Sites on Calmodulin That Interact with the C-terminal Tail of Cav1.2 Channel

John A. Putkey,Quinn K. Kleerekoper,Susan L. Hamilton,Liangwen Xiong,Rong He

doi:10.1074/jbc.m410558200

Abstract

Two fragments of the C-terminal tail of the alpha(1) subunit (CT1, amino acids 1538-1692 and CT2, amino acids 1596-1692) of human cardiac L-type calcium channel (Ca(V)1.2) have been expressed, refolded, and purified. A single Ca(2+)-calmodulin binds to each fragment, and this interaction with Ca(2+)-calmodulin is required for proper folding of the fragment. Ca(2+)-calmodulin, bound to these fragments, is in a more extended conformation than calmodulin bound to a synthetic peptide representing the IQ motif, suggesting that either the conformation of the IQ sequence is different in the context of the longer fragment, or other sequences within CT2 contribute to the binding of calmodulin. NMR amide chemical shift perturbation mapping shows the backbone conformation of calmodulin is nearly identical when bound to CT1 and CT2, suggesting that amino acids 1538-1595 do not contribute to or alter calmodulin binding to amino acids 1596-1692 of Ca(V)1.2. The interaction with CT2 produces the greatest changes in the backbone amides of hydrophobic residues in the N-lobe and hydrophilic residues in the C-lobe of calmodulin and has a greater effect on residues located in Ca(2+) binding loops I and II in the N-lobe relative to loops III and IV in the C-lobe. In conclusion, Ca(2+)-calmodulin assumes a novel conformation when part of a complex with the C-terminal tail of the Ca(V)1.2 alpha(1) subunit that is not duplicated by synthetic peptides corresponding to the putative binding motifs.

Highlights

Calmodulin (CaM),1 a ubiquitous Ca2ϩ sensor, directly or indirectly regulates excitation-contraction coupling and other important physiological functions in cardiac myocytes [3,4,5,6,7]
Nuclear Magnetic Resonance Spectroscopy (NMR) amide chemical shift perturbation mapping shows the backbone conformation of calmodulin is nearly identical when bound to CT1 and CT2, suggesting that amino acids 1538 –1595 do not contribute to or alter calmodulin binding to amino acids 1596 –1692 of CaV1.2
Peterson et al [22] found a four amino acid cluster (VVTL) within the EF hand to be essential for Ca2ϩ-dependent inactivation (CDI)

Summary

Introduction

Calmodulin (CaM), a ubiquitous Ca2ϩ sensor, directly or indirectly regulates excitation-contraction coupling and other important physiological functions in cardiac myocytes [3,4,5,6,7]. Cardiac L-type Ca2ϩ channels (CaV1.2) are modulated by the interaction of the channel ␣1 subunit C-terminal tail with CaM [8], such that CaM binding to this region is required for both. Peterson et al [22] and De Leon et al [23] identified critical determinants of CDI within the consensus Ca2ϩ binding motif (the EF hand) of the cardiac L-type Ca2ϩ channel. Zhou et al [24] and Qin et al [13] maintained that CDI of CaV1.2 did not require the EF hand motif. Additional data to define the CaM binding site on the Cterminal tail of the ␣1 subunit and to elucidate the mechanisms of CDI and CDF of CaV1.2 are needed. We describe the expression of fragments of the ␣1 subunit C-terminal domain and provide new details of its interaction with CaM

Methods

Results

Conclusion