Research Article

Structure of the voltage-gated calcium channel Cav1.1 complex

+ See all authors and affiliations

Science  18 Dec 2015:
Vol. 350, Issue 6267, aad2395
DOI: 10.1126/science.aad2395

You are currently viewing the abstract.

View Full Text

A complex channel comes into focus

Voltage-gated calcium (Cav) channels are activated in response to membrane potential to initiate calcium-mediated signaling pathways and are associated with diseases such as cardiac arrhythmia and epilepsy. Cav1.1 couples changes in membrane potential to cardiac muscle contraction. It comprises a core subunit and three auxiliary subunits. Wu et al. isolated the Cav1.1 complex from rabbit skeletal muscle and determined its structure by single-particle electron cryomicroscopy using direct electron detection and advanced image processing. The detailed architecture of the pseudotetrameric eukaryotic Cav channel in complex with its auxiliary subunits provides an important framework for understanding the function and disease mechanisms of related channels.

Science, this issue p. 10.1126/science.aad2395

Structured Abstract

INTRODUCTION

Voltage-gated Ca2+ (Cav) channels activate in response to membrane potential changes and initiate Ca2+-mediated cellular signaling cascades. The L-type, high voltage–activated Cav channel Cav1.1 is specialized for the excitation-contraction (E-C) coupling that causes contraction of skeletal muscles in response to changes in membrane potential. The action potential–induced conformational changes of Cav1.1 activate the type 1 ryanodine receptor (RyR1), which releases Ca2+ from the sarcoplasmic reticulum and subsequently triggers muscle contraction. The Cav1.1 complex consists of the pore-forming subunit α1 and auxiliary subunits α2δ, β, and γ that modulate the membrane trafficking, current kinetics, and gating properties of the Cav channels. The Cav channels represent important drug targets because of their association with diseases such as hypokalemic periodic paralysis, cardiac arrhythmia, and epileptic seizure. The α2δ-1 subunit is directly targeted by the gabapentinoid drugs used for conditions such as epilepsy and neuropathic pain. In contrast to the homotetrameric Kv channels, the α1 subunit of eukaryotic Cav and Nav channels consists of one single polypeptide chain that is organized into four repeated domains (I to IV) of six transmembrane helices (S1 to S6). The large size, pseudo-symmetry, and heavy glycosylation of the Cav and Nav channels are major impediments to the use of x-ray crystallography for structural elucidation.

RATIONALE

We purified endogenous Cav1.1 complex from rabbit skeletal muscle membranes with the use of glutathione S-transferase (GST)–fused β1a, which is an exclusive auxiliary subunit of Cav1.1. Structural determination was achieved using single-particle cryo–electron microscopy (cryo-EM). The structural model was generated by homology modeling, rigid-body docking, and de novo model building. The identification of the four homologous repeats in the α1 subunit was achieved through analysis of the distinctive extracellular loops of the pore domain, unique sequence patterns, and mass spectrometric analysis of cross-linked samples.

RESULTS

The EM density map for the rabbit Cav1.1 complex was reconstructed to 4.2 Å resolution according to the gold-standard Fourier shell correlation (FSC) 0.143 criterion. The overall structure is approximately 170 Å in height and 100 Å in the longest dimension of the width. The α1 subunit folds into four homologous repeats, each exhibiting the voltage-gated ion channel fold formed by S1 to S6. The S5 and S6 segments from the four repeats constitute the pore domain, whereas the S1 to S4 segments in each repeat form the voltage-sensing domain (VSD). The four-fold symmetry of the pore domain is disrupted by the distinct extracellular loops (designated L5 loops for those between S5 and the P1 helix, and L6 loops for those connecting the P2 helix and S6) and by the slightly different conformations of the S5 and S6 segments among the four repeats. The S6IV segment is immediately followed by a cytosolic C-terminal domain. The selectivity filter is constituted by the side chains of four critical Glu residues and the carbonyl oxygen atoms of the two preceding residues in each repeat. The clockwise arrangement of the four repeats in the extracellular view may be conserved in all eukaryotic Cav and Nav channels.

The four transmembrane helix–containing γ subunit, which exhibits structural similarity to claudins, interacts with VSDIV, whereas the cytosolic β subunit is located adjacent to VSDII of α1. On the extracellular side, the α2 subunit interacts with the extracellular loops of repeats I to III through its von Willebrand A (VWA) and Cache1 domains. The α2 residue Arg243, which has been implicated in binding to the drug pregabalin, is in the Cache1 domain and covers a central pocket that may accommodate ligands.

CONCLUSION

Our study reveals the detailed architecture of a pseudo-tetrameric eukaryotic Cav channel in complex with its auxiliary subunits. Although molecular understanding of ion selectivity and voltage gating will require improved resolution, the present structural analysis provides an important framework for understanding the function and disease mechanisms of related Cav and Nav channels. The intersubunit interfaces identified in the complex structure provide the basis for molecular interpretation of α1 modulation by the auxiliary subunits.

The cryo-EM structure of the rabbit voltage-gated Ca2+ channel Cav1.1 complex at a nominal resolution of 4.2 Å.

The overall EM density map on the left is colored according to different subunits. The structure model on the right is domain-colored. The glycosyl moieties are shown as sticks. The putative Ca2+ is shown as a green sphere.

Abstract

The voltage-gated calcium channel Cav1.1 is engaged in the excitation-contraction coupling of skeletal muscles. The Cav1.1 complex consists of the pore-forming subunit α1 and auxiliary subunits α2δ, β, and γ. We report the structure of the rabbit Cav1.1 complex determined by single-particle cryo–electron microscopy. The four homologous repeats of the α1 subunit are arranged clockwise in the extracellular view. The γ subunit, whose structure resembles claudins, interacts with the voltage-sensing domain of repeat IV (VSDIV), whereas the cytosolic β subunit is located adjacent to VSDII of α1. The α2 subunit interacts with the extracellular loops of repeats I to III through its VWA and Cache1 domains. The structure reveals the architecture of a prototypical eukaryotic Cav channel and provides a framework for understanding the function and disease mechanisms of Cav and Nav channels.

View Full Text