Intramolecular loop/tail interactions are essential for connexin 43‐hemichannel activity

Abstract

Connexin-assembled gap junctions (GJs) and hemichannels coordinate intercellular signaling processes. Although the regulation of connexins in GJs has been well characterized, the molecular determinants controlling connexin-hemichannel activity are unresolved. Here we investigated the regulation of Cx43-hemichannel activity by actomyosin contractility and intracellular [Ca(2+)] ([Ca(2+)](i)) using plasma membrane-permeable TAT peptides (100 μM) designed to interfere with interactions between the cytoplasmic loop (CL) and carboxy-terminal (CT) in primary bovine corneal endothelial cells and HeLa, C6 glioma, and Xenopus oocytes ectopically expressing Cx43. Peptides corresponding to the last 10 CT aa (TAT-Cx43CT) prevented the inhibition of Cx43-hemichannel activity by contractility/high [Ca(2+)](i), whereas a reverse peptide (TAT-Cx43CTrev) did not. These effects were independent of zonula occludens-1, a cytoskeletal-associated Cx43-binding protein. In contrast, peptides corresponding to CL (TAT-L2) inhibited Cx43-hemichannel responses, whereas a mutant peptide (TAT-L2(H126K/I130N)) did not inhibit. In these assays, TAT-Cx43CT acted as a scaffold for TAT-L2 and vice versa, a finding supported by surface plasmon resonance measurements. Loop/tail interactions appeared essential for Cx43-hemichannel activity, because TAT-Cx43CT restored the activity of nonfunctional hemichannels, consisting of either Cx43 lacking the C-terminal tail (Cx43(M239)) or intact Cx43 ectopically expressed in Xenopus oocytes. We conclude that intramolecular loop/tail interactions control Cx43-hemichannel activity, laying the basis for developing hemichannel-specific blockers.

At the cross-point of connexins, calcium, and ATP: blocking hemichannels inhibits vasoconstriction of rat small mesenteric arteries

“…Control experiments on isolated SMC-pairs L2-peptide (see Supplementary material online, Figure S1) prevents CL-CT loop-tail interactions, thereby inhibiting hemichannel ATP release 10,32 while slightly favouring the GJ open state. 33 We characterized L2 effects on hemichannel unitary currents recorded in HeLa cells exogenously expressing Cx43 with whole-cell patch clamp ( Figure 3A).…”

“…Hemichannels are permeable to Ca 2þ and may thus contribute to cellular Ca 2þ entry 4 and diffusive ATP release. 5 Moreover, hemichannel opening is [Ca 2þ ] i -dependent in a bimodal manner, [6][7][8][9][10] with moderate [Ca 2þ ] i elevation favouring opening and above $500 nM [Ca 2þ ] i inhibiting opening. The bimodal Ca 2þ -dependence of hemichannels resembles the Ca 2þ -dependence of inositol-trisphosphate receptor (IP 3 R) channels, which are Ca 2þ release channels in the endoplasmic reticulum (ER) that play a central role in generating Ca 2þ oscillations in diverse cell types including SMCs.…”

“…25,26 In contrast to this, L2 inhibits Cx43-hemichannels as concluded from indirect measurements of hemichannel function. 10 CT9 corresponds to the last 9 CT amino acids of Cx43 and dye uptake studies suggested it prevents Cx43 hemichannel closure at micromolar [Ca 2þ ] i 9,10 , thereby promoting hemichannel opening. Thus, L2-inhibition of hemichannels and CT9-prevention of high [Ca 2þ ] i hemichannel closure makes these peptides interesting tools to determine the role of Cx43-hemichannels in Ca 2þ oscillations.…”

Green Fluorescent Protein Changes the Conductance of Connexin 43 (Cx43) Hemichannels Reconstituted in Planar Lipid Bilayers

“…However, the characterization was restricted to Cx43 and Cx43-eGFP in HeLa cells. Other studies elucidate the essential role of the C-terminal tail of Cx43 and its interaction with the L2 region (amino acid residues 119 -144) of the intracellular loop to maintain protein activity (16).…”

Function and Regulation of Pannexin 1 Channels in the Vascular Endothelium

“…In addition to Gap19, a second mimetic peptide designed to interrupt intracellular loop:Ctail interactions called TAT-L2 has been reported to specifically inhibit endogenous Cx43 36 hemichannel activity in endothelial cells and hemichannel activity in ectopic expression systems [195]. These new peptides have shed light on the gating of Cx43 hemichannels which appears to be tightly regulated by interactions between the intracellular domains of Cx43.…”