An electrostatic mechanism for Ca2+-mediated regulation of gap junction channels

Abstract

Gap junction channels mediate intercellular signalling that is crucial in tissue development, homeostasis and pathologic states such as cardiac arrhythmias, cancer and trauma. To explore the mechanism by which Ca2+ blocks intercellular communication during tissue injury, we determined the X-ray crystal structures of the human Cx26 gap junction channel with and without bound Ca2+. The two structures were nearly identical, ruling out both a large-scale structural change and a local steric constriction of the pore. Ca2+ coordination sites reside at the interfaces between adjacent subunits, near the entrance to the extracellular gap, where local, side chain conformational rearrangements enable Ca2+chelation. Computational analysis revealed that Ca2+-binding generates a positive electrostatic barrier that substantially inhibits permeation of cations such as K+ into the pore. Our results provide structural evidence for a unique mechanism of channel regulation: ionic conduction block via an electrostatic barrier rather than steric occlusion of the channel pore.

Mechanism of gating by calcium in connexin hemichannels

“…The recent and novel Ca 2+ -bound crystal structure for Cx26 gap junction channels showed Ca 2+ ions located within the pore and coordinated by residues E42, E47, and G45 (18). This only partially agrees with our mutagenesis studies indicating that E47 and D50 are likely part of the binding site in Cx26 hemichannels.…”

“…The equivalent residues in Cx46 were shown to have similar roles. D50 and E47 likely form a Ca 2+ -bound gating ring at the entrance of the pore, as supported by the novel crystal structure of hCx26 gap junction channels obtained in the presence of Ca 2+ (18). Nevertheless, our accessibility studies show that such a Ca 2+ -bound gating ring does not prevent access of ions or small molecules to the inner pore, indicating that the gate (physical or electrostatic) is located deeper into the pore.…”

“…Recent computational analysis from the Ca 2+ -bound crystal structure revealed that Ca 2+ binding produces a positive electrostatic barrier that inhibits occupancy or permeation of cations in the junctional pore (18). Here we tested whether a Ca 2+ -bound gating ring serves as an electrostatic or physical gate that prevents passage of ions and small molecules in Cx26 hemichannels.…”

“…A recent crystal structure of Cx26 gap junction channels (docked hemichannels) also indicates that E47 residues directly participate in Ca 2+ coordination and the formation of a Ca 2+ -bound sensing ring at the entrance of the pore (18). Calculations based on this structure indicated that the Ca 2+ -bound sensing ring forms an electrostatic gate that restricts occupancy of the channel by cations in gap junction channels (18). (17)].…”

“…This type of Ca 2+ -binding domain at the extracellular face of connexin hemichannels is not feasible, because it would interfere with the formation of intercellular gap junction channels. Most likely, Ca 2+ sensing is predominantly mediated by motifs that lie within or are exposed to the pore lumen (18).…”