SUMMARY1. The ryanodine receptor protein of sheep cardiac muscle sarcoplasmic reticulum membranes functions as a ligand-regulated ion channel following solubilization with the zwitterionic detergent CHAPS (3-[(3-cholamidopropyl)-dimethylammonio]-1-propane sulphonate); purification by density gradient centrifugation, reconstitution into proteo-liposomes and incorporation into planar phospholipid bilayers.2. In the absence of divalent cations, measurable conductance is observed with the group la cations and with some larger organic cations. In symmetric 210 mM solutions the following conductance sequence was determined: K+ > Rb+ NH4+ > Na+ = Cs+ > Li+ > Tris+.3. Other organic cations, e.g. TEA+, do not produce measurable current under these conditions. 4. Single-channel conductance saturates with increasing ionic activities of K+, Na+ and Li+. Saturation curves are described by Michaelis-Menten kinetic schemes with the following values of maximal conductance and apparent dissociation constant: K+ 900 pS, 19-9 mM; Na+ 516 pS, 17-8 mM; Li' 248 pS, 9-1 mM.5. The channel displays only minor differences in permeability amongst the group la cations. Relative permeability, monitored under bi-ionic conditions, yields the following sequence: Na+, 1-15 > K+, 1-00 = Li+, 0 99 > Rb+, 0-87 > Cs+, 0-61. Under similar conditions the permeability ratio of NH4+ to K+ was found to be 1-32 and that for Tris+ to K+ was 0-22.6. The K+ conductance is reduced by low concentrations of the impermeant cation TEA+. Block appears as a smooth reduction in single-channel current amplitude and the degree of block is dependent upon applied voltage. These observations are consistent with a single-site blocking scheme in which TEA+ has access to a site within the voltage drop of the channel from only the cytosolic face of the channel protein and interacts with a site located approximately 90% of the electrical distance across the channel. The zero-voltage dissociation constant for TEA+ block is 50 mM.7. Single-channel conductance measurements in mixtures of K+-Na+ and K+-Li+ reveal no anomalous behaviour as the mole fraction of the ions is varied.8

We have investigated the basic properties of a predominantly anion-selective channel derived from highly purified human platelet surface membrane. Single channels have been reconstituted into planar phospholipid bilayers by fusion of membrane vesicles and recorded under voltage-clamp conditions. The channel is found to have the following properties: (i) Channel activity occurs in bursts of openings separated by long closed periods. (ii) The current-voltage relationship is nonlinear. Channel current is seen to rectify, with less current flowing at positive than at negative voltages. Rectification may be due to asymmetric block by HEPES/Tris buffers. In 450 mM KCl, 5 mM HEPES/Tris, pH 7.2, the single channel conductance at -40 mV is approximately 160 pS and at +40 mV is approximately 90 pS. (iii) The conductance-concentration relationship follows a simple saturation curve. Half maximal conductance is achieved at a concentration of approximately 1000 mM KCl, and the curve saturates at a conductance of approximately 500 pS. (iv) Reversal potentials interpreted in terms of the Goldman-Hodgkin-Katz equation indicate a Cl: K permeability ratio of 4:1. (v) The channel accepts all of the halides as well as a number of other anions. The following sequence of relative anion permeabilities (in the presence of K+) is obtained: F- less than acetate- less than gluconate- less than Cl- less than Br- less than I- less than NO3- less tha SCN-.(vi) Cations as large as TEA+ are permeant. (vii) Current through the channel is blocked in the presence of DIDS, SITS and ATP, but not by Zn2+.

SUMMARY1. Anion-selective channels from the apical membrane of respiratory epithelia are involved in the secretion of chloride into the airway lumen. In cystic fibrosis (CF) there is an abnormality of phosphorylation-regulated chloride transport in this tissue, whilst a calcium-dependent pathway appears to function normally.2. Using incorporation of apical membrane vesicles into planar phospholipid bilayers, we have characterized the most commonly seen anion-selective channel from sheep tracheal epithelium. and complete block at higher concentrations.7. ATP produced a dose-related reduction in P. with effects at 1 /AM and complete closing at 1 mm. These effects were only seen with addition to the cis chamber.8. The catalytic subunit of protein kinase A, either when incubated with vesicles prior to incorporation into bilayers, or when added directly to either chamber, produced no effect.9. Channels with very similar properties were seen from transfected human tracheo-bronchial cells. 10. Recent whole-cell patch-clamp studies have suggested a distinct calcium-MS 9064 138 C1-CHANNELS FROM SHEEP TRACHEAL EPITHELIUM activated chloride current in secretory epithelia. The described channel has properties in common with this current and may be a candidate for its single-channel basis.

Our research interest is to understand structure/function relationships of membrane proteins, membrane-associated proteins and large macromolecular complexes of biomedical importance. These systems, usually refractory to structural analysis by most methods, are ideally suited for high-resolution investigation using electron cryo-microscopy. We describe our findings on four systems as follows that will be discussed in full detail. 1) Structural studies of cellular immune receptors such as MHC molecules, T cell receptor (TCR) and TCR/MHC complexes have been carried out with recombinant soluble forms of the extra cytoplasmic domain of these glycoproteins. The important role of the membrane bilayer in T cell recognition and antigen presentation has become increasingly obvious with the description of lipid micro domains where these molecules segregate. However, the interactions and orientation of these receptors at the lipid bilayer are unknown. We have used H-2K b , a major-histocompatibility (MHC) class I molecule, and tethered its soluble domain to a lipid bilayer via a surrogate connecting peptide to reveal the disposition of MHC molecule on the membrane surface. We demonstrate that the long axis of the MHC molecule is approximately parallel to the plane of the membrane with the peptide-binding pocket close to the membrane surface. This orientation is orthogonal to the commonly held textbook depiction and potentially has implications in the important interactions with accessory molecules. This result was determined by analysing 4.5Å resolution electron crystallographic projection data from frozenhydrated 2-dimensional crystals. Ionic interactions between the lipid head group and the protein appear to be responsible for this orientation.2) Anthrax toxin (AT), secreted by Bacillus anthracis, is a three-protein cocktail of lethal factor (LF, 90 kD), edema factor (EF, 89 kD) and the protective antigen (PA, 83 kD). PA is a member of a unique family of soluble proteins that form channels in lipid bilayer membranes. Steps in 280