Structural studies of N-terminal mutants of Connexin 26 and Connexin 32 using (1)H NMR spectroscopy

Abstract

Alterations in gap junctions underlie the etiologies of syndromic deafness (KID) and Charcot-Marie Tooth disease (CMTX). Functional gap junctions are composed of connexin molecules with N-termini containing a flexible turn around G12, inserting the N-termini into the channel pore allowing voltage gating. The loss of this turn correlates with loss of Connexin 32 (Cx32) function by impaired trafficking to the cell membrane. Using (1)H NMR we show the N-terminus of a syndromic deafness mutation Cx26G12R, producing "leaky channels", contains a turn around G12 which is less structured and more flexible than wild-type. In contrast, the N-terminal structure of the same mutation in Cx32 chimera, Cx32*43E1G12R shows a larger constricted turn and no membrane current expression but forms membrane inserted hemichannels. Their function was rescued by formation of heteromeric channels with wild type subunits. We suggest the inflexible Cx32G12R N-terminus blocks ion conduction in homomeric channels and this channel block is relieved by incorporation of wild type subunits. In contrast, the increased open probability of Cx26G12R hemichannels is likely due to the addition of positive charge in the channel pore changing pore electrostatics and impairing hemichannel regulation by Ca(2+). These results provide mechanistic information on aberrant channel activity observed in disease.

Keywords: Connexin; Deafness; NMR; Peptides; Structure-function.

Figure 1

The two-dimensional ¹H NMR spectrum of the Cx32G12R mutant peptide showing labeled NOE interactions between aliphatic and aromatic side chain protons.

Figure 2

A summary of NOE restraints for the Cx26 WT, Cx26G12R and Cx32G12R mutant N-terminal peptides. The horizontal lines indicate backbone NOES with the thickness representing intensity.

Figure 3

Plots of ¹³C_α secondary chemical shifts (Δd¹³C = δ_obs - δ_randomcoil,, ppm) for Cx26, Cx26G12R, Cx32 and Cx32G12R peptides.

Figure 4

a) Plots of temperature coefficients vs. residue number for the Cx26 WT (top) and mutants Cx26G12R (middle) and Cx32G12R (lower). Temperature coefficients were only calculated for residues whose NH proton was observable at all temperatures. b) Temperature coefficients of residues 11–15 for the wild-type Cx26, mutant Cx26G12R and Cx32G12R peptides. The error bars represent errors of 0.15 ppb/K.

Figure 4

a) Plots of temperature coefficients vs. residue number for the Cx26 WT (top) and mutants Cx26G12R (middle) and Cx32G12R (lower). Temperature coefficients were only calculated for residues whose NH proton was observable at all temperatures. b) Temperature coefficients of residues 11–15 for the wild-type Cx26, mutant Cx26G12R and Cx32G12R peptides. The error bars represent errors of 0.15 ppb/K.

Figure 5

The 20 lowest energy conformers of the Cx26 WT, Cx26G12R and Cx32G12R mutants are shown. Superpositions of the backbone atoms within residues 3–15 are shown for all the structures.

Figure 6

Representative ribbon diagrams of the Cx26 WT, Cx26G12R, Cx32G12R and Cx32 N-terminal peptides.

Figure 7

Expression of G12R in Xenopus oocytes. Panel A. Western blot of Xenopus oocyte membrane proteins. Lane A. N2E Cx32*43E1 247 6 His. Lane B. Cx32*43E1 (wild type). Lane C. Cx32*43E1 G12R. Lane D. Oocytes coexpressing N2E Cx32*43E1 247 6 His and Cx32*43E1 G12R. Cx32*43E1 is a Cx32 chimera in which the first extracellular loop (E1, residues 41–70) has been replaced with that of Cx43 [46]. N2E Cx32*43E1 247 6 His denotes a Cx32*43E1 construct to which a hexa-histidine tag was attached at residue 247. Wild type Cx32 has an electrophoretic mobility that corresponds to ~ 27 kDa; the his-tagged truncation to ~ 23 kDa. Xenopus oocyte yolk proteins that bind trace amounts of either primary and/or secondary antibodies are marked by asterisks. The western blot in lane D corresponds to a “pull down” assay of heteromeric channels containing G12R subunits by IMAC affinity purification of channels containing his-tagged “wild type” (N2E Cx32*43E1 247 6 His) subunits. The presence of full length Cx32 (G12R) protein indicates formation of heteromeric hemichannels. Panel B. Representative current traces obtained from oocytes voltage clamped to 0 mV and stepped from +50 to −100 mV in 10 mV increments. The currents shown in the left panel correspond to endogenous currents in oocytes expressing Cx32*43E1 G12R protein. No currents attributable to connexin channels are observed. The center panel shows currents from an oocyte co-injected with equal amounts of G12R and N2E Cx32*43E1 247 6 His RNA. The right panel shows currents from an oocyte expressing N2E Cx32*43E1 247 6 His protein. Currents elicited by depolarization from 0 mV are colored red (0 to 50 mV). Current relaxations at potentials more positive than 30 mV in the center and left panels result from closure of voltage-dependent V_j-gates and demonstrate expression of N2E Cx32*43E1 247 6 His subunits (see text). Current relaxations at inside negative potentials correspond to loop-gate closure in homomeric N2E Cx32*43E1 247 6 His channels (right panel).