Keyword Search:

This Site Medical Ctr. All of Yale

Clifford L. Slayman Professor of Cellular & Molecular Physiology A.B. 1958 Kenyon College Ph.D. 1963 Rockefeller University

Deconstruction and Modeling of Microbial Ion Transporters

The molecular machines which mediate small-molecule traffic across biological membranes almost all either create or dissipate electric fields within those membranes, and the resulting changes of transmembrane voltage or current can be used not only to monitor the underlying traffic, but also to characterize changes in the molecular structure of the machines themselves. Microorganisms offer a vast resource for such measurements and analysis: because of their abundance and variety, and because many of them maintain very large transmembrane voltages--in the range of 150 to 300 mV (c.f. ~80 mV in animal cells). Such voltages are used to drive the accumulation of nutrients, the expulsion of wastes and toxins, and in some cases the release of useful secretory products.

Our laboratory is presently using electrophysiological techniques (especially patch recording) as the primary assay of ion-transport processes in three different microorganisms: Saccharomyces (baker’s yeast), Neurospora (bread mold), and Candida (a human pathogen); and is using site-directed mutagenesis to manipulate the structures of a variety of membrane proteins: potassium channels (TOK1), potassium trans­porters (TRK1,2) and proton pumps (PMA1). And we are continuing to catalogue surprises, a significant recent one being the ability of certifiable potassium-uptake proteins, ScTrk1p and ScTrk2p in yeast, to mediate large, adventitious chloride currents.

Such behavior can be modeled as a function either of gated channels or of dual-barrier chemical reactions, but the latter coincides especially well with a theoretical atomic structure for the TRK proteins, which features a double hour-glass pathway formed amid an intramembranal cluster of four protein molecules, and permeable principally to chaotropic ions. This general structure has been proposed as an undifferentiated “primitive” anion channel. It may be archetypical for higher eukaryotic transporters which also mediate adventitious chloride currents: especially the excitatory amino-acid and amine transporters in neural tissues.

Figure caption:

Structural model of an assembled tetramer of yeast TRK protein, SpTrk1p (from Schizosaccharomyces). A) Perspective of the mem­brane components of the whole tetramer. White (back­­ground) = stick-figure representation of six transmembrane helices in each of the four monomers. Magenta = clustered loops form the functional K + pathways. Red & Green = clustering of the remaining 2 x 4 trans­mem­brane helices, to form a central pore. B, D) Ribbon diagrams of the eight clustered helices, designated M1d (inner) and M2d (outer), viewed (B) down the axis of the central pore from the intracellular surface of the yeast plasma membrane; and viewed (D) in axial (longitudinal) section from within the plane of the mem­brane. C, E) Space-filling diagrams (H atoms omitted) corres­pond­ing to B,D. Green = neutral amino acids, Red = basic amino acids, Blue = acidic amino acids. Blue balls indicate the putative pathway for chloride transit. Coordinates from H.R. Guy & S.R. Durell (Biophys. J. 77:789, 1999). Drawing by A. Rivetta (Biophys. J. 89:2412, 2005).

Selected publications:

Bertl, A., Bihler, H. Kettner, C., & Slayman, C.L., 1998. Electrophysiology in the eukaryotic model cell, Saccharomyces cerevisiae. Pflügers Archiv Eur. J. Physiol. 436: 999-1013. PMID: 9799419

Zeng, G.-F., Pypaert, M., & Slayman, C.L., 2004. Epitope tagging of the yeast K +-carrier, TRK2, demonstrates folding which is consistent with a channel-like structure. J. Biol. Chem. 279: 3003-3013. PMID: 14570869

Baev, D., Rivetta, A., Vylkova, S., Sun, J.N., Zeng, G.-F., Slayman, C.L., & Edgerton, M., 2004. The TRK1 potassium transporter is the critical effector for killing of Candida albicans by the cationic protein, Histatin 5. J. Biol. Chem. 279:55060-55072. PMID: 15485849

clifford.slayman@yale.edu