Charged Residues at the First Transmembrane Region Contribute to the Voltage Dependence of the Slow Gate of Connexins

Bernardo I Pinto,Isaac E García,Amaury Pupo,Mauricio A Retamal,Agustín D Martínez,Ramón Latorre,Carlos González

doi:10.1074/jbc.m115.709402

Bernardo I Pinto, Isaac E García + Show 5 more

Open Access

https://doi.org/10.1074/jbc.m115.709402

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Abstract

Connexins (Cxs) are a family of membrane-spanning proteins that form gap junction channels and hemichannels. Connexin-based channels exhibit two distinct voltage-dependent gating mechanisms termed slow and fast gating. Residues located at the C terminus of the first transmembrane segment (TM-1) are important structural components of the slow gate. Here, we determined the role of the charged residues at the end of TM-1 in voltage sensing in Cx26, Cx46, and Cx50. Conductance/voltage curves obtained from tail currents together with kinetics analysis reveal that the fast and slow gates of Cx26 involves the movement of two and four charges across the electric field, respectively. Primary sequence alignment of different Cxs shows the presence of well conserved glutamate residues in the C terminus of TM-1; only Cx26 contains a lysine in that position (lysine 41). Neutralization of lysine 41 in Cx26 increases the voltage dependence of the slow gate. Swapping of lysine 41 with glutamate 42 maintains the voltage dependence. In Cx46, neutralization of negative charges or addition of a positive charge in the Cx26 equivalent region reduced the slow gate voltage dependence. In Cx50, the addition of a glutamate in the same region decreased the voltage dependence, and the neutralization of a negative charge increased it. These results indicate that the charges at the end of TM-1 are part of the slow gate voltage sensor in Cxs. The fact that Cx42, which has no charge in this region, still presents voltage-dependent slow gating suggests that charges still unidentified also contribute to the slow gate voltage sensitivity.

Highlights

The voltage sensor domain of Cxs has not been clearly established, there is evidence suggesting that residues lining the pore of the channel are involved in transjunctional voltage dependence in gap junctions (GJs) [11]
Cx26 GJ closes at positive potentials, and Cx32 closes at negative potentials
This study specially focused on charges of the N terminus and TM-1 regions that were previously associated with voltage dependence and gating of Cxs (Fig. 1) [17, 22]

Summary

Introduction

The voltage sensor domain of Cxs has not been clearly established, there is evidence suggesting that residues lining the pore of the channel are involved in transjunctional voltage dependence in GJs [11]. In slow gating the channel transits through several intermediate conductances between the open and the fully closed states [14] This has been related to a turn of the 310-helix region at the end of TM-1 [3, 15]. Insertion of negatively charged residues up to the 10th position of the N terminus of Cx32 can reverse the polarity of the fast gate, suggesting that in this region of the. The slow gating mechanism in Cx26 hemichannels has been associated with a conformational change of the TM-1/EL-1 region of the protein This change is most likely a rotation of the helix, which would close the channel [22]. Mutations in this region are known to affect the voltage dependence of GJCs, but their particular effects on slow gating are unknown [10, 18]

Results

Discussion

Conclusion