Effects of domain structure on in-plane reactions and interactions

The existence of an in-plane domain structure in biological membranes raises the question of the physiological function, if any, of this structure. One important function may be to enhance or limit the equilibrium poise and rates of in-plane reactions through control by the cell of the percolation properties of the domain system. At low average domain occupancy by reactants or interactants, which must be the case for most biological membrane components, moving the domain system from connection to disconnection has marked effects on the apparent equilibrium poise and the rates of membrane-confined reactions. This conclusion is based on computer modelling of the effects of disconnection/connection of nine types of bimolecular in-plane reactions. Using the phase structure and percolation properties of two-component, two-phase phospholipid bilayers, it is possible to examine experimentally homo- and heterodimeriztion reactions, and enzyme-catalysed reactions in-plane as well as the effects of a transmembrane peptide on these systems. These theoretical and experimental studies suggest that percolation effects may be physiologically important in biological membranes. Whether this is in fact the case remains to be demonstrated.     

Tetanus toxin channel in phosphatidylserine planar bilayers: conductance states and pH dependence

Tetanus toxin (TeTx) forms ionic channel in phosphatidylserine bilayers. TeTx channels exhibit different modes of channel bursting activity, from a closed state to well defined open states of different amplitudes. At positive applied voltages, TeTx channels flicker continuously between a closed state and the various distinct open states. Furthermore, fast transitions into subconductance states are discernible within the bursts of channel activity. Elementary conductance steps submultiple of the open states were not identified in single channel records owing to rapid transitions between different states. However, statistical analysis shows that conductances cluster with amplitudes multiple of an elementary value: e.g. 25–30 pS at neutral pH. Single channel current amplitudes decrease with the pH of the bulk electrolyte solution. Conductance decrements can be accounted for by the relative decrease of permeant cation concentration at the membrane-water interface, by a relative enrichment of protons that block the channel or by the stabilization of a conformational state of the channel protein. Offprint requests to: F. Gambale     

Colicin N forms voltage- and pH-dependent channels in planar lipid bilayer membranes

The protein antibiotic colicin N forms ion-permeable channels through planar lipid bilayers. Channels are induced when positive voltages higher than +60 mV are applied. Incorporated channels activate and inactivate in a voltage-dependent fashion. It is shown that colicin N undergoes a transition between an “acidic” and a “basic” channel form which are distinguishable by different voltage dependences. The single-channel conductance is non-ohmic and strongly dependent on pH, indicating that titratable groups control the passage of ions through the channel. The ion selectivity of colicin N channels is influenced by the pH and the lipid composition of the bilayer membrane. In neutral membranes the channel undergoes a transition from slightly cation-selective to slightly anion-selective when the pH is changed from 7 to 5. In lipid membranes bearing a negative surface charge the channel shows a more pronounced cation selectivity which decreases but does not reverse upon lowering the pH from 7 to 5. The high degree of similarity between the channel characteristics of colicin A and N suggests that the channels share common features in their molecular structure. Offprint requests to: F. Pattus     

Characterization of a partially degraded Na+ channel from urinary tract epithelium

Summary The mammalian urinary bladder contains in its apical membrane and cytoplasmic vesicles, a cation-selective channel or activating fragment which seems to partition between the apical membrane and the luminal (or vesicular space). To determine whether it is an activating fragment or whole channel, we first demonstrate that solution known to contain this moiety can be concentrated and when added back to the bladder causes a conductance increase, with a percent recovery of 139±25%. Next, we show that using tip-dip bilayer techniques (at 21°C) and a patch-clamp recorder, the addition of concentrated solution resulted in the appearance of discrete current shots, consistent with the incorporation of a channel (as opposed to an activating fragment) into the bilayer. The residency time of the channel in the bilayer was best described by the sum of two exponentials, suggesting that the appearance of the channel involves an association of the channel with the membrane before insertion. The channel is cation selective and more conductive to K⁺ than Na⁺ (by a factor of 1.6). It has a linearI–V relationship, but a singlechannel conductance that saturates as KCl concentration is raised. This saturation is best described by the Michaelis-Menten equation with aK _m of 160mm KCl and aG _max of 20 pS. The kinetics of the channel are complex, showing at least two open and two closed states.Since the characteristics of this channel are similar to a channel produced by the degradation of amiloride-sensitive Na⁺ channels by the proteolytic enzyme kallikrein (which is released by the cortical collecting duct of the kidney), we suggest that this channel then is not synthesized by the cell but is rather a degraded form of the epithelial Na⁺ channel.     

Properties of Voltage-gated Ion Channels Formed by Syringomycin E in Planar Lipid Bilayers

Using the planar lipid bilayer technique we demonstrate that the lipodepsipeptide antibiotic, syringomycin E, forms voltage-sensitive ion channels of weak anion selectivity. The formation of channels in bilayers made from dioleoylglycerophosphatidylserine doped with syringomycin E at one side (1–40 μg/ml) was greatly affected by cis-positive voltage. A change of voltage from a positive to a negative value resulted in (i) an abrupt increase in the single channel conductance (the rate of increase was voltage dependent) simultaneous with (ii) a closing of these channels and an exponential decrease in macroscopic conductance over time. The strong voltage dependence of multichannel steady state conductance, the single channel conductance, the rate of opening of channels at positive voltages and closing them at negative voltages, as well as the observed abrupt increase of single channel conductance after voltage sign reversal suggest that the change of the transmembrane field induces a significant rearrangement of syringomycin E channels, including a change in the spacing of charged groups that function as voltage sensors. The conductance induced by syringomycin E increased with the sixth power of syringomycin E concentration suggesting that at least six monomers are required for channel formation. Received: 3 April 1995/Revised: 24 August 1995     

The Permeation of Ammonium through a Voltage-independent K+ Channel in the Plasma Membrane of Rye Roots

Nitrogen is available to the plant in the form of NH⁺ ₄ in the soil solution. Here it is shown that a voltage-independent K⁺ channel in the plasma membrane of rye (Secale cereale L.) roots is permeable to NH⁺ ₄. The channel was studied following its incorporation into planar 1-palmitoyl-2-oleoyl phosphatidyl ethanolamine bilayers. The unitary conductance of the channel was greater when assayed in the presence of 100 mm NH₄Cl than 100 mm KCl. However, the probability of finding the channel open (P _o ) was lower in the presence of 100 mm NH₄Cl (P _o = 0.63) than in 100 mm KCl (P _o = 0.8), suggesting that P _o can be regulated by the (permeant) ions present in solution. When assayed in equimolar concentrations of NH₄Cl (cis) and KCl (trans), the zero-current (reversal) potential for the channel (E _rev) exhibited a complex concentration dependence. At low cation concentrations, the apparent permeability of NH⁺ ₄ relative to K⁺ (P_NH4/P_K) was greater than 1.0. However, as the cation concentration was increased, P_NH4/P_K initially decreased to a minimum of 0.95 at 3 mm before increasing again to a maximum of 1.89 at 300 mm. At cation concentrations above 300 mm, P_NH4/P_K decreased slightly. This implies that the pore of the channel can be occupied by more than one cation simultaneously. Ammonium permeation through the pore was simulated using a model which is composed of three energy barriers and two energy wells (the ion-binding sites). The model (3B2S) allowed for single-file permeation, double cation occupancy, ion-ion repulsion within the pore and surface potential effects. Results indicated that energy peaks and energy wells were situated asymmetrically within the electrical distance of the pore, that cations repel each other within the pore and that the vestibules to the pore contain negligible surface charge. The energy profile obtained for NH⁺ ₄ is compared with ones obtained for K⁺ and Na⁺. This information allows the fluxes through the K⁺ channel of the three major monovalent cations present in the soil solution to be predicted. Received: 16 October 1995/Revised 12 March 1996     

Structure and function of amiloride-sensitive Na+ channels

A new molecular biological epoch in amiloride-sensitive Na⁺ channel physiology has begun. With the application of these new techniques, undoubtedly a plethora of new information and new questions will be forthcoming. First and foremost, however, is the question of how many discrete amiloride-sensitive Na⁺ channels exist. This question is important not only for elucidating structure-function relationships, but also for developing strategies for pharmacological or, ultimately, genetic intervention in such diseases as obstructive nephropathy, Liddle's syndrome, or salt-sensitive hypertension where amiloride-sensitive Na⁺ channel dysfunction has been implicated [17, 62].Epithelia Na⁺ channels purified from kidney are multimeric. However, it is not yet clear which subunits are regulatory and which participate directly as a part of the Na⁺ conducting core and what is the nature of the gate. The combination of electrophysiologic techniques such as patch clamp and the ability to study reconstituted channels in planar lipid bilayers along with molecular biology techniques to potentially manipulate the individual subunits should provide the answers to questions that have puzzled physiologists for decades. It seems clear that the robust versatility of the channel in responding to a wide range of differing and potentially synergistic regulatory inputs must be a function of its multimeric structure and relation to the cytoskeleton. Multiple mechanisms of regulation imply multiple regulatory sites. This hypothesis has been validated by the demonstration that enzymatic carboxyl methylation and phosphorylation have both individual and synergistic effects on the purified channel in planar lipid bilayers.     

Staphylococcus aureus alpha-toxin-induced pores: Channel-like behavior in lipid bilayers and patch clamped cells

The conductance of pores induced by Staphylococcus aureus -toxin in Lettre cells has been compared to that in bilayers composed of synthetic lipids or Lettre cell membrane constituents. Previously described characteristics of toxin-induced conductance changes in lipid bilayers, namely rectification, voltage-dependent closure, and closure at low pH or in the presence of divalent cations (Menestrina, 1986) are displayed also in bilayers prepared from Lettre cell membranes and in patch clamped Lettre cells. It is concluded that endogenous proteins do not affect the properties of -toxininduced channels significantly and that the relative lack of ion channels in Lettre cells makes them ideal for studies of pore-forming toxins by the patch clamp technique.Dr. Sviderskaya is on leave of absence from the Physiology Institute, University of St. Petersburg, RussiaWe are grateful to Dr. J.P. Arbuthnott and Dr. K. Hungerer for gifts of S. aureus -toxin, to Dr. T.B. Bolton for collaboration with patch clamped cells and to Dr. J.M. Graham for help with the preparation of Lettre cell plasma membranes. This study was supported by the Cell Surface Research Fund, Medical Research Council, Science and Engineering Research Council, UNESCO (Molecular and Cell Biology Network) and The Wellcome Trust.     

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 Å. 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.The authors wish to thank Dr. F.S. Cohen for making available unpublished data and for helpful comments. This work was supported by National Institutes of Health grant GM 37396 and by the Howard Hughes Medical Institute Undergraduate Biological Sciences Education Initiative (E.R.K.)     

Formation of ion channels in lipid bilayers by a peptide with the predicted transmembrane sequence of botulinum neurotoxin A.

Synthetic peptides patterned after the predicted transmembrane sequence of botulinum toxin A were used as tools to identify an ion channel-forming motif. A peptide denoted BoTxATM, with the sequence GAVILLEFIPEIAI PVLGTFALV, forms cation-selective channels when reconstituted in planar lipid bilayers. As predicted, the self-assembled conductive oligomers express heterogeneous single-channel conductances. The most frequent openings exhibit single-channel conductance of 12 and 7 pS in 0.5 M NaCl, and 29 and 9 pS in 0.5 M KCl. In contrast, ion channels are not formed by a peptide of the same amino acid composition as BoTxATM with a scrambled sequence. Conformational energy calculations show that a bundle of four amphipathic alpha-helices is a plausible structural motif underlying the measured pore properties. These studies suggest that the identified module may play a functional role in the ion channel-forming activity of intact botulinum toxin A.