Cx43 hemichannels reconstituted in PC bilayers
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To measure the intrinsic permeability of a variety of fluorescent probes across homo- and hetero- oligomeric gap junction (GJ) channels in transfected cells.
We will explore the dependence of permeability on channel
composition. Absolute rates will be determined by simultaneous
electrical (patch-clamp) and fluorescent measurements. Transfer rates of
probes will delimit the bounds on the permeation rates of messenger
molecules with similar size and charge.
To build artificial double-bilayer systems, incorporate whole GJ
channels, and study their properties in controllable external conditions.
In collaboration with Dr. S. Tripathi (TIFR, Bombay), we have (i)
incorporated GJ hemichannels (half a GJ channel) in a single bilayer,
mimicking previous work (ii) tried various methods to form artificial
double bilayers, including multiple patch pipette membranes picked from
bilayer “bubbles” and multiple “tip dips”, with some success already. We
have also expressed Cx43 channels with a 6-His tag in Sf9 cells through
the Bac-to-Bac system and have affinity-purified them. These channels
again show activity in tip-dip bilayers (see figure below). We will next
attempt to incorporate intact GJ channels in double bilayers.
Modeling vascular smooth muscle cells
Vascular smooth muscle cells form the basis for the
myogenic response. A model that describes the steady state response of
vascular smooth muscle cells to arterial pressure (Knot & Nelson,
1998) would be important in discriminating between different mechanisms
that set myogenic tone. The only smooth muscle cell model reported so
far is the kinetic model by Yang et al. (2003). This model, while
successful in describing the timecourse of the fast vascular response,
seems unable to mimic steady state cellular behavior (preliminary
observations).
We have developed a model of the steady state
response that accounts for ion balance for K, Na and Ca. The model
differs from that of Yang et al.'s (2003) model in that it specifically
considers processes with longer time constants ( e. g.
inactivation of delayed rectifiers with a time constant ~3 s) that may
be important in setting the basal tone. The three ionic balance
equations are coupled in two ways: (a) through the Na-K pump and the
Na-Ca exchanger and (b) the maxi K channel, whose gating activity is
very sensitive to [Ca], and which putatively provides a negative
feedback mechanism that stabilizes the steady state. The
predictions of the model are stable against small parameter changes and
seem to account for the experimental results reported in the
literature. A specific outcome from the model is that cellular Na
concentration varies five fold over the range of experimentally applied
pressures (10-100 mm Hg).
We are working on incorporating gap junctions in a tissue model,
where each cell is described by the model above. We will use this to
model calcium wave spread. The next goal is to incorporate
receptor-mediated processes to complete a model of vascular smooth
muscle tissue, perhaps incorporating tension development as well.
Gap junctions in suicide gene therapy (with S. Bagavathi; please follow link)
Macromolecular permeation through nanopores (with B. M. Jaffar
Ali; please follow link)
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Life Sciences Division,
AU-KBC Research Center
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MIT,
Chromepet, Chennai 600044
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(tel)
+91-44-2-223-4885 (fax) +91-44-2-223-1034
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