Forty-First Annual Meeting March 2-6, 1997 Ernest N. Morial Convention Center New Orleans, Louisiana. Tuesday Symposia and Posters, Part III
The mscL gene from E. coli encodes a peptide of 136 amino acids which forms a homohexameric complex that constitutes a functional mechanosensitive channel (MscL). This channel is thought to sense rapid decreases in environmental osmolarity and respond by release of intracellular small molecules and ions. To study the relationship between the structure and function of this channel, we have randomly mutagenized the mscL gene and isolated mutants that, when expressed, slow or halt growth. These
... gainof-function mutants since the mscL knockout shows no detectable plate phenotype. Thus far, over 20 mutants with single amino acid substitutions have been isolated. The majority and most severe mutations occur between amino acids 13 and 31, indicating the importance of this region in forming a proper channel. This region contains the first part of the first of two predicted transmembrane domains. Characterization of these mutants by whole cell physiology is now allowing us to sort them into different classes. For example, one class of mutants has lost the ability to retain the major osmolyte K+ when the mutated gene is expressed, while another class increases K+ efflux in response to osmotic shock. Preliminary electrophysiological data of mutants tested indicate changes in channel kinetics. Our results identify amiino acid residues that are crucial for the proper functioning of this mechanosensitive channel. These data may also provide helpful information in elucidating the physiological role of the channel. (This study is supported by NIH GM47856) Staphylococcal a-Hemolysin (aHL) is a 34 kDa water soluble protein that forms pores in native and artificial membranes. The pores are believed to form when membrane-bound monomers convert into membrane-embedded oligomers during a multistep process. The oligomers, long thought to be hexamers, were recently shown to be heptamers on the basis of X-ray diffraction and gel shift experiments [Gouaux et al. PNAS 2L 12828 (1994)]. We have adapted a procedure which is frequently used to grow 2D crystals in electron microscopy to prepare aHL-loaded supported bilayers suitable for high resolution Atomic Force Microscopy in solution. Although Fourier transforms of the samples indicate better than 1 nm resolution, the size, shape, and subunit stoichiometry of the membrane-associated oligomers can be radily discemed even without applying image analysis techniques. Complexes of short-chain poly(3-hydroxybutyrate) (PHB; MW-12,000) and inorganic polyphosphate (PPi; MW-5000), extracted from Escherichia coli plasma membranes, form ion channels in planar lipid bilayers that display many of the signal characteristics of calcium channels: voltage-activation, selectivity for divalent over monovalent cations, permeant to Ca+2, Sr+2, Ba+2, and block by La+3, Co+2, Cd+2, and Mg+2 in that order (Reusch et al. 1995. Biophys. J. 69:754). The channel complexes have also been reconstituted from synthetic PHB128 and calcium polyphosphate65 (manuscript in preparation). The channels show a multiple number of substates and unique gating properties. Conductance of the most frequently observed open state is -104 pS with fiequent long openings of the order of several seconds. The channel complexes are impermeable to sodium and potassium. Pure synthetic PHB can form non-selective ion channels only at 100-1000 fold higher weight ratio in lipid (Seebach et al. 1996. Helv. Chim. Acta 79:507). Analysis of open and closed time distributions reveal complex gating kinetics of the channel with multiple open and closed states. The single-channel characteristics in planar lipid bilayers of the biological and synthetic complexes are nearly indistinguishable. Tu-PM-H1O The immunosuppressant agent WIN 17317-3 (WIN) has been reported to selectively block the lymphocyte potassium channel Kvl.3 with a selectivity factor of about 100 fold over other homologous members of the Kvl.x family of channels (Biophys J. 70 A446, 1996). We have examined more closely the channel selectivity of WIN against human Kvl.3 and Kvl.5 channels heterologously expressed in CHO cells as well voltage-gated Na channels. In contrast to previous reports, we have observed that the potency of block (at +10 mV) of WIN against Kvl.3 and Kv1.5 is similar with IC,0 s of 0.1 ILM. Block of both channel types is time dependent suggesting a dependence on channel activation. WIN is also a potent inhibitor of tetrodotoxin-sensitive voltage-gated sodium currents in GH3 cells and CHO cells expressing cloned p1 Na channels. WIN produces both tonic and use-dependent block (at +10 mV) exceeding 90o/. at 3 piM. Following treatment with chloramine-T to remove inactivation, 3 PM WIN was found to produce a time dependent block (>90%/o) of Na currents in GH5 cells. The results suggest that WIN is a potent blocker of both voltage-gated K' and Nae channels which might limit the clinical utility ofthis compound as an immunosuppressant. We investigated the action of verapamil and N-methyl-verapanil (1)575) on the mKvl.3 channel and a mutation derived from the SS/S6 linker, H404T, to obtain structural and functional information about voltage-gated potassium channels. The whole-cell and inside-out configuration of the patch-clump technique wan used to examine the current properties obtained by injection of in vitro transcribed mRNA in RBL cells. Measurements in the wholecell configuration were done in mammalian Ringer's solution with patch-pipettes containing KF. In inside-out patches the bath solution consists of K-aspartate solution with pipettes containing mammalian Ringer's solution. The action of extraor intracellularly applied verapamil on wildtype mKvl.3 currents had two different properties: 1) acceleration of the rate of current decay during depolarizing pulses, and 2) reduction of steady-state peak current. The membrane impermeable D575 affected current through mKvl.3 wildtype channels similar to verapamil only if applied to the intracellular side of the membrane. The H404T mutation in the outer vestibule decreased the ability of extra-and intracellularly applied verapamil and intracellularly applied N-met-verapamil to reduce steady-state peak current about 25-fold, whereas the acceleration of current decay caused by these compounds was nearly unaffected. Substances known to interact with the extracellular site of the channel, like extracellularly applied tetraethylammonium, [TEA+t. or kaliotoxin did not compete with extracellularly applied verapamil on blocking steady-state peak current, wheras intracellularly applied [TEA'], known to interact with the intracellular site of the channel, was able to reduce the effect of extracellularly applied verapanil to block steady-state peak current suggesting competition for a common binding site between [TEA'l] and verapamil. The results from the competition experiments as well as from the mutation in the outer vestibule of mKvl.3 are compatible with the idea that veapamil, if applied extracellularly, pass through the membrane to reach its internal binding site on the mKvl.3 channel. (Supported by a grant from Pfizer Inc, CT and the DFG (Gr 848/4-1)).