Lukas K. Buehler

 

CURRICULUM VITAE

 

 

POSITIONS:

 

1997 - present R.W. Johnson Pharmaceutical Research Institute

1996 - present Adjunct Lecturer in Biology, University of California, San Diego

1994 - 1995 Research Associate at The Scripps Research Institute, Dept. of Chemistry, with M.R. Ghadiri.

1992 - 1994 Research Associate at The Scripps Research Institute, Dept. of Cell Biology, with N.B. Gilula.

1989 - 1992 Post-doctoral Fellow at the University of California, San Diego, Depts. of Physics, with M. Montal.

 

EDUCATION:

 

1986 - 1989 Dr. Phil. II (Ph.D.) in Biochemistry at the University of Basel, Basel, Switzerland, comparing ion channel properties of different porins of E.coli B and K-12 strains for their lipid and glycolipid dependence in planar lipid bilayers.

1987 EMBO lecture course on cell signaling and signal transduction, Pavia, Italy.

1985 EMBO Short Term Fellowship in the laboratory of Dr. H.Schindler, Institute of Biophysics, University of Linz, Linz, Austria, aimed at the study of the in vitro synthesis (with R. Zimmermann, University of Munich, Germany) and membrane insertion of bacterial (pro-)porin into planar lipid bilayers.

1980 - 1985 Diploma 'Biology II' in Biophysics at the University of Basel, Switzerland. Diploma thesis on the effect of Mg++ on the activity of porin from E.coli B

1976-79 Gymnasium (high school), Liestal, Switzerland

 

CONSULTING

 

1997 The Scripps Research Institute; consulting service for Dr. M. Reza Ghadiri in the general area of electrophysiology and single channel ion conductance measurements

 

 

 

 PUBLICATIONS

 

Research Papers:

Buehler, L.K., J.R. Granja, and M.R. Ghadiri. Quantized conductance in self-assembled peptide transmembrane nanotubes. submitted

Buehler, L.K. Porin and Gap Junction Channels: Analogous function from different structures. in preparation

Clark, T. D., L.K. Buehler, and M.R. Ghadiri. 1998. Cyclic b -peptide nanotubes as artificial transmembrane ion channels. J.Am.Chem.Soc. 120:651-656.

Falk, M.M, L.K. Buehler, N.M. Kumar, and N.B. Gilula. 1997. Cell-free synthesis and regulated assembly of connexins into functional gap junction membrane channels. EMBO J. 16:2703-2716.

Buehler, L.K., K.A. Stauffer, N.B. Gilula, and N.M. Kumar. 1995. Single channel behavior of recombinant b 2 gap junction protein reconstituted into planar lipid bilayers. Biophys.J. 68:1767-75.

Ghadiri, M.R., J.R. Granja, and L.K. Buehler. 1994. Self assembling peptide nanotubes designed to function as transmembrane ion channels. Nature 369:301-4.

Oblatt-Montal, M., L. Buehler, T. Iwamoto, J.M. Tomich, and M. Montal. 1993. Synthetic peptides and four helix bundle proteins as model systems for the pore-forming structure of channel proteins. I. Transmembrane segment M2 of the nicotinic cholinergic receptor channel is a key pore lining structure. J.Biol.Chem. 268:14601-7.

Buehler, L.K. and J.P. Rosenbusch. 1993. Single channel behavior of matrix porin of E.coli. Biochem.Biophys.Res.Comm. 190:624-9.

Buehler, L.K., S. Kusumoto, H. Zhang, and J.P. Rosenbusch. 1991. Plasticity of E.coli porin channels: dependence of their conductance on strain and lipid environment. J.Biol.Chem. 266:24446-50.

 

Abstracts/Correspondence/Books :

Buehler, L.K. J.R. Granja, and M.R. Ghadiri. 1995. Permeation properties of self-assembling peptide nanotubes that form ion channels in planar lipid bilayers. 39th Annual Biophysical Soc. Meeting. Biophys.J. 68:A371

Buehler, L.K. 1994. Gap junctions and intracellular communications. [letter, comment]. Science 265:1018-9; discussion 1019-20.

Buehler, L.K., N.M. Kumar, and N.B. Gilula. 1993. Reconstitution of b 2 gap junction protein into planar lipid bilayers. Abstract of the 11th International Biophysics Congress, 25-30 July, in Budapest, Hungary.

 

General Interest :

Buehler, L.K. America's (and the world's) scientists. (Opinion) San Diego Union Tribune G-3, July 6, 1997.

Buehler, L.K. Nur Rhetorik (Just rhetoric) Die Weltwoche, February 20, 1997 (German language newspaper).

 

 

RESEARCH EXPERIENCE

 

Research

In the laboratories at the R.W.Johnson Pharmaceutical Research Institute, and the Scripps Research Institute with M.R. Ghadiri and N.B. Gilula, I organized and assembled electrophysiology assays for patch-clamp and double-electrode voltage clamp experiments and reconstitution assays for single channel recordings of proteins and pore forming peptides in planar lipid bilayers (BLMs):

 

Challenge

Approach

Result

______________________________________________________________________

 

Develop drug screening assay for novel ion channel and receptor proteins

electrophysiology: use Xenopus oocytes and mammalian cell lines as heterologous expression system and establish robust ion-uptake assays useful for high throughput screening in multi-array plate assays;

characterization of acid sensitive Na-channel from dorsal root ganglion cells in Xenopus oocytes; characterization of a novel 5HT3 serotonin subtype receptor in Xenopus oocytes;

training of senior scientist in voltage- and patch-clamp experiments and current recording analysis; test Cl- channels in neuroblastoma cell lines using fluorescence dyes; SPQ, MQAE (cyto-fluorometer)

test Ca-channels in neuroblastoma cell lines with Fluo3, Ca-green (FLIPR)

 

 

 

evaluation of novel high throughput screening assays for ion channels

establishing contacts with research laboratories

evaluated silicon chip based biosensors using impedance measurements of whole cells (H. Vogel, EPFL, Lausanne, Switzerland; G. Kovacs, Stanford);

evaluate FlaSh: protein based fluorescence sensor for membrane potentials using a hybrid K-channel-GFP construct (M.Siegel, Caltech)

 

 

 

Characterize single channel behavior and selectivity of peptide (with M.R. Ghadiri)

Establish reconstitution assay and single channel recording using the patch-pipette technique and Montal-Mueller technique to form synthetic planar lipid bilayers

Characterize single channel activity of cyclic octapeptide nanotubes (Ghadiri et al., 1994) and analyze conductance variability and selectivity of this peptide self-assembly system (Buehler et al., in prep.); train two graduate students in electrophysiology techniques and analysis

 

 

 

Characterize gap junction hemi-channels (with N.B. Gilula)

Establish reconstitution assay and single channel recording using the patch-pipette technique and Montal-Mueller technique to form synthetic planar lipid bilayers;

double-cell patch clamp recordings with gap junction proteins expressed in BHK cell lines; determine electrical coupling of paired cells and correlate with dye coupling (carboxy-fluorescent)

Characterize single channel activity of Cx26 (Buehler et al., 1995), Cx43 (unpublished results), and in vitro synthesized Cx26, 32, and 43 channels (Falk et al., 1997), as well as a 7 subunit of the neuronal nicotinic Acetylcholine receptor (unpublished results)

 

 

 

 

Biochemistry, Biophysics. and Molecular Biology of Membrane Proteins

Experimental experience in expressing, isolating, purifying, and reconstituting membrane proteins. Cell systems used are E.coli, baculovirus/insect cell cultures and Xenopus oocytes. Experience with an in vitro protein biosynthesis assay using either E.coli cell free lysate (porins) or rabbit reticulocyte lysate (gap junction proteins and neuronal nAChR alpha7 subunit) including the preparation of dog pancreatic microsomes for translocation assays. In vitro transcription of mRNA for cell free and oocyte expression of ion channels. Purification of membrane proteins using ion exchange and gel filtration chromatography (including FPLC), reconstitution of the proteins into membranes (vesicles and/or planar membranes) using non-ionic detergents. Isolation of phospholipids from native sources.

 

Functional Analysis of Ion Channels

Single channel and macroscopic current recordings and analysis, subunit dependence, and pharmacology of serotonin receptors and acid sensing channels in Xenopus oocytes. Reconstitution of bacterial porins into planar lipid bilayers to compare the function of two osmo-regulated, alternately expressed porins OmpF and OmpC. Characterization of the plasticity of their unit pore conductance in dependence of the lipid environment. Identification of conditions to study single pore behavior (in distinction to the triple pore) that induces strong, asymmetric rectification at voltage ranges of physiologically important Donnan-potentials across the outer membrane of E.coli.

 

Functional Analysis of Pore Forming Peptides (model channels)

1) Characterization of the ion channel activity of synthetic peptides that mimic the pore forming structure of the Torpedo nicotinic acetylcholine receptor. Tethered 4 a -helical bundles were reconstituted into planar lipid bilayers and their channel properties compared with those of the authentic receptor channels. In addition, the peptide activity was compared with anti-microbial peptides Magainin from frog skin.

 

2) Cyclic peptides (nanotubes) that stack to form channels of defined diameters in hydrophobic solvents (but stay monomeric in aqueous phase) were reconstituted into planar lipid bilayers and the resulting channel characteristics (conductance, selectivity, voltage dependence) are being determined

 

Theoretical Analysis of Ion Channels

1) The gap junction channel serves as a model system to understand the influence of the relative distribution of fixed charges within the channel on conductance properties and transmembrane potential profiles in its two configurations as either full channel (spanning two membranes) or hemi-channel (spanning a single membrane). In collaboration with R.S.Eisenberg and D.P.Chen at the Rush Medical College, Dept. of Physiology, Chicago, IL.

 

2) Understanding structure-function relationships of the voltage gating properties of porins and gap junction channels. Both channels show indistinguishable voltage-gating behavior, although their structures are non related, one being a -helical (GJ) and the other having a b -barrel conformation. Evolutionary aspects of the relation between structural differences and sequence dissimilarities leading to identical functional properties are being addressed.