Research Results II
Protein phosphorylation has been shown to alter the unitary conductance and permeability of gap junction channels formed by Cx43. When connexins like Cx43 and Cx45 form heteromeric channels, their gating and permeability properties are altered. We have already shown that heteromerization is enough to change the phosphorylated level of Cx43. During this project, conducted by Dr. Osman Ahmed, we use of mutants of Cx43 in trying to determine the molecular regions where phosphorylation responsible of changes in permeability occurs. In Figure 5 we present a simple experiment where the permeability of Cx43 is shown to be reduced after adding 300nM TPA. If serines at the carboxyl tail are substituted by alanines, TPA has no effect (botom line). The technique used for this experiments is called cell drop. Cells in which their membranes were labeled with DiI (red) and their cytoplasms was loaded with Calcein-AM, were cocultured with nonlabeled homotypic cells. After 3-6 hours, when gap junctions are formed, free calcein diffuses into those coupled cells. These experiments together with iontophoretic micro-injections of fluorescent dyes, has been possible to elucidate the impact of phosphorylation in heteromeric channel permeability and selectivity.
In terms of cardiac repair, we have characterized the propagation of electrical activity in cardiocyte monolayers obtained from mouse neonatal ventricular cells (Vasquez et al, 2009). One of our long term goals is to be able to design pluripotential cells that can be implanted, and with them, the capacity of regulating cell to cell communication and excitability in order to correct defects in the metabolic and electrical coupling of cardiovascular tissues (See Figure 6). In particular, we are currently interested in the interaction of cardiocytes and fibroblasts, where mathematical modeling predicts robust changes in conduction parameters and action potential morphology (Sachse et al, 2007, 2008).
Our laboratory has recently initiated a collaboration with Dr. Ken Spitzer from the CVRTI and Dr. Richard Vaughan-Jones from the University of Oxford. This collaboration is related to the biophysical properties of cardiac gap junction channel at low and high intracelular pH. We are trying to determine if gating by intracellular pH (during acidosis and alkalosis) share the same molecular mechanisms (Zhuang et al, 2009; Figure 7 ).