Thaddeus A. Bargiello

Different subsets of connexin proteins are expressed in almost all mammalian tissues and organ systems where they form intercellular gap junction channels. Gap junction channels are formed by the head-to-head docking of two hemichannels in adjacent cells to provide a direct communication pathway for intercellular electrical and chemical signaling. This signaling plays a vital physiological function in many organs including heart and brain, where gap junctions form electrical synapses; and in non-excitable tissues such as liver and pancreas, where they synchronize cellular activity and secretion. Subsets of hemichannels not docked with apposed hemichannels mediate plasma membrane currents and molecular flux in native tissues and exogenous expression systems. These channels are termed undocked or unapposed hemichannels and recent evidence suggests that they play important roles in normal and pathophysiology. Mutations of human connexin genes are the pathophysiological basis for seven major disorders including: myelin and neuronal disorders, nonsyndromic and syndromic deafness, skin diseases, cataracts, oculodentodigital dysplasia, and idiopathic atrial fibrillation.

Current projects include determining the structure-function correlates of mutations in two connexin genes, Cx32 and Cx26, which underlie respectively the X-linked form of Charcot-Marie-Tooth disease and syndromic and non-syndromic hearing loss. These studies utilize a combination of NMR structural determinations, cell biology, computational and biophysical methods to establish how structural defects caused by mutations lead to the diseased state.

A second project is directed towards defining the mechanism of voltage-dependent gating at the atomic level. Membrane voltage is an important parameter regulating the opening and closing of connexins. All connexin channels display two distinct voltage gating mechanisms, termed Vj- and loop-gating. A major objective of the lab is to define the conformational path that links open and closed states. The strategy we employ utilizes a variety of experimental approaches including cysteine scanning for metal bridge formation, chemical crosslinking and fluorescence spectroscopy to define open and closed states and temperature dependence of gating kinetics to define the height of energy barriers. Computational methods, including all atom molecular dynamics are used to refine structural models of open and closed states and to define the most probable path that links open and closed states. These include target and steered molecular dynamics simulations and brute force simulations with the application of voltage to an all atom explicit membrane system on a new supercomputer, Anton that allows long simulations in the order of hundreds of microseconds to milliseconds. Open state models are evaluated by comparison of computational derived permeabilities obtained with Brownian Dynamics simulation or umbrella sampling methods to experimental data. The strategy that we employ involves a synergistic and reciprocal relationship between computer simulations and experiments. The simulations guide experimental design and interpretation; experimental outcomes help to refine the models and guide future simulations.

A third project stems from our recent computational and experimental studies of ion permeation of Cx26 hemichannels indicating that acetylation plays an important role in determining ionic charge selectivity and by extension, permeation of charged second messengers. We are determining the role of differential acetylation in reported cell specific differences in the permeability of Cx26 intercellular channels to anionic and cationic molecules reported in the literature. Regulated acetylation would have important physiological consequences by tuning the permeability of connexin channels to optimize the flux of specific classes of second messengers and other signaling molecules. In this way, acetylation could regulate the physiology of cellular networks coupled by gap junctions, and in a cell/tissue-specific manner.

Selected Publications