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AU-KBC RESEARCH CENTRE  Life Sciences Research Faculty S. V. Ramanan

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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.

Cx43 hemichannels reconstituted in PC 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
MIT, Chromepet, Chennai 600044
(tel) +91-44-2-223-4885   (fax) +91-44-2-223-1034
S. V. Ramanan