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Neuroscience Graduate Program at UCSF

Faculty - Jeff Lansman, Ph.D.

Biophysics of Mechanosensation


Research Description

The mechanisms that allow cells to sense and respond to mechanical forces are the least understood of all known sensory processes. These include the sense of touch, hearing, and proprioception, as well as the ability to sense pressure and shear stresses in blood vessels and other organs. Mechanical forces act at the cell membrane to cause opening of specific ion channels. The coupling of mechanical forces to ion channels embedded in the membrane, however, involves rather complex interactions that depend upon the membrane lipid composition and bulk properties, organization of the subcellular cytoskeleton, and the localization of scaffolding and adaptor proteins that organize and regulate membrane spatial domains. Mechanosensation is an emergent property of a complex membrane-protein interactome.

My lab studies the biophysics of mechanotransduction by ion channels and the role of cytoskeletal and scaffolding proteins in the biophysical processes involved in mechanical force sensing. We use mouse mutants that lack specific components of the dystrophin glycoprotein complex that functions to prevent contraction-induced membrane damage by connecting the intracellular actin network to the extacellular matrix. The mdx mouse lacks the cytoskeletal protein, dystrophin, the absence of which causes human Duchenne muscular dystrophy. We have shown that MS channels in muscle from mdx mice have abnormally long open times that contribute to the high levels of intracellular calcium responsible for muscle death. Our results also show that MS channels in the cytoskeletal mutants are likely heteromeric proteins composed of subunits that can open and close indepedently

1) Biophysical analysis of the muscle mechanosome. Dystrophin-deficient mdx mice have only a very mild skeletal myopathy. Utrophin, a dystrophin homolog, is thought to compensate for the absence of dystrophin in the mdx mouse. Double knockout mice lacking both proteins have severe muscle pathology similar to humans with Duchenne dystrophy. Recently, we have shown using the dystrophin/utrophin DKO greatly prolonged single-channel openings (Tan & Lansman, 2014). On a time scale much longer than that of individual activations, MS channel opening in DKO muscle is periodic, rather than stochastic, and often involves simultaneous opening of more than one channel (Lansman, 2015).  We have proposed a model in which dystrophin/utrophin acts as a gating spring to maintain the structure of local membrane domains; disruptions in local domain structure produce variations in channel opening. Present experiments include: 1) Identification of the subunit composition of heteromeric TRPV4-containing MS channels in skeletal muscle; 2) Deletion of other components of the dystrophin-glycoprotein complex using RNA interference to determine their role in mechanotransduction; 3) Reconstitution of a simple mechanotransduction system in CHO cells; and 4) Investigation of elementary mechanosome events (EMEs) and their relation to calcium signaling in muscle.

2) Pharmacology of MS channels.  We have found that specific TRP channel inhibitors block MS currents in skeletal muscle. We have discovered that some compounds, such as aminoglycosides and ruthenium red, act as “partial antagonists,” in that the cause partial occlusion of the channel pore.  Partial antagonists are of interest since they would block pathological calcium entry, while allowing physiological calcium fluxes.  We also maintain an interest in neurotoxins derived from marine snails of the Conus species. There are 500-700 species of cone snail. Cone snails use complex venoms to capture prey and venoms consist of many small peptides each encoded by separate genes. In collaboration with the Biodiversity Initiative and the University of Papua New Guinea, we are identifying novel Conus species in the remote north coastal region of Oro Province, with the goal of developing novel therapeutics for skeletal and cardiac myopathies. 

3) Mechanically-induced, exo- and endocytosis in muscle. We have detected transient fusion of vesicles with the surface membrane in single-channel recordings from membrane patches that may be related to membrane repair mechanisms following mechanical disruption of the skeletal muscle membrane.  We use measurements of membrane capacitance to detect discrete changes in membrane surface area in cell-attached patches to understand the repair process in the different mouse models of muscular dystrophy. These exocytotic events may also involve vesicles carrying glucose transporters to the membrane and understanding their trafficking in real time is relevant to understanding insulin action in diabetes.

Voltage-gated calcium channels
We have a long-standing interest in neuronal voltage-gated calcium channels and their role in neural development and degeneration. Neurons in the brain possess many different types of voltage-gated calcium channels. In earlier studies, I identified the L- and T-type calcium channels in cardiac muscle.  A major focus of our work is to understand the complex functional diversity of L-type channels in neurons, which are involved in many functions, including gene expression, neuronal survival, growth, motility, development and cell death.  Our initial work characterized the whole-cell and single-channel Ca2+ currents in central neurons using granule cells cultured from mouse cerebellum. The L-type current in granule cells is blocked by dihydropyridines, increased by intracellular cyclic AMP, and inhibited by hydrolysis-resistant cyclic GTP analogues. Subsequent studies revealed functionally distinct L-type channels: type 1 channels that re-open at negative membrane potentials during recovery from inactivation; and type 2 channels, in which channel openings during a voltage clamp step were prolonged by a pre-pulse to a positive voltage. The analysis of L-type channels that reopen after repolarization showed the mechanism involved dissociation of an intracellular blocking molecule at negative voltages. The discovery of reopening and facilitating L-type channels expanded the physiological range over which L-type channels control Ca2+ entry in neurons
There are two ongoing projects:

1) Voltage-gated calcium channels and congenital neurological disease.  Mutations in the P/Q-type calcium channel alpha 1A subunit gene cause migraine and cerebellar ataxia in humans. In mice, the leaner mutation occurs at a splice site in the alpha 1A subunit gene and produces absence epilepsy and cerebellar ataxia and degeneration. Patch clamp recordings from cerebellar granule cells from leaner mice, show the selective reduction of a specific class of Q-type calcium channel. Reduction in Q-type channels leads to homeostatic changes in the expression of L-type channels and NMDA receptor channels. These changes in ion channel expression are of interest since granule cells selectively undergo early and massive cell death in the leaner cerebellum. Whole-cell patch clamp methods are used to understand the compensatory changes in excitability. Optogenetic activation and inhibition of specific cerebellar neurons is used to understand the changes in cerebellar circuit behavior that contribute to granule cell death during early development.

2) Store-coupled calcium entry pathways in neurons. In addition to conventional electrical excitability, neurons possess a mechanism for intracellular excitability that uses a regenerative intracellular calcium mobilizing system.  A major question is the source for refilling intracellular calcium stores to maintain regenerative intracellular calcium signals.  In cerebellar granule cells from mice, we have found that group I metabotropic glutamate receptors produce an IP3 and protein kinase C-independent facilitation of L-type current. Facilitation of the L-type current is coupled to the activation of intracellular caffeine- and ryanodine-sensitive calcium stores in the absence of metabotropic glutamate receptor activation. We believe that activation of intracellular calcium stores is directly coupled to movement of the L-type channel voltage sensor, analogous to excitation-coupling in skeletal muscle.  We use whole-cell recordings with combined photometric measurements of fura-2 fluorescence to measure intracellular calcium ion levels with millisecond time resolution and flash photolysis to release intracellularly-trapped caged calcium and other signaling molecules to probe mechanisms of intracellular calcium signaling.

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Selected Publications

Link to Publications via PubMed

Lansman, J.B. (2007)  Mechanosensitive ion channels in dystrophic muscle.  In, “Current Topics in Membranes” 59: 467-484  ed, Hamill, O.P.

Vasquez, I., Tan, N., Boonyasampant, M, Koppitch, K., and Lansman, J.B. (2012) Partial opening and subconductance gating of mechanosensitive ion channels in dystrophic muscle. Journal of Physiology 590.23:6167-6185

Ho, T.C., Horn, N.A., Huyhn, T., Kelava, L. and Lansman, J.B. (2012) Evidence TRPV4 contributes to mechanosensitive ion channels in mouse skeletal muscle fibers. Channels 6(4):1-9

Tan, N. and Lansman, J.B. (2014) Utrophin regulates modal gating of mechanosensitive ion channels in dystrophic skeletal muscle Journal of Physiology 592.15:3303-3323

Lansman, J.B.  (2015) Utrophin suppresses low frequency oscillations and coupled gating of mechanosensitive ion channels in dystrophic skeletal muscle. Channels May 4;9(3):145-60

Lansman, J.B. (2015) Elementary mechanosome events and the conductance variability of mechanosensitive ion channels in dystrophic skeletal muscle. (submitted)

   

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Jeff Lansman, Ph.D.



Email

jeff.lansman@ucsf.edu

Phone

415-476-1322

Office Address

UCSF MC 0450 513 Parnassus
513 Parnassus Avenue, Room S-1272
San Francisco, CA 94143

Other Websites

PIBS Website

Cellular and Molecular Pharmacology