Molecular interaction and functional regulation of connexin50 gap junctions by calmodulin

Abstract

Cx50 (connexin50), a member of the α-family of gap junction proteins expressed in the lens of the eye, has been shown to be essential for normal lens development. In the present study, we identified a CaMBD [CaM (calmodulin)-binding domain] (residues 141-166) in the intracellular loop of Cx50. Elevations in intracellular Ca2+ concentration effected a 95% decline in gj (junctional conductance) of Cx50 in N2a cells that is likely to be mediated by CaM, because inclusion of the CaM inhibitor calmidazolium prevented this Ca2+-dependent decrease in gj. The direct involvement of the Cx50 CaMBD in this Ca2+/CaM-dependent regulation was demonstrated further by the inclusion of a synthetic peptide encompassing the CaMBD in both whole-cell patch pipettes, which effectively prevented the intracellular Ca2+-dependent decline in gj. Biophysical studies using NMR and fluorescence spectroscopy reveal further that the peptide stoichiometrically binds to Ca2+/CaM with an affinity of ~5 nM. The binding of the peptide expanded the Ca2+-sensing range of CaM by increasing the Ca2+ affinity of the C-lobe of CaM, while decreasing the Ca2+ affinity of the N-lobe of CaM. Overall, these results demonstrate that the binding of Ca2+/CaM to the intracellular loop of Cx50 is critical for mediating the Ca2+-dependent inhibition of Cx50 gap junctions in the lens of the eye.

Figure 1. Cxs membrane topology and the putative CaM-binding sites

The α-class of Cxs are composed of four transmembrane segments, two extracellular loops, one cytoplasmic loop, a short N-terminus and a much longer C-terminal tail. The predicted CaM-binding sites are located in the second half of the intracellular loop between TM2 and TM3. The numeric score (1–9) represents the probability of an accurate prediction of high-affinity CaM-binding sites. The CaM-binding sequences identified in the α-class Cxs are similar to those of CaMKII. All of the aligned sequences fit the 1–5–10 CaM-binding mode subclass, where each number represents the presence of a hydrophobic residue. m, mouse; h, human; s, sheep; r, rat; #, hydrophobic residues (highlighted in grey); B, basic residues (underscored).

Figure 2. Ca²⁺- and CaM-dependence of Cx50 gap junction uncoupling

(A) Cx50-N2a cell pair superfusion with 1 μM ionomycin bath saline with or without 1.8 mM CaCl₂ demonstrates a time-dependent uncoupling of Cx50 gap junctions that requires 1.8 mM [Ca²⁺]_o. (B) Pre-treatment with 2 mM CDZ for 10–15 min before patch-clamp analysis completely prevented the 1.8 mM [Ca²⁺]_o-dependent uncoupling response. (C) Inclusion of 1 μM Cx50p^141–166 in both whole-cell patch pipettes prevented 80 % of the Ca²⁺/CaM-dependent decline in Cx50 G_j. In contrast, experiments performed with SCx50p prevented only 20 % of the 1.8 mM [Ca²⁺]_o–CaM-dependent reduction in Cx50 G_j. The average ± S.E.M. initial g_j values for all experimental groups are displayed in Table 1.

Figure 3. Mechanistic basis for Ca²⁺/CaM-dependent Cx50 gap junction uncoupling

(A) The product of the number of open channels (N) and open probability (P _o) from two poorly coupled Cx50-N2a cell pairs is plotted as a function of time and 1 μM ionomycin +1.8 mM [Ca²⁺]_o superfusion. N · P_o declined from an average control value of 1.21 open channels to 0 within 2 min of ionomycin/CaCl₂ saline superfusion. (B) Cx50 gap junction channel current recordings from one experiment are shown for the time points indicated by the circles in (A). The number and duration of the open channels declined progressively to 0 without any apparent reduction in the single-channel conductance (314 pS), indicative of closure of a Ca²⁺–CaM-dependent gate without a block of the ion-permeation pathway.

Figure 4. Monitoring the interaction between CaM and Cx50p^141–166 by (¹H,¹⁵N)-HSQC spectroscopy

(A) An overlay of HSQC spectra of holo-CaM (black) with the spectrum of the holo-CaM–Cx50p^141–166 (grey). (B) An overlay of the HSQC spectra of holo-CaM (black) with the spectrum of the holo-CaM–SCx50p (grey). (C) Chemical-shift perturbation in CaM induced by addition of 2-fold molar excess of Cx50p^141–166. The weight-average chemical-shift change (Δδ) was calculated using eqn (5). Residues with Δδ> 0.1 p.p.m. were mapped to the three-dimensional structure of holo-CaM (PDB code 3CLN). (D) The chemical-shift change of Gly³³ during titration of holo-CaM with Cx50p^141–166. The disappearance of the peak (free form) was accompanied by the appearance of the corresponding peak (bound form) at a new position. (E) Structural basis of the difference in directionality of chemical shift change. The structure of the CaM–CaMKII complex (PDB code 1CDM) is shown with the peptide (inside) and the CaM residues (outside). Leu¹¹⁶ and Met¹⁴⁵ of CaM are within 5 Å of Lys²⁹⁸p and Lys³⁰⁰p of the peptide respectively. These peptide positions correspond to Lys¹⁴⁶p and Arg¹⁴⁸p for Cx43, and Arg¹⁴⁹p and Glu¹⁵¹p for Cx50, as can be seen in the superposition of their sequences. In Cx50, Glu¹⁵¹p may be pulled away from Met¹⁴⁵ by Lys¹⁴⁷p. In contrast, in Cx43, Arg¹⁴⁸p may be pushed towards Met¹⁴⁵ by Lys¹⁴⁴p. These differences in peptide sequences can cause changes of chemical shifts in different directions.

Figure 5. Hydrodynamic radii of CaM–Cx50p^141–166 complex determination by pulse-field gradient NMR

The NMR signal decay of holo-CaM (○) and holo-CaM–Cx50p^141–166 complex (●) as a function of field strength. The calculated hydrodynamic radii of the CaM and complexes are indicated on the top.

Figure 6. CD studies of the interaction between CaM and Cx50p^{141 – 166}

(A) Far-UV CD spectra of CaM in the presence of 5 mM EGTA (○, apo-CaM) or CaCl₂ (□, holo-CaM), and a CaM–Cx50p^141–166 (1:1) mixture with 5 mM EGTA (●, apo-CaM–Cx50p^141–166) or CaCl₂ (■, holo-CaM–Cx50p^141–166). (B) Far-UV spectra of the synthetic peptide Cx50p^141–166 with addition of TFE (0–80 %). The inset shows calculated α-helical content as a function of TFE concentration for the peptides Cx50p^141–166 (■), Cx44p^129–150 (▲) and Cx43p^136–158 (●). (C) Far-UV spectra of the synthetic peptide Cx50p^141–166 with (●) or without (○) addition of CaM.

Figure 7. Interaction of Cx50p^141–166 with D-CaM monitored by steady-state fluorescence

(A) The titration curve of D-CaM (2 μM) with Cx50p^141–166 in the presence of 5 mM Ca²⁺ in a buffer consisting of 50 mM Tris/HCl (pH 7.5) and 100 mM KCl. The inset shows the fluorescence spectrum of D-CaM in the absence (broken line) or the presence (continuous line) of an equivalent molar concentration of Cx50p^141–166. (B) Titration curve of D-CaM (2 μM) with Cx50p^141–166 in the presence of 5 mM EGTA in a buffer consisting of 50 mM Tris/HCl (pH 7.5) and 100 mM KCl. The inset shows the fluorescence spectrum of D-CaM in the absence (broken line) or the presence (continuous line) of an equivalent molar concentration of Cx50p^141–166. (C) pH-dependence of Cx50p^141–166 and Cx44p^129–150 binding to CaM. The binding affinities were derived from the peptide titration curves of D-CaM at various pH values. All experiments were conducted in triplicate.

Figure 8. Domain-specific equilibrium Ca²⁺ titration of CaM (○) and CaM in complex with Cx50p^141–166 (●)

(A) The intrinsic phenylalanine fluorescence (λ_ex = 250 nm; λ_em = 280 nm) was monitored to report the equilibrium Ca²⁺-binding constants of the N-lobe of CaM. (B) The intrinsic tyrosine fluorescence (λ_ex = 277 nm; λ_em = 320 nm) was monitored to report the equilibrium Ca²⁺-binding constants of the C-lobe of CaM. The free ionized Ca²⁺ concentration was measured using the Ca²⁺ indicator dye Oregon Green 488 BAPTA-5N. All experiments were conducted in triplicate in 50 mM Hepes (pH 7.5), 100 mM KCl, 5 mM NTA and 0.5 mM EGTA.