Monica M. Lurtz

Identification of the Calmodulin Binding Domain of Connexin 43

Calmodulin (CaM) has been implicated in mediating the Ca2+-dependent regulation of gap junctions. This report identifies a CaM-binding motif comprising residues 136–158 in the intracellular loop of Cx43. A 23-mer peptide encompassing this CaM-binding motif was shown to bind Ca2+-CaM with 1:1 stoichiometry by using various biophysical approaches, including surface plasmon resonance, circular dichroism, fluorescence spectroscopy, and NMR. Far UV circular dichroism studies indicated that the Cx43-derived peptide increased its α-helical contents on CaM binding. Fluorescence and NMR studies revealed conformational changes of both the peptide and CaM following formation of the CaM-peptide complex. The apparent dissociation constant of the peptide binding to CaM in physiologic K+ is in the range of 0.7–1 μm. Upon binding of the peptide to CaM, the apparent Kd of Ca2+ for CaM decreased from 2.9 ± 0.1 to 1.6 ± 0.1 μm, and the Hill coefficient nH increased from 2.1 ± 0.1 to 3.3 ± 0.5. Transient expression in HeLa cells of two different mutant Cx43-EYFP constructs without the putative Cx43 CaM-binding site eliminated the Ca2+-dependent inhibition of Cx43 gap junction permeability, confirming that residues 136–158 in the intracellular loop of Cx43 contain the CaM-binding site that mediates the Ca2+-dependent regulation of Cx43 gap junctions. Our results provide the first direct evidence that CaM binds to a specific region of the ubiquitous gap junction protein Cx43 in a Ca2+-dependent manner, providing a molecular basis for the well characterized Ca2+-dependent inhibition of Cx43-containing gap junctions.

Calmodulin mediates the Ca2+-dependent regulation of Cx44 gap junctions.

We have shown previously that the Ca2+-dependent inhibition of lens epithelial cell-to-cell communication is mediated in part by the direct association of calmodulin (CaM) with connexin43 (Cx43), the major connexin in these cells. We now show that elevation of [Ca2+](i) in HeLa cells transfected with the lens fiber cell gap junction protein sheep Cx44 also results in the inhibition of cell-to-cell dye transfer. A peptide comprising the putative CaM binding domain (aa 129-150) of the intracellular loop region of this connexin exhibited a high affinity, stoichiometric interaction with Ca2+-CaM. NMR studies indicate that the binding of Cx44 peptide to CaM reflects a classical embracing mode of interaction. The interaction is an exothermic event that is both enthalpically and entropically driven in which electrostatic interactions play an important role. The binding of the Cx44 peptide to CaM increases the CaM intradomain cooperativity and enhances the Ca2+-binding affinities of the C-domain of CaM more than twofold by slowing the rate of Ca2+ release from the complex. Our data suggest a common mechanism by which the Ca2+-dependent inhibition of the alpha-class of gap junction proteins is mediated by the direct association of an intracellular loop region of these proteins with Ca2+-CaM.

Molecular interaction and functional regulation of connexin50 gap junctions by calmodulin.

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.

Ligand binding methods for analysis of ion channel structure and function.

Publisher Summary The nicotinic acetylcholine receptor (AChR) is an ion channel that is opened by the binding of two molecules of acetylcholine on its extracellular surface. On prolonged exposure to acetylcholine, the channel desensitizes and acquires high affinity for acetylcholine. The equilibrium binding to the two sites appears weakly cooperative, but the two sites are distinct, as shown by the binding of various antagonists that preferentially bind one site versus the other. The sites are allosterically linked to a binding site located within the channel pore itself, the noncompetitive antagonist site. Binding to this site can alter the conformation in favor of either the resting conformation or the desensitized conformation, depending on the ligand. Desensitization, as induced by noncompetitive antagonists, is also marked by increased affinity for agonist binding to the acetylcholine sites. The ability of many cholinergic ligands to bind all three sites further complicates analyses of the linkage. An increased awareness of these phenomena has permitted binding to the AChR to be understood in more detail, and binding assays are finding greater use in elucidating the structure of the binding sites. This chapter describes several ligand binding techniques: radioligand binding by centrifugation assay, radioligand binding by DE-81 filter binding, and fluorescent ligand binding. In addition, the chapter discusses the way direct binding measurements, indirect binding by competition, and noncompetitive allosteric effects can be analyzed.