulation by pH and membrane composition.

Colicin E1 is a plasmid-encoded bacteriocidal protein which, though water soluble when secreted by its host bacterium, spontaneously interacts with planar lipid bilayers to form voltage-gated ion channels. In asolectin bilayers, the preference for anions over cations exhibited by these channels at low pH can be reversed by raising the pH on either side of the membrane. When incorporated into membranes composed of either of the two zwitterionic lipids, bacterial phosphatidylethanolamine and diphytanoyl phosphatidylcholine, colicin E1 channels were nearly ideally anion selective in the limit of low pH and moderately cation selective at the high pH limit. In phosphatidylcholine membranes, however, the response of these channels to changes in pH exhibited a pattern of behavior peculiar to this lipid. If the side of the membrane on which the protein had been introduced (the cis side) was exposed to pH 4.0, all the channels in the bilayer, whether opened or closed, became refractory to further changes in pH. This irreversibility has been interpreted as evidence that the selectivity of colicin E1 is under the control of a pH-sensitive conformational change. Protonation of groups on the cis side of the membrane appear to be essential to the conversion to the anion-selective state. These groups are rendered kinetically inaccessible to the aqueous phase when the transition takes place in phosphatidylcholine membranes.

Formation of ion channels by colicin B in planar lipid bilayers.

The gene for the antibacterial peptide colicin B was cloned and transformed into a host background where it was constitutively overexpressed. The purified gene product was biologically active and formed voltage-dependent, ion-conducting channels in planar phospholipid bilayers composed of asolectin. Colicin B channels exhibited two distinct unitary conductance levels, and a slight preference for Na+ over Cl-. Kinetic analysis of the voltage-driven opening and closing of colicin channels revealed the existence of at least two conducting states and two nonconducting states of the protein. Both the ion selectivity and the kinetics of colicin B channels were highly dependent on pH. Excess colicin protein was readily removed from the system by perfusing the bilayer, but open channels could be washed out only after they were allowed to close. A monospecific polyclonal antiserum generated against electrophoretically purified colicin B eliminated both the biological and in vitro activity of the protein. Membrane-associated channels, whether open or closed, remained functionally unaffected by the presence of the antiserum. Taken together, our results suggest that the voltage-independent binding of colicin B to the membrane is the rate-limiting step for the formation of ion channels, and that this process is accompanied by a major conformational rearrangement of the protein.

Ion transport mediated by copolymers composed of polyoxyethylene and polyoxypropylene.

Block copolymers composed of polyoxyethylene and polyoxypropylene were found to increase the influx of Na+ and the efflux of K+ from human erythrocytes. They were, however, ineffective at promoting the transport of Ca2+. The size of the ion fluxes induced by the copolymers correlated with their efficacy in stimulating inflammation. These compounds were also found to induce conductance increases in planar lipid bilayers in a nonvoltage dependent and nonstepwise manner. In both experimental systems, ion transport was facilitated only under temperature and ionic-strength conditions in which the polymers form aggregates in aqueous solution. In neither system did the concentration dependence of transport activity exhibit a pronounced cooperativity. These observations are consistent with the view that aqueous monomers of these surface active agents partition into the membrane, where they facilitate the conductive movement of monovalent cations by means of a carrier type mechanism. As a novel class of ionophores, these substances are of practical interest because they can be water soluble and are potentially reversible.

Octyl glucoside promotes incorporation of channels into neutral planar phospholipid bilayers. Studies with colicin Ia.

Colicin Ia forms voltage-dependent channels in planar phospholipid bilayers containing acidic phospholipids. Addition of the neutral detergent octyl glucoside, at concentrations three orders of magnitude below its critical micelle concentration (CMC), greatly increases channel-forming activity without altering the properties of the channels themselves. Further, octyl glucoside promotes formation of channels by colicin Ia in membranes containing only neutral lipids, making it possible to study the biophysical properties of the channel undistorted by the effects of negative surface charge. In neutral membranes, the macroscopic currents are biphasic with time, the fast component is voltage-dependent and the slow component voltage-independent. The single-channel conductance in 1 M NaCl is 31 pS and the channel is slightly anion selective. The mechanism by which the detergent facilitates channel formation is discussed.

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.

Solvent substitution as a probe of channel gating in Myxicola. Effects of D2O on kinetic properties of drugs that occlude channels.

The effects of solvent substitution on the steady-state and kinetic properties of drugs (gallamine triethiodide) and ions (nonyltriethylammonium and Ba++) known to occlude Na+ and K+ channels have been examined and compared with the effects of D2O on unmodified channels. In general, we observed large isotope effects on the kinetics of occlusion at temperatures of 5 degrees C, but only minor effects at 15 degrees C, consistent with processes involving significant solvent interaction. Steady-state behavior was not affected. In the case of gallamine, where a dual effect on INa is evident, although both processes were D2O sensitive, only the occlusion phase had a significant temperature dependence.

Solvent substitution as a probe of channel gating in Myxicola. Differential effects of D2O on some components of membrane conductance.

Careful examination of effects of solvent substitution on excitable membranes offers the theoretical possibility of identifying those aspects of the gating and translocation processes which are associated with significant changes in solvent order. Such information can then be used to develop or modify moire detailed models. We have examined the effects of heavy water substitution in Cs+-and K+-dialyzed Myxicola giant axons. At temperatures of 4-6 degrees C, the rates of Na+, K+, and Na+ inactivation during a maintained depolarization were all showed by approximately 50% in the presence of D2O. In contrast, the effects of solvent substitution on the time-course of prepulse inactivation and reactivation were much larger, with slowing averaging 160%. Studies at higher temperatures yielded Q10's for Na+ activation and K+ activation which were essentially comparable (0.72) and slightly but significantly smaller than that for inactivation during a maintained depolarization (0.84). In contrast, the Q10 for the D2O effect on prepulse inactivation was approximately 0.48. Heavy water substitution decrease Gk to a significantly greater extent than G(Na), while the decrease in the conductance of the Na+ channel caused by D2O was independent of whether the current-carrying species was Na+ or Li+. Sodium channel selectivity to the alkali metal cations and NH4+ was not changed by D2O substitution.

Modifications of sodium channel gating in Myxicola giant axons by deuterium oxide, temperature, and internal cations.

In dialyzed Myxicola axons substitution of heavy water (D2O) externally and internally slows both sodium and potassium kinetics and decreases the maximum conductances. Furthermore, this effect is strongly temperature dependent, the magnitude of the slowing produced by D2O substitution decreasing with increasing temperature over the range 3-14 degrees C with a Q10 of approximately 0.71. The relatively small magnitude of the D2O effect, combined with its strong temperature dependence, suggests that the rate limiting process producing a conducting channel involves appreciable local changes in solvent structure. Maximum conductances in the presence of D2O were decreased by approximately 30%, while the voltage dependences of both gNa and gK were not appreciably changed. In contrast to the effects of heavy water substitution on the ionic currents, membrane asymmetry currents were not altered by D2O, suggesting that gating charge movement may preceed by several steps the final transformation of the Na+ channel to a conducting state. In Myxicola axons the effect of temperature alone on asymmetry current kinetics can be well described via a simple temporal expansion equivalent to a Q10 of 2.2, which is somewhat less than the Q10 of GNa activation. The integral of membrane asymmetry current, representing maximum charge movement, is however not appreciably altered by temperature.

Immobilization of intramembrane charge in Myxicola giant axons.

1. Immobilization of gating charge was examined in Myxicola giant axons dialysed with Cs+. 2. With increasing pulse durations the ratio QOFF/QON decreases to as little as 0.25 with a time constant of 3.2 msec determined from composite data. Na inactivation time constants measured in the same axons ranged from 0.8 to 1.5 msec. A slow component of QOFF tends to appear as QOFF/QON decreases. 3. Both QON and QOFF may be decreased by a prepulse with no change in their time course. In this case the time course of the decrease is similar to that observed for INA. 4. The normalized steady-state charge immobilization curve is shifted in the depolarized direction by 15--20 mV from the normalized Na inactivation curve.

Internal cesium alters sodium inactivation in Myxicola.

When Myxicola giant axons are internally dialyzed with Cs+ as the sole cation, the time-course of prepulse inactivation is selectively accelerated compared to its rate with K+ dialysis in the same axons. This decrease in tauph occurs without any change in the magnitude or time-course of INa during step depolarizations and results in tauph/taush ratios near unity over most of the potential range in Cs+ dialyzed axons.

Combined voltage-clamp and dialysis of Myxicola axons: behaviour of membrane asymmetry currents.

4. A new technique for the simultaneous internal dialysis and voltage-clamp of Myxicola axons is described and shown to control the internal composition of the axon adequately. 2. The permeability ratio (PNa/PK) of the sodium channel is 15.5 in axons dialysed with sodium-free solutions, somewhat higher than observed in perfused axons. 3. Asymmetry currents are easily recorded in dialysed Myxicola axons. At 4--5 degrees C for pulse durations sufficiently short Qon = Qoff with a maximum charge displacement of 10 nC/cm2 and half the charge being displaced by a step to -32 mV. Maximum slope of the Q(V) curve is 15 mV/e-fold change in charge displaced. The time constant tau on reaches a maximum of 325 microseconds at a potential of -23 mV. 4. The time course of sodium activation cannot be adequately accounted for by (Q/Q infinity) X using any single value of X for potentials between -40 and 40 MV. 5. Both asymmetry currents and INa are inactivated by the same amount when Myxicola axons are repetitively depolarized at frequencies from 0.1 to 50 Hz.

Characteristics of sodium tail currents in Myxicola axons. Comparison with membrane asymmetry currents.

Sodium currents after repolarization to more negative potentials after initial activation were digitally recorded in voltage-clamped Myxicola axons compensated for series resistance. The results are inconsistent with a Hodgkin-Huxley-type kinetic scheme. At potentials more negative than -50 mV, the Na+ tails show two distinct time constants, while at more positive potentials only a single exponential process can be resolved. The time-course of the tail currents was totally unaffected when tetrodotoxin (TTX) was added to reduce gNa to low values, demonstrating the absence of any artifact dependent on membrane current. Tail currents were altered by [Ca++] in a manner consistent with a simple alteration in surface potential. Asymmetry current "off" responses are well described by a single exponential. The time constant for this response averaged 2.3 times larger than that for the rapid component of the Na+ repolarization current and was not sensitive to pulse amplitude or duration, although it did vary with holding potential. Other asymmetry current observations confirm previous reports on Myxicola.