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Department of Physiology and Biophysics

University of Colorado Department of Physiology and Biophysics
 

Kurt Beam, PhD

Professor


Department of Physiology & Biophysics

UCD Anschutz Medical Campus
RC-1 North Tower, P18-7132
Mail Stop 8307
Tel (303) 724-4542
Fax (303) 724-4501

E-mail: kurt.beam@ucdenver.edu


A major focus of my laboratory is excitation-contraction coupling in skeletal muscle, which depends upon functional interactions between voltage-gated calcium channels (dihydropyridine receptors, DHPRs) in the transverse tubular (TT) membrane and calcium release channels in the sarcoplasmic reticulum (SR), as indicated schematically in Fig. 1.  To study the interactions of these proteins in a near-native environment, we use cultured muscle cells which are genetically null for DHPR subunits and/or RyR1.  These muscle cells are injected with cDNA encoding native or altered calcium channels, followed by whole-cell patch clamp measurement of membrane currents, and photometric measurements of intracellular calcium.  The work has shown that there is specialized, bi-directional signaling between the DHPR and RyR1 and identified regions of both the DHPR and RyR1 that are important for this signaling.  However, these techniques do not establish whether the functionally important regions represent actual sites of physical contact between the DHPR and RyR1.  For this reason, we have implemented approaches that could allow us to identify such interaction sites within intact muscle cells.

One approach we currently use is to express cDNAs which encode calcium channels with fluorescent protein inserted at targeted sites, (Link to movie) which permits the use of fluorescence resonance energy transfer (FRET), a technique that provides a sensitive measure of whether proteins are close (<10 nm) to one another.  These experiments indicate that RyR1 may come into close apposition with two regions of the DHPR.

A second approach makes use of the expression of cDNAs which encode calcium channels in which the biotin acceptor domain (“BAD”) is attached to the targeted sites, resulting in the enzymatic addition of biotin at those sites.  Subsequent exposure to fluorescent streptavidin reveals whether the accessibility of this large molecule to the site is influenced by the presence or absence of RyR1 (the results are in overall good agreement with the approach of using fluorescent proteins).  In collaboration with Drs. Claudio Perez and Paul Allen, we are producing transgenic mice expressing DHPRs with site-directed biotinylation (Fig. 2).  These mice allow us to purify DHPRs for single particle reconstruction, to carry out site-directed cross-linking, and to use gold particles attached to streptavidin to directly assess the orientation of specific DHPR sites with respect to RyR1.

The long term goal of our work is to determine regions of close proximity between the proteins (i.e., potential sites of interaction) within the TT/SR junction and also to determine whether those regions undergo dynamic changes during physiological function.   The sorts of approaches we are developing may be of general use for analyzing the molecular machinery underlying important functions in many cell types.


Selected Publications

Tanabe, T., Beam, K.G., Powell, J.A. and Numa, S. Restoration of excitation-contraction coupling and slow calcium current in dysgenic muscle by dihydropyridine receptor complementary DNA.  Nature 336:134-139, 1988. pdf

Tanabe, T., Beam, K.G., Adams, B.A., Niidome, T. and Numa, S.  Regions of the skeletal muscle dihydropyridine receptor critical for excitation-contraction coupling.  Nature 346:567-569, 1990. pdf

Mynlieff, M. and Beam, K.G. Characterization of voltage-dependent calcium currents in mouse motoneurons.  J. Neurophysiol. 68:85-92, 1992. pdf

Takekura, H., Bennett, L., Tanabe, T., Beam, K.G. and Franzini-Armstrong, C.F.  Restoration of junctional tetrads in dysgenic myotubes by dihydropyridine receptor cDNA.  Biophysical J. 67:793-803, 1994. pdf

Dirksen, R.T. and Beam, K.G. Single calcium channel behavior in native skeletal muscle.  J. Gen. Physiol., 105:227-247, 1995. pdf

Nakai, J., Dirksen, R.T., Nguyen, H.T., Pessah, I.N., Beam, K.G. and Allen, P.D.  Enhanced dihydropyridine receptor channel activity in the presence of ryanodine receptor.  Nature 380:72-75, 1996. pdf

Lorenzon, N.M., Lutz, C.M., Frankel, W.N., and Beam, K.G.  Altered calcium channel currents in Purkinje cells of the neurological mutant mouse leaner.  J. Neurosci. 18:4482-4489, 1998. pdf

Wilkens, C.M., Nicole Kasielke, N., Bernhard E. Flucher, B.E., Beam, K.G. and Grabner, M. Excitation-contraction coupling is unaffected by drastic alteration of the sequence surrounding residues L720-L764 of the a1S II-III loop. Proc. Natl. Acad. Sci. U.S.A., 98:5892-5897, 2001. pdf

Proenza, C., O’Brien, J.J., Nakai, J., Mukherjee, S., Allen, P.D. and Beam, K.G.  Identification of a region of RyR1 that participates in allosteric coupling with the a1S (CaV1.1) II-III loop. J. Biol. Chem.277:6530-6535, 2002. pdf

Protasi, F., Paolini, C., Nakai, J., Beam, K.G., Franzini-Armstrong, C., and Allen, P.D.  Two separate regions of RyR1 participate in the functional and structural interactions with DHPRs that allow skeletal type e-c coupling.  Biophys. J. 83:3230-3244, 2002. pdf

Papadopoulos, S, Leuranguer, V., Bannister R. and Beam, K.G. Mapping sites of potential proximity between the DHPR and RyR1 in muscle using a CFP-YFP tandem as a FRET probe. J. Biol. Chem., 279:44046-44056, 2004. pdf

Lorenzon, N.M, Haarmann, C.S., Norris, E., Papadopoulos, S. and Beam, K.G. Metabolic biotinylation as a probe of supramolecular structure of the triad junction in skeletal muscle.  J. Biol. Chem., 279:44057-44064, 2004. pdf

Cherednichenko, G., Hurne, A.M., Fessenden, J.D., Lee, E.H., Allen, P.D., Beam, K.G. and Pessah, I.N.  Conformational activation of Ca2+ entry by depolarization of skeletal myotubes.  Proc. Natl. Acad. Sci. U.S.A. 101:15793-8, 2004. pdf

Leuranguer, V., Papadopoulos, S, and Beam, K.G.  Organization of calcium channel b1a subunits in triad junctions in skeletal muscle.  J. Biol. Chem. 281: 3521-3527, 2006. pdf

Sheridan, D.C., Takekura, H. Franzini-Armstrong, C., Beam, K.G., Allen, P.D. and Perez, C.F. Bi-directional signaling between calcium channels of skeletal muscle requires multiple, direct and indirect, interactions.  Proc. Natl. Acad. Sci. U.S.A. 103: 19760-19765, 2006. pdf


Latest Publications in PubMed


​​Hicham Bichraoui, Ph.D.
Postdoctoral Fellow

Email: Hicham.Bichraoui@ucdenver.edu​

Ong Moua
Professional Research Assistant​

​E-mail: Ong.Moua@ucdenver.edu

Study the cycling of neurotransmitter-containing synaptic vesicles in nerve-muscle synapses during nerve activity.​

Alex Polster
Postdoctoral Fellow

E-mail: Alexander.Polster@ucdenver.edu

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