Research Results I
At this time we have characterized the effect of oligomerization in the permeability and gating properties of some connexin combinations, but many others are needed to explored (Zhong et al 2009; see Figures 1 and 2). We have participated in determining the molecular nature of metabolites that differentially cross gap junction channels (Goldberg et al, 2004) and determine the relevance of accessory molecules involved in protein phosphorylation necessary to gate connexin43 channels during c-SRC activation (Goldberg et al, 2007).
In our laboratory, the technique of double whole cell voltage clamp is one of the most used (Del Corso et al, 2006). We have also participated in the development of a system where we can simultaneously record intercellular currents and the difusion of fluorescent dyes across established gap junctions.
The movie in Figure 3, represents a typical approach of two micropipettes to a pair of cultured HeLa cells. Here we also present an example on how we determine dye permeability across gap junctions. First, we load a micropipette with this dye and then perform a double voltage clamp technique. This allow us to have a close idea on how different combinations of channels participate in regulating the diffusion of metabolites across a tissue. Further on in the movie we present the movement of Lucifer Yellow dye accross connexin channel formed by Cx43 and Cx45. The participation of Cardiology fellows from the University of Utah like Nazem Akoum and Osman Ahmed and graduate students from Biotechnology like Daniel Appadurai has been unique.
To understand these differences in permeability, we have generated a mathematical model. Abhijit Mondal, a graduate student at the lab is a specialist in robotics and is currenlty developing a molecular mathematical model that can help us understanding how different molecular factors affect diffusion across heterotypic gap junction channels. As shown in Figure 4, with a 3D model is possible to describe the structure and geometry of the channels, and also the movement of particles (resembling dyes used to test gap junction permeability) and their interactions with other molecules and the pore walls.