Biological Sciences

Mauricio Montal Professor of Biology, UCSD e-mail: montal@biomail.ucsd.edu

      Current research projects are focused on three main questions:

·  Structure-function relationships in channel proteins and channel dysfunction in disease mechanism, especially concerning inherited epilepsy.
      The thrust of our program aims to understand the fundamental principles underlying the sequence-structure determinism, a major unsolved issue in contemporary biology. Membrane channels, the field of inquiry, are oligomeric proteins organized as symmetric or pseudosymmetric arrays around a central aqueous pore. The ultimate function of the protein is to allow the selective and regulated diffusion of ions across the membrane lipid bilayer. We have developed a hierarchical strategy to elucidate sequence-structure determinants by investigating autonomously folded modules in absence of the entire channel protein. This endeavor has led to the determination of the first high-resolution structure in membranes of the channel lining segments of neurotransmitter-gated channels using a combination of solution and solid-state NMR spectroscopy. A complementary approach is based on the identification, from bacterial genome sequences, of a rich repertoire of voltage-gated channels, the size of which is within the practical range of NMR spectroscopy. These advances set us on a solid framework to proceed to the determination of the structure of an intact channel protein in membranes by NMR spectroscopy. Concerning channel dysfunction, we identified a missense mutation in the voltage-gated Na⁺ channel Na_v1.2 in patients with febrile and afebrile seizures, and a de novo nonsense mutation in patients with intractable epilepsy. These findings implicate Na_v1.2 in a human disease and identify an impairment of channel inactivation as a molecular defect underlying the hyperexcitability phenotype characteristic of epilepsy.

·  Neuronal survival and the connection between electrical excitability and apoptotic cell death.

      Neural activity is crucial for cell survival and fine patterning of neuronal connectivity during neurodevelopment. To investigate the role in vivo of action potential Na⁺ channels in these processes we generated knockout mice deficient in brain Na_v1.2. Null mice die perinatally with severe hypoxia and massive neuronal apoptosis, notably in the brainstem. The current aim is to identify changes in the regulation of gene expression of apoptosis-related genes in neurons and to use this information to dissect the connection between electrical excitability and neuronal survival. The strategy implemented takes advantage of the powerful DNA microarray technology and is likely to provide novel and timely clues about the signaling cascades impacted by the deletion of Na_v1.2
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·  Botulinum neurotoxins (BoNT) and mechanism of synaptic vesicle exocytosis.

      During the past two decades, my group has been engaged in dissecting the molecular steps in the mode of action of Clostridium botulinum neurotoxins. Recently, we showed that the heavy chain of BoNT acts as a transmembrane chaperone for the light chain to ensure a translocation competent conformation during its transit from the acidic endosome into the cytosol - its site of action. The light chain is a metalloprotease that cleaves the protein components involved in synaptic vesicle fusion with the neuronal membrane, thereby abrogating synaptic transmission at presynaptic motor nerve endings. To accomplish this task, the heavy chain operates as a transmembrane protein-conducting channel: the channel is occluded by the light chain during transit and open after completion of translocation and release of cargo. Our findings suggest that the BoNT channel is a potential target for intervention to attenuate BoNT neurotoxicity which we pursue by screening synthetic combinatorial libraries to identify selective blockers. A second facet involves the concept that the peptide products of substrate proteolysis by BoNTs uncouple excitation from secretion pointing to new means of intervention.

      Ferrer-Montiel, A.V., J.M. Merino, S.E. Blondelle, E. Perez-Paya, R.A. Houghten and M. Montal. (1998). Novel peptide leads tageted to the NMDA receptor channel protect neurons against excitotoxic death. Nature Biotechnol. 16:286-291.

      Opella, S.J., F.M. Marassi, J.J. Gesell, A.P. Valente, Y. Kim, M. Oblatt-Montal and M. Montal. (1999). Structures of the M2 channel-lining segments from nicotinic acetylcholine and NMDA receptors by NMR spectroscopy. Nature Struct. Biol. 6:374-379.

      Yao, Y., A.V. Ferrer-Montiel, M. Montal and R.Y. Tsien. (1999). Activation of store-operated calcium-current in Xenopus oocytes requires SNAP-25 but not a diffusible messenger. Cell 98:475-485.

      Marassi, F.M., C. Ma, H. Gratkowski, S.K. Strauss, K. Strebel, M. Oblatt-Montal, M. Montal and S.J. Opella. (1999). Correlation of the structural and functional domains in the membrane protein Vpu from HIV-1. Proc. Natl. Acad. Sci. USA 96:14336-14341.

      Planells-Cases, R., M. Caprini, J. Zhang, E.M. Rockenstein, R.R. Rivera, C. Murre, E. Masliah and M. Montal. (2000). Neuronal death and perinatal lethality in voltage-gated sodium channel a2-deficient mice. Biophys. J. 78:2878-2891.

      Tai, K.K., S.E. Blondelle, J.M. Ostresh, R.A. Houghten and M. Montal. (2001). An N-methyl-D-aspartate receptor channel blocker with neuroprotective actitivity. Proc. Natl. Acad. Sci. USA 98:3519-3524.

      Sugawara, T. et al. A missense mutation of the Na⁺ channel a2 subunit gene Na_v1.2 in a patient with febrile and afebrile seizures causes channel dysfunction. Proc. Natl. Acad. Sci. USA, 98:6384-6389.

      Caprini, M., S. Ferroni, R. Planells-Cases, J. Rueda, C. Rapisarda, A. Ferrer-Montiel and M. Montal. (2001). Structural compatibility between the putative voltage sensor of Kv channels and the prokaryotic KcsA channel. J. Biol. Chem. 21070-21076.

      Montal, M. and Opella, S.J. (2002). The structure of the M2 channel-lining segment from the nicotinic acetylcholine receptor. Biochim. Biophys. Acta 1565: 287-293.

      Koriazova, L. and Montal, M. (2003). Translocation of botulinum neurotoxin light chain protease through the heavy chain channel. Nature Struct. Biol. 10:13-18.

      Fischer, A. and Montal, M. (2006). Characterization of Clostridial botulinum neurotoxin channels in neuroblastoma cells. Neurotoxicity Res. 9:93-100.
 
      Santos, J.S., Lundby, A., Zazueta, C. and Montal, M. (2006). Molecular template for a voltage sensor in a novel K⁺ channel. I. Identification and functional characterization of KvLm, a voltage-gated K⁺channel from Listeria monocytogenes. J. Gen. Physiol. 128:283-292.
 
      Lundby, A., Santos, J.S., Zazueta, C. and Montal, M. (2006). Molecular template for a voltage sensor in a novel K⁺ channel. II. Conservation of a eukaryotic sensor fold in a prokaryotic K⁺ channel. J. Gen. Physiol. 128:293-300.