Connexin 43 hemichannels mediate the Ca²⁺ influx induced by extracellular alkalinization

Abstract

Although alkaline pH is known to trigger Ca(2+) influx in diverse cells, no pH-sensitive Ca(2+) channel has been identified. Here, we report that extracellular alkalinization induces opening of connexin 43 hemichannels (Cx43 HCs). Increasing extracellular pH from 7.4 to 8.5, in the presence of physiological Ca(2+)/Mg(2+) concentrations, rapidly increased the ethidium uptake rate and open probability of HCs in Cx43 and Cx43EGFP HeLa transfectants (HeLa-Cx3 and HeLa-Cx43EGFP, respectively) but not in parental HeLa cells (HeLa-parental) lacking Cx43 HCs. The increase in ethidium uptake induced by pH 8.5 was not affected by raising the extracellular Ca(2+) concentration from 1.8 to 10 mM but was inhibited by a connexin HC inhibitor (La(3+)). Probenecid, a pannexin HC blocker, had no effect. Extracellular alkalinization increased the intracellular Ca(2+) levels only in cells expressing HCs. The above changes induced by extracellular alkalinization did not change the cellular distribution of Cx43, suggesting that HC activation occurs through a gating mechanism. Experiments on cells expressing a COOH-terminal truncated Cx43 mutant indicated that the effects of alkalinization on intracellular Ca(2+) and ethidium uptake did not depend on the Cx43 C terminus. Moreover, purified dephosphorylated Cx43 HCs reconstituted in liposomes were Ca(2+) permeable, suggesting that Ca(2+) influx through Cx43 HCs could account for the elevation in intracellular Ca(2+) elicited by extracellular alkalinization. These studies identify a membrane pathway for Ca(2+) influx and provide a potential explanation for the activation of cellular events induced by extracellular alkalinization.

Permeation of Calcium through Purified Connexin 26 Hemichannels

“…For this, it is essential to use a purified and reconstituted preparation of HCs where there is no possibility of Ca 2ϩ transport through parallel unidentified pathways. Our results support previous interpretations that suggested a role of Ca 2ϩ fluxes through GJCs and HCs in physiological and pathophysiological conditions (17,21,(25)(26)(27)(28). Ca 2ϩ permeability is relatively high, similar to that for Na ϩ .…”

“…Although most cells have fewer HCs than GJCs at the plasma membrane, the much higher extracellular [Ca 2ϩ ] suggests that HCs can have a significant role in Ca 2ϩ influx, at least under certain circumstances (17,21,(25)(26)(27)(28) or in disease-causing mutants that display higher Ca 2ϩ permeability (G45E mutant associated with keratitis ichthyosis deafness syndrome) (29). In summary, our results showed that Cx26 HCs are permeable to Ca 2ϩ and suggest that HCs can be a pathway for Ca 2ϩ influx into cells under conditions such as ischemia.…”

“…However, HCs can be activated in the presence of millimolar extracellular [Ca 2ϩ ] under a number of conditions, including ischemia, inflammation, connexin dephosphorylation, and extracellular alkalinization (25)(26)(27)(28). Understanding Ca 2ϩ permeation through Cx26 HCs is also important because increased Ca 2ϩ permeability may constitute the bases * This work was supported, in whole or in part, by National Institutes of Health for the association of the G45E mutant with the keratitis ichthyosis deafness syndrome (29).…”

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

“…Accumulating evidence suggests that hemichannels can be opened by various messengers and conditions, thereby providing a conduit that allows the passage of ions and molecules with MW up to 1.5 kDa. 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.…”

Involvement of Gap Junction Proteins in Infectious Diseases Caused by Parasites

“…In addition, Cx26, Cx32 and Cx43 HCs are permeable to Ca 2+ [73][74][75][76] and might be involved in initiation of intracellular rise in Ca 2+ signals. In protozoan infections, a key process in early stages of invasion is the rise in cytosolic Ca 2+ concentration [77].…”