Head of Mechanosensory Biophysics Laboratory
Molecular Cardiology and Biophysics Division
| Telephone: | +61-2-9295 8743 |
| Fax: | +61-2-9295 8770 |
| Email: | b.martinac@victorchang.edu.au |
Research Focus
The molecular basis of sensing odorants, hormones and neurotransmitters is well understood. In comparison, little is known about how living cells detect mechanical forces acting upon them. What is known however is that at the basis of sensory inputs in all living organisms, including mechanosensory transduction, are integral membrane proteins such as ion channels that involved in a large variety of physiological functions and currently constitute about 60% of pharmaceutical drug targets. Structural studies of these proteins using traditional methods such as X-ray crystallography or NMR is quite challenging, and even when successful, provides only a snap-shot view of the proteins. Moreover, in many of these methods, the proteins have to be removed from their natural lipid environment and it can be difficult to trap the protein in specific functional states. Therefore, spectroscopic methods such as electron paramagnetic resonance (EPR) and FRET spectroscopy are important for revealing conformational dynamics and structural mechanisms of protein function as they function in natural lipid environments.
As the primary molecular transducers of mechanical force in living cells mechanosensitive (MS) ion channels have been implicated in various mechanosensory physiological processes in living cells. Testing the key hypothesis that mechanical force is delivered through the lipid bilayer to MS ion channels in membranes of prokaryotic and eukaryotic cells is central to the research in our laboratory. By studying structure and function of mechanosensitive channels of prokaryotes and eukaryotes the research in our laboratory is aiming to elucidate the general physical principles underlying the biophysics and physiology of mechanosensory transduction using a multidisciplinary approach including molecular biology, protein biochemistry, patch clamp recording and spectroscopic techniques
Bacterial mechanosensitive channels
Mechanosensitive (MS) channels are an important class of membrane proteins that function as mechano-electrical switches sensing the physical state (i.e. pressure, tension) of cellular membranes and are involved in mechanosensory processes such as touch and pain sensation, hearing, blood pressure control, micturition, cell volume regulation, tissue growth or turgor control. Specifically, the bacterial MS channels function as emergency release valves that respond to sudden hypo-osmotic changes in environmental osmolarity, by opening large, fairly non-selective pores to relieve membrane tension. Experimental work over the years have firmly established that bacterial MS channels directly sense membrane tension in the lipid bilayer caused by external mechanical force applied to cell membrane.
Among the bacterial MS channels, the best characterized are the channels of large conductance, MscL? which opens a pore with a conductance of ~3 nS; and small conductance, MscS, which opens a pore of ~1 nS conductance. Both MscL and MscS are homo-multimeric proteins ? heptamer in case of MscS and pentamer in case of MscL, whose 3D structures have been obtained by X-ray crystallography and which have been further characterized by biochemical, electrophysiological, spectroscopic studies and theoretical modeling (Fig 1). These studies indicate that both proteins undergo large scale conformational changes in response to bilayer tension.
Fig. 1. Bacterial MS channels. (A) The structure of the pentameric MscL channel (left) and a channel monomer (right) from M. tuberculosis according to the 3D structural model. (B) A current trace of a single MscL channel reconstituted into azolectin liposomes (w/w protein/lipid of 1:2000) recorded at +30 mV pipette potential. The channel gated more frequently and remained longer open with increase in negative pressure applied to the patch-clamp pipette (trace shown below the channel current trace). (C) 3D structure of the MscS homoheptamer (left) and a channel monomer (right) from E. coli. (D) Current traces of two MscS channels reconstituted into azolectin liposomes (w/w protein/lipid of 1:1000) recorded at +30 mV pipette voltage. Increase in pipette suction (trace shown below the channel current trace) caused an increase in the activity of both channels. C and On denote the closed and open state of the n number of channels.
Eukaryotic mechanosensitive channels
MS ion channels are also found in membranes of a variety of eukaryotic cells. Touch, hearing, proprioception, gravitropism and control of cellular turgor are examples of MS channel involvement in mechanosensory transduction in eukaryotic cells. Despite much electrophysiological information, molecular characterization of the MS channel role in mechanotransduction in eukaryotes has been slow compared with the progress made in the research on bacterial MS channels. Recent evidence however, suggests that similar to prokaryotic channels eukaryotic MS ion channels can also be gated by the bilayer mechanism In that regard TRP-type MS channels belonging to phylogenetically diverse familes of ion channels, are of particular interest to research in our laboratory since they seem to play a crucial role in muscular dystrophy, polycystic kidney disease as well as in cardiac arrythmias. The research on molecular principles of mechanotransduction is a research area of fundamental importance because better understanding the mechanisms of mechanosensation at a structural level is likely to assist in understanding the pathology of diseases of which malfunction of MS channels may be a cause.
Lab Members:
Paul Rohde, BSc MMedSc (Laboratory Manager)
Evgeny Petrov, PhD (Postdoctoral Fellow)
Andrew Battle, PhD (Postdoctoral Fellow)
Takeshi Nomura, PhD (Postdoctoral Fellow)
Charles Cranfield, PhD (Postdoctoral Fellow)
Maryrose Constantine, BSc (Research Assistant)
Selected Publications:
Martinac, B., Buechner, M., Delcour, A.H.., Adler, J. & Kung, C. Pressure-sensitive ion channel in Escherichia coli. Proc. Natl. Acad. Sci. USA 84: 2297-2301, 1987.
Gustin, M. C., Zhou, X. L., Martinac, B. and Kung, C. A mechanosensitive ion channel in the yeast plasma membrane. Science 242: 762-765, 1988.
Martinac, B., Adler, J. & Kung, C. Mechanosensitive ion channels of E. coli activated by amphipaths. Nature 348: 261-263, 1990.
Sukharev, S.I., Blount, P., Martinac, B., Blattner, F.R., & Kung, C. mscL alone encodes a functional large-conductance mechanosensitive channel in E. coli. Nature, 368: 265-268, 1994.
Hamill, O. P. and Martinac, B. Molecular basis of mechanotransduction in living cells. Physiol. Rev. 81: 685-740, 2001.
Martinac, B. and Hamill, O.P. Gramicidin A channels can switch between stretch-activation and stretch-inactivation depending upon bilayer thickness. Proc. Natl. Acad. Sci. USA 99: 4308-4312, 2002.
Perozo, E., Kloda, A., Cortes, D.M. and Martinac, B. Physical principles underlying the transduction of bilayer deformation forces during mechanosensitive channel gating. Nature Struct. Biol. 9: 696-703, 2002.
Perozo, E., Cortes, D.M., Sompornpisut, P. Kloda, A. and Martinac, B. Structure of MscL in the open state and the molecular mechanism of gating in mechanosensitive channels. Nature 418: 942-948, 2002.
Maroto, R., Raso, A., Wood, T.G., Kurosky, A., Martinac, B. and Hamill, O.P. TRPC1 forms the stretch-activated cation channel in vertebrate cells. Nature Cell Biology 7(2): 179-185, 2005.
Corry, B., Rigby, P., Liu, Z.-W. and Martinac, B. Conformational changes involved in MscL channel gating measured using FRET spectroscopy. Biophys. J. 89: L49-L51, 2005.
Gottlieb, P., Folgering, J., Maroto, R., Raso, A., Wood, T.G., Kurosky, A., Bowman, C., Bichet, D., Patel, A., Sachs, F., Martinac, B., Hamill, O.P. and Honor?, E.: Revisiting TRPC1 and TRPC6 mechanosensitivity. Pflugers Archiv-European Journal of Physiology 455: 1097-1103, 2008.
Martinac, B., Saimi, Y. & Kung, C. (2008) Ion channels in microbes. Physiol. Rev. 8: 1449-1490, 2008.
Recent Publications (2005-present):
Primary Papers
Khan, R.N., Martinac, B., Madsen, B.W., Milne, R.K., Yeo, G.F. and Edeson, R.O. Hidden Markov analysis of mechanosensitive channel gating. Mathematical Biosciences 193: 139-158, 2005.
Norman, C., Zhen-Wei Liu, Z-W., Rigby, R, Raso, A., Petrov, Y. and Martinac, B. Visualisation of MscL in Bacteria Using Confocal Microscopy. Eur. Biophys. J. 34: 396-403, 2005.
Tsai, I-J., Zhen-Wei Liu, Z-W., Rayment, J, Norman, C., McKinley, A. and Martinac, B. The role of the Periplasmic Loop Residue Glutamine 65 for MscL Mechanosensitivity. Eur. Biophys. J. 34: 403-413, 2005.
Nguyen, T., Clare, B. and Martinac, B. The Effects of Parabens on the Mechanosensitive Channels. Eur. Biophys. J. 34: 389-396, 2005.
Macdonald, A.G. and Martinac, B. Effect of high hydrostatic pressure on the bacterial mechanosensitive channel MscS. Eur. Biophys. J. 34: 434-442, 2005.
Hughes, S., El Haj, A., Jon Dobson, J. and Martinac, B. The Influence of Static Magnetic Fields on Mechanosensitive Ion Channel Activity in Artificial Liposomes. Eur. Biophys. J. 34: 461-469, 2005.
Maroto, R., Raso, A., Wood, T.G., Kurosky, A., Martinac, B. and Hamill, O.P. TRPC1 forms the stretch-activated cation channel in vertebrate cells. Nature Cell Biology 7(2): 179-185, 2005.
Corry, B., Rigby, P., Liu, Z.-W. and Martinac, B. Conformational changes involved in MscL channel gating measured using FRET spectroscopy. Biophys. J. 89: L49-L51, 2005.
Kloda, A. and Martinac, B. C-terminal charged cluster of MscL, RKKEE, functions as a pH sensor. Biophys. J. 90: 1992-1998, 2006.
Corry, B., Jayatilaka, D., Martinac, B. and Rigby, P. Determination of the orientational distribution and orientation factor for transfer between membrane bound fluorophores using a confocal microscope. Biophys. J. 91: 1032-1045, 2006.
Meyer, G.R., Gullingsrud, J., Schulten, K. and Martinac, B. Molecular Dynamics study of MscL interactions with a curved lipid bilayer. Biophys. J. 91: 1630-1637, 2006.
Kloda, A., Lua, L., Hall, R., Adams, D.J. and Martinac, B. Liposome reconstitution and modulation of recombinant N-methyl-D-aspartate receptor channels by membrane stretch. Proc. Natl. Acad. Sci. USA 104: 1540-1545, 2007.
Petrov, E. and Martinac, B. Modulation of channel activity and gadolinium block of MscL by static magnetic fields. Eur. Biophys. J. 36: 95-105, 2007.
Petrov, E., Rohde, P.R., Macdonald, A.G. and Martinac, B. Effect of high hydrostatic pressure and voltage on gating of the bacterial mechanosensitive channel of small conductance. Proceedings of the 4th International Conference on High Pressure Bioscience and Biotechnology Vol. 1, No.1 (Eds. F. Abe and A. Suzuki) pp.20-27, 2007.
Chen R., Yan H., Zhao K-N., Martinac B. and Liu G.B. Comprehensive analysis of prokaryotic mechanosensation genes: Their characteristics in codon usage. DNA Sequence 18 (4): 269-278, 2007.
Gottlieb, P., Folgering, J., Maroto, R., Raso, A., Wood, T.G., Kurosky, A., Bowman, C., Bichet, D., Patel, A., Sachs, F., Martinac, B., Hamill, O.P. and Honor?, E.: Revisiting TRPC1 and TRPC6 mechanosensitivity. Pflugers Archiv-European Journal of Physiology 455: 1097-1103, 2008.
Martinac, B. Open channel structure of MscL: A patch-clamp and spectroscopic study. Appl. Magn. Resonance. 2009 (in press).
Teichman, M.D.H., Wagner, F.V., Fink, R.h.A., Chamberlain, J.S., Launikonis, B.S., Martinac, B. and Friedrich, O.: Inhibitory control over Ca2+ sparks via mechanosensitive channels is disrupted in dystrophin deficient muscle but restored by mini-dystrophyn expression. PLoS Journal, Vol. 3, Issue 11/e3644:1-16, 2008.
Hurst, A.C., Gottlieb, P.A. and Martinac, B. Concentration dependent effect of GsMTx4 on mechanosensitive channels of small conductance in E. coli spheroplasts. Eur. Biophys. J. 38: 415-425, 2009.
Battle, A.R., Petrov, E., Pal, P. and Martinac, B. Rapid and improved reconstitution of bacterial mechanosensitive ion channel proteins MscS and MscL into liposomes using a modified sucrose method. FEBS Letters 583: 407-412, 2009.
Reviews
Martinac, B. Structural plasticity in MS channels. Nature Struct. Mol. Biol. 12(2): 104-105, 2005. (NSMB News and Views)
Kloda, A., Martinac, B. and Adams, D. J.: Polymodal regulation of NMDA receptor channels. Channels, 1(5): 334-343, 2007.
Kloda, A., Petrov, E., Meyer, G.R., Nguyen, T., Hurst, A., Hool, L and Martinac. B. MscL, bacterial mechanosensitive channel of large conductance. Intern. J. Biochem. Cell Biol. 40(2): 164-169, 2008.
Hurst, A.C., Petrov, E., Kloda, A., Nguyen, T., Hool, L., Martinac, B. MscS, the bacterial mechanosensitive channel of small conductance. Intern. J. Biochem. Cell Biol. 40(4): 581-585, 2008.
Corry, B.A. and Martinac, B. Bacterial mechanosensitive channels: experiment and theory. Biochim. Biophys. Acta, 1778: 1859-1870, 2008.
Martinac, B., Saimi, Y. and Kung, C.: Ion channels in microbes. Physiol. Rev. 88: 1449-1490, 2008.
Books
Bacterial ion channels and their eukaryotic homologues, (Kubalski, A.and Martinac, B., editors), ASM Press, Washington D.C., 2005.
Sensing with ion channels, (Martinac, B., editor), Springer Series in Biophysics 11, Springer Verlag, Berlin, Heidelberg, New York, 2008.
Book chapters
Kubalski, A. and Martinac, B. Ion channels of prokaryotes: Towards the understanding of structure and function of ion channels. In: Bacterial ion channels and their eukaryotic homologues, (Kubalski A. and Martinac, B., editors). ASM Press, Washington D.C. 2005.
Martinac, B. Mechanosensitive channels. In: Biological Membrane Ion Channels: Dynamics, Structure, and Applications (eds. S. H. Chung, O. S. Andersen and V. Krishnamurthy). Springer, New York. Chapter 10, pp. 369-398, 2006.
Martinac, B. 3.5 billion years of mechanosensory transduction: structure and function of mechanosensitive channels in prokaryotes. In: Current Topics in Membranes 58: 25-57, 2007. (O.P. Hamill, Ed.), Elsevier Inc., San Diego, CA.
Corry, B. and Martinac, B. Computational studies of the bacterial mechanosensitive channels: In: Mechanosensitivity in Cells and Tissues (A. Kamkin and I. Kiseleva, Eds.) Springer-Verlag, pp. 103-116, 2007.
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