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Molecular mechanisms of ion channels and neurotoxins.
Our research aims to understand how channel
proteins work by exploiting naturally occuring neurotoxins as molecular
probes of channel structure and function. Two ion channels under current
investigation are the voltage-gated Na+-channel and the Ca2+-activated
K+ channel which are both expressed in many different types of electrically
excitable cells. A key question in the Na+-channel field is: How does
this channel selectively discriminate among inorganic cations such as
Na+, K+ and Ca2+? We are beginning to obtain molecular insights to this
mechanism by analyzing channel mutations that are defective in ionic
selectivity. In the case of the K(Ca) channel, we are studying how intracellular
domains of the channel protein control ion permeation and gating. We
have found that a class of small proteins (Kunitz inhibitors), which
include mamba snake dendrotoxins and bovine pancreatic trypsin inhibitor,
bind to an internal site on the channel protein and induce discrete
subconductance events at the single-channel level. In our work, we use
a diverse combination of techniques that include single-channel analysis,
planar bilayer, whole-cell and patch recording, as well as molecular
biological approaches. Our work has also led to the discovery of an
interesting saxitoxin-binding protein called saxiphilin. Saxiphilin
is a homolog of transferrin that does not bind Fe3+. It is also a potent
inhibitor of cysteine proteinases such as papain and cathepsins B and
L. The long term goal of this project is to determine the physiological
function of saxiphilin.
Figure caption:
Single sodium channels from calf heart were
inserted into planar lipid bilayers in the presence of various symmetrical
concentrations of NaCl from 0.1 to 3.0 M as indicated External ZnCl2
in the range of 20 to 640 mM was added to include a convenient frequency
of flickering events. Closed (c) and open (o) levels of the single-channel
current are indicated by dashed lines. The intermediate subconductance
level reveals the magnitude of Na+ current flowing through the channel
with a single Zn2+ ion bound in its external vestibule.
Recent publications:
Ni
YG, Chen JG, Androutsellis-Theotokis A, Huang CJ, Moczydlowski E, Rudnick
G. A
lithium-induced conformational change in serotonin transporter alters
cocaine binding, ion conductance, and reactivity of cys-109. J Biol
Chem. 2001 Aug 17;276(33):30942-7.
Bian S, Favre I, Moczydlowski E. Ca2+-binding
activity of a COOH-terminal fragment of the Drosophila BK channel involved
in Ca2+-dependent activation. Proc Natl Acad Sci U S A. 2001 Apr
10;98(8):4776-81.
Huang CJ, Moczydlowski E. Cytoplasmic
polyamines as permeant blockers and modulators of the voltage-gated
sodium channel. Biophys J. 2001 Mar;80(3):1262-79.
Krishnan G, Morabito MA, Moczydlowski E. Expression
and characterization of Flag-epitope- and hexahistidine-tagged derivatives
of saxiphilin for use in detection and assay of saxitoxin. Toxicon.
2001 Feb-Mar;39(2-3):291-301.
edward.moczydlowski@yale.edu
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