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
Nav1.2 in patients with febrile and afebrile
seizures, and a de novo nonsense mutation in patients with
intractable epilepsy. These findings implicate Nav1.2
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 Nav1.2. 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 Nav1.2
.
· 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 Nav1.2 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.
Mauricio Montal received his Ph.D. from the University of Pennsylvania
and his M.D. from the National University of Mexico. He is the recipient
of a K.C. Cole Award of the Biophysical Society for outstanding
contributions in Membrane Biophysics and a Research Scientist Award
from NIMH.
