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Neuroscience Graduate Program at UCSF
Biophysics of mechanosensitive and voltage-gated calcium channels
Calcium ions act as a signal inside cells to turn on many essential processes, such as secretion of neurotransmitters, regulation of gene expression, axon outgrowth, cell motility, and cell death. We are interested in how specific types of calcium channels contribute to normal and pathophysiological processes in neurons and other excitable cells. We use electrophysiological and optical methods to study biophysical mechanism controlling calcium channel activity and intracellular calcium accumulation in single cells. Our work falls into two general areas:
Mechanosensitive ion channels. The mechanisms that allow cells to sense and respond to mechanical forces are the least understood of all known sensory mechanisms. Mechanical forces act at the membrane to cause opening of specific ion channels. The coupling of mechanical forces to ion channels embedded in the membrane involves rather complex interactions that depend upon the membrane lipid composition, the organization of the subcellular cytoskeleton, and the shape of the membrane surface. We use a simple genetic approach to study the biophysical properties of mechanosensitive ion channels in skeletal muscle from mice with mutations that cause a loss of specific cytoskeletal and other membrane proteins involved in mechanical force transduction. One model, the mdx mouselacks the cytoskeletal protein, dystrophin. An absence of dystrophin leads to degeneration of skeletal and cardiac muscle in human Duchenne muscular dystrophy. We have shown that mechanosensitive ion channels in muscle from mdx mice have abnormally long open times that contribute to the high levels of intracellular calcium responsible for muscle death. We are interested in both the biophysical mechanisms of mechanical transduction by ion channels and the role of these channels in degenerative diseases of skeletal and cardiac muscle.
Current projects:
1) Molecular identity of the mechanosensitive channel in muscle. The molecular identity of vertebrate mechanosensitive channels remains unknown. They are likely to be heteromeric channels formed from several gene products. We are using transgenic mice and RNA interference to delete specific members of the TRP (transient receptor potential) family of cation channels to identify the gene products that contribute to the biophysical properties of single mechanosensitive channels.
2) Analysis of the gating mechanisms of mechanosensitive channels in skeletal muscle from mice with cytoskeletal abnormalities. We use hidden Markov models to model the conductance transitions of single mechanosensitive channels. This analysis constitutes a complete description of the channel’s kinetic behavior and provides rate constants for all conformational changes of the channel protein. The goal is to understand the role of membrane mechanical properties in the mechanism of mechanotransduction by ion channels.
3) Analysis of ion channels in dystrophin/utrophin double knockout mice. Utrophin is a dystrophin homolog that is thought to compensate for an absence of dystrophin in the mdx mouse. The double knock out mice have severe muscle pathology more similar to humans with Duchenne dystrophy than the mdx mouse. We are analyzing the biophysical properties of mechanosensitive channels in the DKO mice and the developmental changes in channel expression in relation to disease pathogenesis.
4) Studies of the biophysical basis of drug interaction with mechanosensitive channels. We have found that TRP ion channel inhibitors cause discrete blocking events in recordings of single-channel activity from skeletal muscle. We are analyzing the detailed kinetic mechanisms of the interaction between a single drug molecule and single channel.
5) Analysis of mechanically-induced, non-secretory exo- and endocytosis in normal and dystrophic muscle. We have detected transient fusion of vesicles with the surface membrane in single-channel recordings from cell-attached 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 cytoskeletal mutants.
6) Drug discovery. In collaboration with the Department of Pharmacology, University of Papua New Guinea, we are identifying novel neurotoxins from cone snails that specifically block mechanosensitive ion channels and other TRP family members. The goal is develop novel therapeutics for skeletal and cardiac myopathies. Cone snails use complex venoms to capture prey and venoms consist of many small peptides each encoded by a separate gene. There are some 500 species of cone snail. Venom from species in the remote coastal regions of Papua New Guinea have not been studied. I have developed relationships with klans in this region, including the Yari-Yari in the Tufi peninsula, who have taught me about local species.
Neuronal voltage-gated calcium channels
Neurons in the brain possess many different types of voltage-gated calcium channels. They are generally closed in resting neurons, but open transiently in response to changes in the membrane potential and contribute the wide variety of electrical impulses in the brain. Many years ago, we first identified the L- and T-type calcium channels in cardiac muscle. A major focus of our work now is understanding the complex functional diversity of the L-type channel in neurons which is involved in many functions, including gene expression, neuronal survival, development and death. There are currently two projects in this area.
1) Voltage-gated calcium channels and neurological disease: Mutations in the P/Q-type calcium channel alpha 1A subunit cause migraine and cerebellar ataxia in humans. In mice, the leaner mutation occurs at a splice site in the P/Q-type channel 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 is associated with homeostatic changes in the expression of other types of ion channels responsible for electrical excitability and synaptic transmission, particularly the L-type channel which is up-regulated two-fold in mutant granule cells. The changes in ion channel expression are of interest since, in the leaner cerebellum, granule cells selectively undergo early and massive cell death. Current experiments are focused on elucidating the compensatory changes in excitability that occur in neurons possessing mutant P/Q-type channels and the mechanisms that contribute to the wide spread cell death during early development of the granule cell population.
2) Store-coupled calcium entry pathways in neurons. In addition to conventional electrical membrane excitability, neurons possess a second mechanism for intracellular excitability that utilizes a regenerative calcium mobilizing system. A major question is the source for refilling intracellular calcium stores to maintain regenerative intracellular calcium signals. In cultured 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. The facilitation of the L-type current is coupled to the activation of intracellular caffeine- and ryanodine-sensitive calcium stores in the absence of mGluR 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 are using whole-cell recordings with combined photometric measurements of fura-2 fluorescence to measure intracellular calcium ion levels and flash photolysis of intracellularly-trapped caged calcium-releasing compounds to determine the coupling mechanism between intracellular stores and L-type calcium channels and whether this pathway provides a mechanism for store refilling during repetitive electrical activity.
Link to Publications via PubMed
Winegar, B., Haws, C.M. and Lansman, J.B. (1996) Subconductance block of mechanosensitive ion channels in skeletal muscle fibers by aminoglycoside antibiotics. J. Gen. Physiol. 107:433-443
Chavis, P., Fagni, L, Lansman, J.B, and Bockaert, J. (1996) Functional coupling between ryanodine receptors and L-type calcium channels in neurons. Nature 382:719-722
Franco-Obregon, A., and Lansman, J.B. (2002) Changes in mechanosensitive channel gating following mechanical stimulation in skeletal muscle myotubes from the mdx mouse. Journal of Physiology 539.2:391-407
Lansman, J.B. and Franco-Obregon, A. (2005) Stretch-inactivated channels in skeletal muscle. In, “Mechanosensitivity of Cells and Tissues.” Ed, Kamkin, A. Moscow: Academia Press
Lansman, J.B. and Franco-Obregon, A. (2006) Mechanosensitive ion channels in skeletal muscle: a link in the membrane pathology of muscular dystrophy. Clin. Exp. Physiol. Pharmacology 33(7): 649-56
Lansman, J.B. (2007) Mechanosensitive ion channels in dystrophic muscle. In, “Current Topics in Membranes” 59: 467-484 ed, Hamill, O.P.
Vasquez, I, Boonaypasant, M, Koppitch, K, and Lansman, J.B. A critical period for mechanosensitive and leak channel expression in skeletal muscle fibers from the mdx mouse J. Physiol. (in press)
Ho, T., Horn, N., Huynh, T,, Kelava, L, and Lansman, J.B. An analysis of the specificity and mechanism of the block of mechanosensitive channels in skeletal muscle by TRP channel antagonists. J Gen Physiol. (in press)
Tan, N and Lansman. Ion channel abnormalities in dystrophin-utrophin double knock-out mice: comparison with the mdx mouse J. Physiol (submitted)
Jeff Lansman, Ph.D.
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415-476-1322
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