Calcium-mediated changes in gap junction structure: evidence from the low angle X-ray pattern.

Abstract

Rat liver gap junctions were isolated in Ca2+-free media and analyzed in controlled environments by x-ray diffraction of partially oriented pellets. Different treatments of the same preparations were compared. The ordered hexagonal lattices gave rise to detail that was sensitive to low Ca 2+ concentrations (0.05 mM), but not to Mg 2+ (up to 0.16 mM) or pH (between 6.0 and 8.0). The major Ca2+-mediated responses were reductions in the intensity of the (1, 0) peak and in the off-equatorial contributions to the (2, 1) peak, and changes of scale equivalent to a decrease (,-,2%) in lattice dimension, but an increase (---4%) in the dimension perpendicular to the lattice. A simple structural interpretation of these findings is that Ca 2+ induces the subunits of the channel-forming assembly, the connexon, to align more nearly parallel to the channel, thereby causing the connexon to become slightly longer and more radially compact. The rearrangement is of the same nature as one found under less physiological circumstances by electron microscopy (Unwin, P. N. T., and G. Zampighi, 1980, Nature (Lond.)., 283:545-549), and may be part of a coordinated mechanism by which the channel closes.The gap junction is a region of contact between neighboring animal cells that allows the exchange of small molecules and ions between their interiors (4, 6). The channels responsible for this exchange lie along the hexad axes of hexameric membrane proteins called connexons (2), which link, in pairs, the apposed plasma membranes. In living tissue the connexon responds to local changes in intracellular free Ca 2+ concentration (10, 11), and/or pH (11, 13) and membrane potential (3, 9), thereby regulating the channel's permeability. The mechanism of the transition between the open and closed states of the channel is not known. However, studies of isolated, hexagonally ordered, gap junctions suggest it may involve a change in conformation of the connexon in the space between the two plasma membranes (1, 7) or, alternatively, a coordinated rearrangement of the connexon subunits around the channel (14).As a first step toward understanding the physiological significance of these connexon configurations, we examined the structural response of isolated junctions to changes in ionic environment. Response was assessed quantitatively by dividing each preparation in two, exposing the separate portions to different solutions, and then comparing x-ray diffraction patterns recorded from each portion. Pair-wise analysis provided a sensitive measure of structural state, making the effect of small variabilities between preparations relatively unimportant.Results showed that the isolated junctions change their structure on exposure to low Ca 2÷ concentrations. We report on the properties of this transition, its relation to one previously described (14), and its possible relevance to living tissue. MATERIALS AND METHODSChemicals: Lubrol WX and Arsenazo IlI were obtained from Sigma Chemical Co. (St. Louis, MO); sodium deoxycholate from Calbiochem-Behri...

Mechanism of gating by calcium in connexin hemichannels

“…Electron microscopy-based structures of isolated rat liver gap junction channels showed significant differences in pore diameter between Ca 2+ and Ca 2+ -free structures (31). Atomic force microscopy of isolated murine Cx26 hemichannels showed an increase in pore diameter from 2.5 to 5 nm when Ca 2+ is removed, suggesting that Ca 2+ stabilizes the closed conformation of the channels (32).…”

Innexins form two types of channels

“…This is in contrast to channels formed by connexins, which either are non-responsive to increased cytoplasmic calcium or are closed, depending on their protein composition [35][36][37][38].…”

Regulation of Cellular Communication by Signaling Microdomains in the Blood Vessel Wall

“…It has been well documented that connexin hemichannels and gap junctions are sensitive to calcium environments, although the exact mechanisms of the interaction are not fully defined (Harris, 2001;Lurtz and Louis, 2007;Zhou et al, 2007b;Herve et al, 2012;Herve and Derangeon, 2013). The first observation made in the early 1980s using freeze fracture studies indicated that connexins twist the gap junction channel shut in response to supraphysiological levels of Ca 2+ (Peracchia, 1978;Unwin and Ennis, 1983;Bruzzone et al, 1996). Several studies now suggest that this may be regulated through binding of the calcium sensing subunit calmodulin.…”