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Roland Benz - One of the best experts on this subject based on the ideXlab platform.

  • use of nonelectrolytes reveals the Channel size and oligomeric constitution of the borrelia burgdorferi p66 porin
    PLOS ONE, 2013
    Co-Authors: Roland Benz, Sven Bergstrom, Ivan Barcenauribarri, Marcus Thein, Elke Maier, Mari Bonde
    Abstract:

    In the Lyme disease spirochete Borrelia burgdorferi, the outer membrane protein P66 is capable of pore formation with an atypical high single-Channel Conductance of 11 nS in 1 M KCl, which suggeste ...

  • tcda1 of photorhabdus luminescens electrophysiological analysis of pore formation and effector binding
    Biophysical Journal, 2013
    Co-Authors: Alexander E Lang, Janina Konukiewitz, Klaus Aktories, Roland Benz
    Abstract:

    Tc toxins are widely distributed among different gram-negative and gram-positive bacteria, where they act as pathogenicity factors. The toxins are composed of different components that form oligomers for biological activity. Lipid bilayer experiments were performed with the TcdA1 component of the Tc toxin from Photorhabdus luminescens, which preferentially kills insects by actin polymerization. TcdA1 was able to increase the specific Conductance of artificial lipid bilayer membranes by the formation of ion-permeable Channels. The Channels had on average a single-Channel Conductance of 125 pS in 150 mM KCl and were found to be cation selective. The single-Channel Conductance of the TcdA1-Channels was only moderately dependent on the bulk aqueous KCl concentration, which indicated point-charge effects on the Channel properties. Experiments to study the voltage dependence of the TcdA1 Channel demonstrated that it is reconstituted in a fully oriented way when it is added to only one side of the lipid bilayer membrane. A combination of biologically active components (TccC3) and a possible chaperone (TcdB2) blocked the TcdA1-mediated Conductance efficiently in a dose-dependent manner when they were added to the cis side of the membrane. The half-saturation constant for binding of TcdB2-TccC3 to TcdA1 is in the low nanomolar range.

  • identification of the outer membrane porin of thermus thermophilus hb8 the Channel forming complex has an unusually high molecular mass and an extremely large single Channel Conductance
    Journal of Bacteriology, 2001
    Co-Authors: Elke Maier, Georg Polleichtner, Birgit Boeck, Reinhard Schinzel, Roland Benz
    Abstract:

    The outer membrane of the thermophilic bacterium Thermus thermophilus was isolated using sucrose step gradient centrifugation. Its detergent extracts contained an ion-permeable Channel with an extremely high single-Channel Conductance of 20 nS in 1 M KCl. The Channel protein was purified by preparative sodium dodecyl sulfate (SDS)-polyacylamide gel electrophoresis. It has a high molecular mass of 185 kDa, and its Channel-forming ability resists boiling in SDS for 10 min.

  • the cell wall porin of the gram positive bacterium nocardia asteroides forms cation selective Channels that exhibit asymmetric voltage dependence
    Archives of Microbiology, 1999
    Co-Authors: Franziska G Ries, Thomas Lichtinger, A F Yassin, K P Schaal, Roland Benz
    Abstract:

    Detergent-solubilized cell wall extracts of the gram-positive, strictly aerobic bacterium Nocardia asteroides contain Channel-forming activity as judged from reconstitution experiments using lipid bilayer membranes. The cell wall porin was identified as a protein with an apparent molecular mass of about 84 kDa based on SDS-PAGE. The porin was purified to homogeneity using preparative SDS-PAGE. The 84-kDa protein was no longer observed after heating in SDS buffer. The presumed dissociation products were not observed on SDS-polyacrylamide gels. The cell wall porin increased the specific Conductance of artificial lipid bilayer membranes from phosphatidylcholine/phosphatidylserine mixtures by the formation of cation-selective Channels, which had an average single-Channel Conductance of 3.0 nS in 1 M KCl. The single-Channel Conductance was only moderately dependent on the bulk aqueous KCl concentration, which indicated negative point charge effects on the Channel properties. The analysis of the concentration dependence of the single-Channel Conductance using the effect of negative charges on Channel Conductance suggested that the diameter of the cell wall Channel is about 1.4 nm. Asymmetric addition of the cell wall porin to lipid bilayer membranes resulted in an asymmetric voltage dependence. The cell wall Channel switched into substates, when the cis side of the membrane, the side of the addition of the protein, had negative polarity. Positive potentials at the cis side had no influence on the Conductance of the cell wall Channel.

  • biochemical and biophysical characterization of the cell wall porin of corynebacterium glutamicum the Channel is formed by a low molecular mass polypeptide
    Biochemistry, 1998
    Co-Authors: Thomas Lichtinger, Andreas Burkovski, Michael Niederweis, Reinhard Kramer, Roland Benz
    Abstract:

    The cell wall of the Gram-positive bacterium Corynebacterium glutamicum contains a Channel (porin) for the passage of hydrophilic solutes. The Channel-forming protein was identified, by lipid bilayer experiments, in the cell envelope fractions isolated by sucrose-density centrifugations and in organic solvent of whole cells. It was purified to homogeneity by fast-protein liquid chromatography across a Mono-Q column. The pure protein had a rather low molecular mass of about 5 kDa as judged by SDS-PAGE, which suggested that the cell wall Channel is formed by a protein oligomer. The monomer has according to partial sequencing no significant homology to known protein sequences. The purified protein formed large ion-permeable Channels in lipid bilayer membranes from phosphatidylcholine/phosphatidylserine mixtures with a single-Channel Conductance of 5.5 nS in 1 M KCl. Experiments with different salts suggested that the cell wall Channel of C. glutamicum was highly cation-selective caused by negative charges localized at the Channel mouth. The analysis of the single-Channel Conductance data using the Renkin correction factor suggested that the diameter of the cell wall Channel is about 2.2 nm. Channel-forming properties of the cell wall Channel of C. glutamicum were compared with those of mycobacteria. These Channels share common features because they form large and water-filled Channels that contain point net charges.

Vicente M Aguilella - One of the best experts on this subject based on the ideXlab platform.

  • ion transport in confined geometries below the nanoscale access resistance dominates protein Channel Conductance in diluted solutions
    ACS Nano, 2017
    Co-Authors: Antonio Alcaraz, Lidon M Lopez, Maria Queraltmartin, Vicente M Aguilella
    Abstract:

    Synthetic nanopores and mesoscopic protein Channels have common traits like the importance of electrostatic interactions between the permeating ions and the nanoChannel. Ion transport at the nanoscale occurs under confinement conditions so that the usual assumptions made in microfluidics are challenged, among others, by interfacial effects such as access resistance (AR). Here, we show that a sound interpretation of electrophysiological measurements in terms of Channel ion selective properties requires the consideration of interfacial effects, up to the point that they dominate protein Channel Conductance in diluted solutions. We measure AR in a large ion Channel, the bacterial porin OmpF, by means of single-Channel Conductance measurements in electrolyte solutions containing varying concentrations of high molecular weight PEG, sterically excluded from the pore. Comparison of experiments performed in charged and neutral planar membranes shows that lipid surface charges modify the ion distribution and determine the value of AR, indicating that lipid molecules are more than passive scaffolds even in the case of large transmembrane proteins. We also found that AR may reach up to 80% of the total Channel Conductance in diluted solutions, where electrophysiological recordings register essentially the AR of the system and depend marginally on the pore characteristics. These findings may have implications for several low aspect ratio biological Channels that perform their physiological function in a low ionic strength and macromolecule crowded environment, just the two conditions enhancing the AR contribution.

  • analysis of sars cov e protein ion Channel activity by tuning the protein and lipid charge
    Biochimica et Biophysica Acta, 2013
    Co-Authors: Carmina Verdiabaguena, Antonio Alcaraz, Jose L Nietotorres, Marta L Dediego, Luis Enjuanes, Vicente M Aguilella
    Abstract:

    A partial characterization of the ion Channels formed by the SARS coronavirus (CoV) envelope (E) protein was previously reported (C. Verdia-Baguena et al., 2012 [12]). Here, we provide new significant insights on the involvement of lipids in the structure and function of the CoV E protein Channel on the basis of three series of experiments. First, reversal potential measurements over a wide range of pH allow the dissection of the contributions to Channel selectivity coming from ionizable residues of the protein transmembrane domain and also from the negatively charged groups of diphytanoyl phosphatidylserine (DPhPS) lipid. The corresponding effective pKas are consistent with the model pKas of the acidic residue candidates for titration. Second, the change of Channel Conductance with salt concentration reveals two distinct regimes (Donnan-controlled electrodiffusion and bulk-like electrodiffusion) fully compatible with the outcomes of selectivity experiments. Third, by measuring Channel Conductance in mixtures of neutral diphytanoyl phosphatidylcholine (DPhPC) lipids and negatively charged DPhPS lipids in low and high salt concentrations we conclude that the protein-lipid conformation in the Channel is likely the same in charged and neutral lipids. Overall, the whole set of experiments supports the proteolipidic structure of SARS-CoV E Channels and explains the large difference in Channel Conductance observed between neutral and charged membranes.

  • linearity saturation and blocking in a large multiionic Channel divalent cation modulation of the ompf porin Conductance
    Biochemical and Biophysical Research Communications, 2011
    Co-Authors: Elena Garciagimenez, Vicente M Aguilella, Lidon M Lopez, Antonio Alcaraz
    Abstract:

    Measurement of unitary Conductance is a fundamental step in the characterization of a protein ion Channel permeabilizing a membrane. We study here the effect of salts of divalent cations on the OmpF Channel Conductance with a particular emphasis in dissecting the role of the electrolyte itself, the role of the counterion accumulation induced by the protein Channel charges and other effects not found in salts of monovalent cations. We show that current saturation and blocking are not exclusive properties of narrow (single-file) ion Channels but may be observed in large, multiionic Channels like bacterial porins. Single-Channel Conductance measurements performed over a wide range of salt concentrations (up to 3 M) combined with continuum electrodiffusion calculations demonstrate that current saturation cannot be simply ascribed to ion interaction with protein Channel residues.

  • alamethicin Channel Conductance modified by lipid charge
    European Biophysics Journal, 2001
    Co-Authors: Vicente M Aguilella, Sergey M Bezrukov
    Abstract:

    The membrane surface charge modifies the Conductance of ion Channels by changing the electric potential and redistributing the ionic composition in their vicinity. We have studied the effects of lipid charge on the Conductance of a multi-state Channel formed in planar lipid bilayers by the peptide antibiotic alamethicin. The Channel Conductance was measured in two lipids: in a neutral dioleoylphosphatidylethanolamine (DOPE) and a negatively charged dioleoylphosphatidylserine (DOPS). The charge state of DOPS was manipulated by the pH of the membrane-bathing solution. We find that at high salt concentrations (e.g., 2 M NaCl) the effect of the lipid charge is below the accuracy of our measurements. However, when the salt concentration in the membrane-bathing solution is decreased, the surface charge manifests itself as an increase in the Conductance of the first two Channel levels that correspond to the smallest conductive alamethicin aggregates. Our analysis shows that both the salt and pH dependence of the surface charge effect can be rationalized within the nonlinear Poisson-Boltzmann approach. Given Channel Conductance in neutral lipids, we use different procedures to account for the surface charge (e.g., introduce averaging over the Channel aperture and take into account Na+ adsorption to DOPS heads), but only one adjustable parameter: an effective distance from the nearest lipid charge to the Channel mouth center. We show that this distance varies by 0.3–0.4 nm upon Channel transition from the minimal conducting aggregate (level L0) to the next larger one (level L1). This conclusion is in accord with a simple geometrical model of alamethicin aggregation.

  • membrane surface charge titration probed by gramicidin a Channel Conductance
    Biophysical Journal, 1998
    Co-Authors: Tatiana K Rostovtseva, Vicente M Aguilella, Igor Vodyanoy, Sergey M Bezrukov, Adrian V Parsegian
    Abstract:

    We manipulate lipid bilayer surface charge and gauge its influence on gramicidin A Channel Conductance by two strategies: titration of the lipid charge through bulk solution pH and dilution of a charged lipid by neutral. Using diphytanoyl phosphatidylserine (PS) bilayers with CsCl aqueous solutions, we show that the effects of lipid charge titration on Channel Conductance are masked 1) by Conductance saturation with Cs+ ions in the neutral pH range and 2) by increased proton concentration when the bathing solution pH is less than 3. A smeared charge model permits us to separate different contributions to the Channel Conductance and to introduce a new method for "bilayer pKa" determination. We use the Gouy-Chapman expression for the charged surface potential to obtain equilibria of protons and cations with lipid charges. To calculate cation concentration at the Channel mouth, we compare different models for the ion distribution, exact and linearized forms of the planar Poisson-Boltzmann equation, as well as the construction of a "Gibbs dividing surface" between salt bath and charged membrane. All approximations yield the intrinsic pKain of PS lipid in 0.1 M CsCl to be in the range 2.5-3.0. By diluting PS surface charge at a fixed pH with admixed neutral diphytanoyl phosphatidylcholine (PC), we obtain a Conductance decrease in magnitude greater than expected from the electrostatic model. This observation is in accord with the different Conductance saturation values for PS and PC lipids reported earlier (, Biochim. Biophys. Acta. 552:369-378) and verified in the present work for solvent-free membranes. In addition to electrostatic effects of surface charge, gramicidin A Channel Conductance is also influenced by lipid-dependent structural factors.

Donglin Bai - One of the best experts on this subject based on the ideXlab platform.

  • variants with increased negative electrostatic potential in the cx50 gap junction pore increased unitary Channel Conductance and magnesium modulation
    Biochemical Journal, 2018
    Co-Authors: Mary Grace Tejada, Hiroshi Aoyama, Swathy Sudhakar, Brian H Shilton, Nicholas K Kim, Donglin Bai
    Abstract:

    Gap junction (GJ) Channels are oligomers of connexins forming Channels linking neighboring cells. GJs formed by different connexins show distinct unitary Channel Conductance (γj), transjunctional voltage-dependent gating (Vj-gating) properties, and modulation by intracellular magnesium ([Mg2+]i). The underlying molecular determinants are not fully clear. Previous experimental evidence indicates that residues in the amino terminal (NT) and initial segment of the first extracellular (E1) domain influence the γj, Vj-gating, and/or [Mg2+]i modulation in several GJs. Increasing negatively charged residues in Cx50 (connexin50) E1 (G46D or G46E) increased γj, while increasing positively charged residue (G46K) reduced the γj. Sequence alignment of Cx50 and Cx37 in the NT and E1 domains revealed that in Cx50 G8 and V53, positions are negatively charged residues in Cx37 (E8 and E53, respectively). To evaluate these residues together, we generated a triple variant in Cx50, G8E, G46E, and V53E simultaneously to study its γj, Vj-gating properties, and modulation by [Mg2+]i. Our data indicate that the triple variant and individual variants G8E, G46E, and V53E significantly increased Cx50 GJ γj without a significant change in the Vj gating. In addition, elevated [Mg2+]i reduced γj in Cx50 and all the variant GJs. These results and our homology structural models suggest that these NT/E1 residues are likely to be pore-lining and the variants increased the negative electrostatic potentials along the GJ pore to facilitate the γj of this cation-preferring GJ Channel. Our results indicate that electrostatic properties of the Cx50 GJ pore are important for the γj and the [Mg2+]i modulation.

  • the first extracellular domain plays an important role in unitary Channel Conductance of cx50 gap junction Channels
    PLOS ONE, 2015
    Co-Authors: Xiaoling Tong, Hiroshi Aoyama, Swathy Sudhakar, Honghong Chen, Brian H Shilton, Donglin Bai
    Abstract:

    Gap junction (GJ) Channels provide direct passage for ions and small molecules to be exchanged between neighbouring cells and are crucial for many physiological processes. GJ Channels can be gated by transjunctional voltage (known as Vj-gating) and display a wide range of unitary Channel Conductance (γj), yet the domains responsible for Vj-gating and γj are not fully clear. The first extracellular domain (E1) of several connexins has been shown to line part of their GJ Channel pore and play important roles in Vj-gating properties and/or ion permeation selectivity. To test roles of the E1 of Cx50 GJ Channels, we generated a chimera, Cx50Cx36E1, where the E1 domain of Cx50 was replaced with that of Cx36, a connexin showing quite distinct Vj-gating and γj from those of Cx50. Detailed characterizations of the chimera and three point mutants in E1 revealed that, although the E1 domain is important in determining γj, the E1 domain of Cx36 is able to effectively function within the context of the Cx50 Channel with minor changes in Vj-gating properties, indicating that sequence differences between the E1 domains in Cx36 and Cx50 cannot account for their drastic differences in Vj-gating and γj. Our homology models of the chimera and the E1 mutants revealed that electrostatic properties of the pore-lining residues and their contribution to the electric field in the pore are important factors for the rate of ion permeation of Cx50 and possibly other GJ Channels.

John A Peters - One of the best experts on this subject based on the ideXlab platform.

  • molecular determinants of single Channel Conductance and ion selectivity in the cys loop family insights from the 5 ht3 receptor
    Trends in Pharmacological Sciences, 2005
    Co-Authors: John A Peters, Tim G Hales, Jeremy J Lambert
    Abstract:

    The molecular determinants of the ionic selectivity and single-Channel Conductance of the Cys-loop family of transmitter-gated ion Channels are beginning to be understood with increasing precision, in part, as a result of the recent availability of refined ultrastructural information for the archetype of the family, the nicotinic acetylcholine receptor (nAChR). Studies of another member of this family, the 5-HT 3 receptor, have now provided insight into the structure of its Channel pore, the location of its gate and mechanisms of ion selectivity and translocation. The anomaly of the extremely low single-Channel Conductance of the homo-oligomeric 5-HT 3A receptor has recently been solved, revealing that an intracellular domain of the protein is an important determinant of single-Channel Conductance. Such data are interpreted, in this article, in light of the most recent developments in structural characterization of the nAChR.

  • a cytoplasmic region determines single Channel Conductance in 5 ht3 receptors
    Nature, 2003
    Co-Authors: Stephen P Kelley, James I Dunlop, Ewen F Kirkness, Jeremy J Lambert, John A Peters
    Abstract:

    5-Hydroxytryptamine type 3 (5-HT3) receptors are cation-selective transmitter-gated ion Channels of the Cys-loop superfamily1,2,3,4,5,6,7,8,9. The single-Channel Conductance of human recombinant 5-HT3 receptors assembled as homomers of 5-HT3A subunits, or heteromers of 5-HT3A and 5-HT3B subunits, are markedly different, being 0.4 pS (refs 6, 9) and 16 pS (ref. 7), respectively. Paradoxically, the Channel-lining M2 domain of the 5-HT3A subunit would be predicted to promote cation conduction, whereas that of the 5-HT3B subunit would not7. Here we describe a determinant of single-Channel Conductance that can explain these observations. By constructing chimaeric 5-HT3A and 5-HT3B subunits we identified a region (the ‘HA-stretch’)10 within the large cytoplasmic loop of the receptor that markedly influences Channel Conductance. Replacement of three arginine residues unique to the HA-stretch of the 5-HT3A subunit by their 5-HT3B subunit counterparts increased single-Channel Conductance 28-fold. Significantly, ultrastructural studies of the Torpedo nicotinic acetylcholine receptor11 indicate that the key residues might frame narrow openings that contribute to the permeation pathway. Our findings solve the conundrum of the anomalously low Conductance of homomeric 5-HT3A receptors and indicate an important function for the HA-stretch in Cys-loop transmitter-gated ion Channels.

Mark S P Sansom - One of the best experts on this subject based on the ideXlab platform.

  • mechanisms of activation and desensitization of full length glycine receptor in membranes
    bioRxiv, 2019
    Co-Authors: Arvind Kumar, Mark S P Sansom, Sandip Basak, Shanlin Rao, Yvonne Gicheru, Megan L Mayer, Sudha Chakrapani
    Abstract:

    Glycinergic synapses play a central role in motor control and pain processing in the central nervous system. Glycine receptors (GlyR) are key players in mediating fast inhibitory neurotransmission at these synapses. While previous high-resolution structural studies have provided insights into the molecular architecture of GlyR, several mechanistic questions pertaining to Channel function are still unknown. Here, we present Cryo-EM structures of the full-length GlyR protein reconstituted into lipid nanodiscs that are captured in the unliganded (closed) and glycine-bound (open and desensitized) conformations. A comparison of the three states reveals global conformational changes underlying GlyR Channel gating. The functional state assignments were validated by molecular dynamics simulations of the structures incorporated in a lipid bilayer. Observed permeation events are in agreement with the anion selectivity of the Channel and the reported single-Channel Conductance of GlyR. These studies establish the structural basis for gating, selectivity, and single-Channel Conductance of GlyR in a physiological environment.

  • a novel congenital myasthenic syndrome due to decreased acetylcholine receptor ion Channel Conductance
    Brain, 2012
    Co-Authors: Richard Webster, Susan Maxwell, Hayley Spearman, Kaihsu Tai, Oliver Beckstein, Mark S P Sansom, David Beeson
    Abstract:

    Muscle acetylcholine receptor ion Channels mediate neurotransmission by depolarizing the postsynaptic membrane at the neuromuscular junction. Inherited disorders of neuromuscular transmission, termed congenital myasthenic syndromes, are commonly caused by mutations in genes encoding the five subunits of the acetylcholine receptor that severely reduce endplate acetylcholine receptor numbers and/or cause kinetic abnormalities of acetylcholine receptor function. We tracked the cause of the myasthenic disorder in a female with onset of first symptoms at birth, who displayed mildly progressive bulbar, respiratory and generalized limb weakness with ptosis and ophthalmoplegia. Direct DNA sequencing revealed heteroallelic mutations in exon 8 of the acetylcholine receptor e-subunit gene. Two alleles were identified: one with the missense substitution p.eP282R, and the second with a deletion, c.798_800delCTT, which result in the loss of a single amino acid, residue F266, within the M2 transmembrane domain. When these acetylcholine receptor mutations were expressed in HEK 293 cells, the p.eP282R mutation caused severely reduced expression on the cell surface, whereas p.eΔF266 gave robust surface expression. Single-Channel analysis for p.eΔF266 acetylcholine receptor Channels showed the longest burst duration population was not different from wild-type acetylcholine receptor (4.39 ± 0.6 ms versus 4.68 ± 0.7 ms, n =  5 each) but that the amplitude of Channel openings was reduced. Channel amplitudes at different holding potentials showed that single-Channel Conductance was significantly reduced in p.eΔF266 acetylcholine receptor Channels (42.7 ± 1.4 pS, n =  8, compared with 70.9 ± 1.6 pS for wild-type, n =  6). Although a phenylalanine residue at this position within M2 is conserved throughout ligand-gated excitatory cys-loop Channel subunits, deletion of equivalent residues in the other subunits of muscle acetylcholine receptor did not have equivalent effects. Modelling the impact of p.eΔF266 revealed only a minor alteration to Channel structure. In this study we uncover the novel mechanism of reduced acetylcholine receptor Channel Conductance as an underlying cause of congenital myasthenic syndrome, with the ‘low Conductance’ phenotype that results from the p.eΔF266 deletion mutation revealed by the coinheritance of the low-expressor mutation p.eP282R. * Abbreviation : AChR : acetylcholine receptor

  • filter flexibility in a mammalian k Channel models and simulations of kir6 2 mutants
    Biophysical Journal, 2003
    Co-Authors: Charlotte E Capener, Peter Proks, Frances M Ashcroft, Mark S P Sansom
    Abstract:

    The single-Channel Conductance varies significantly between different members of the inward rectifier (Kir) family of potassium Channels. Mutations at three sites in Kir6.2 have been shown to produce Channels with reduced single-Channel Conductance, the largest reduction (to 40% of wild-type) being for V127T. We have used homology modeling (based on a KcsA template) combined with molecular dynamics simulations in a phosphatidycholine bilayer to explore whether changes in structural dynamics of the filter were induced by three such mutations: V127T, M137C, and G135F. Overall, 12 simulations of Kir6.2 models, corresponding to a total simulation time of 27 ns, have been performed. In these simulations we focused on distortions of the selectivity filter, and on the presence/absence of water molecules lying behind the filter, which form interactions with the filter and the remainder of the protein. Relative to the wild-type simulation, the V127T mutant showed significant distortion of the filter such that approximately 50% of the simulation time was spent in a closed conformation. While in this conformation, translocation of K(+) ions between sites S1 and S2 was blocked. The distorted filter conformation resembles that of the bacterial Channel KcsA when crystallized in the presence of a low [K(+)]. This suggests filter distortion may be a possible general model for determining the Conductance of K Channels.

  • a novel method for structure based prediction of ion Channel Conductance properties
    Biophysical Journal, 1997
    Co-Authors: Oliver S Smart, J Breed, Graham Smith, Mark S P Sansom
    Abstract:

    A rapid and easy-to-use method of predicting the Conductance of an ion Channel from its three-dimensional structure is presented. The method combines the pore dimensions of the Channel as measured in the HOLE program with an Ohmic model of Conductance. An empirically based correction factor is then applied. The method yielded good results for six experimental Channel structures (none of which were included in the training set) with predictions accurate to within an average factor of 1.62 to the true values. The predictive r2 was equal to 0.90, which is indicative of a good predictive ability. The procedure is used to validate model structures of alamethicin and phospholamban. Two genuine predictions for the Conductance of Channels with known structure but without reported Conductances are given. A modification of the procedure that calculates the expected results for the effect of the addition of nonelectrolyte polymers on Conductance is set out. Results for a cholera toxin B-subunit crystal structure agree well with the measured values. The difficulty in interpreting such studies is discussed, with the conclusion that measurements on Channels of known structure are required.