Insights into voltage-gated calcium channel regulation from the structure of the CaV1.2 IQ domain-Ca2+/calmodulin complex

Abstract

Changes in activity-dependent calcium flux through voltage-gated calcium channels (Ca(V)s) drive two self-regulatory calcium-dependent feedback processes that require interaction between Ca(2+)/calmodulin (Ca(2+)/CaM) and a Ca(V) channel consensus isoleucine-glutamine (IQ) motif: calcium-dependent inactivation (CDI) and calcium-dependent facilitation (CDF). Here, we report the high-resolution structure of the Ca(2+)/CaM-Ca(V)1.2 IQ domain complex. The IQ domain engages hydrophobic pockets in the N-terminal and C-terminal Ca(2+)/CaM lobes through sets of conserved 'aromatic anchors.' Ca(2+)/N lobe adopts two conformations that suggest inherent conformational plasticity at the Ca(2+)/N lobe-IQ domain interface. Titration calorimetry experiments reveal competition between the lobes for IQ domain sites. Electrophysiological examination of Ca(2+)/N lobe aromatic anchors uncovers their role in Ca(V)1.2 CDF. Together, our data suggest that Ca(V) subtype differences in CDI and CDF are tuned by changes in IQ domain anchoring positions and establish a framework for understanding CaM lobe-specific regulation of Ca(V)s.

Figure 1

Structure of the Ca²⁺/CaM–Ca_V1.2 IQ domain complex. (a) Ribbon diagram of the complex. Green, CaM Ca²⁺/N lobe; blue, Ca²⁺/C lobe; red, IQ domain, with residues Ile1624 and Q1625 from complex A in stick representation; darker shades, complex A; lighter shades, complex C. The complexes were superposed using the Ca²⁺/C lobes and IQ domains. Labels indicate termini of components in complex A. Dashed lines indicate regions absent from the structures. (b) 90° rotation of a. The shift in Ca²⁺/N lobe Ca²⁺ position in EF-hand 2 is indicated. (c) Sequence alignment of IQ regions from each Ca_V1 and Ca_V2 isoform. Zigzag, α-helical regions; straight line, nonhelical residues; dashed lines, residues present in the crystallized construct but absent from the electron density. Residues contacting CaM (≤4 Å) for conformation A and conformation C are highlighted as follows: green, contacts to Ca²⁺/N lobe; cyan, Ca²⁺/C lobe; purple, Ca²⁺/N lobe and Ca²⁺/C lobe. Asterisks indicate principal aromatic anchor positions. Sequences are human Ca_V1.2 1609–1647, human Ca_V1.1 1514–1552, rat Ca_V1.3 1641–1679, human Ca_V1.4 1563–1602, human Ca_V2.1 1947–1985, human Ca_V2.2 1845–1883 and rat Ca_V2.3 1811–1850.

Figure 2

Lobe-specific Ca²⁺/CaM–Ca_V1.2 IQ domain interactions. (a) Ca²⁺/CaM C lobe from complex A bound to the IQ domain. Buried surface area = 1,819 Ų (965 Ų hydrophobic). (b) Ca²⁺/CaM N lobe from complex A bound to the IQ domain. Buried surface area = 1,450 Ų (743 Ų hydrophobic). (c) Ca²⁺/CaM N lobe from complex C bound to the IQ domain. Buried surface area = 1,491 Ų (500 Ų hydrophobic). IQ domain is shown in stick representation with aromatic anchor residues in white. CaM lobes are shown in surface representation with residues that contribute hydrophobic (yellow), negatively charged (red), positively charged (blue) and polar (green) side chain contacts (≤4 Å) to the IQ domain indicated. Select residues are labeled to orient the reader. IQ domain residue labels are boxed.

Figure 3

ITC characterization of Ca²⁺/CaM–Ca_V1.2 IQ domain interactions. (a) 70 μM IQ domain into 7 μM Ca²⁺/C lobe. (b) 500 μM Ca²⁺/N lobe into 50 μM IQ domain. (c) 200 μM Ca²⁺/N lobe into a solution of 20 μM IQ domain and 37 μM C lobe. (d) 60 μM IQ domain F1628A mutant into 6 μM Ca²⁺/C lobe. (e) 500 μM Ca²⁺/N lobe into a solution of 5 μM IQ domain F1628A mutant. Isotherms are fit to a single binding site model for a and d and a double binding site model for b and e. Panels show addition of 10 μl aliquots of titrant into the target solution (top) and binding isotherms (bottom).

Figure 4

Lobe-specific interactions affect CDI and CDF. (a) Voltage-activated Ba²⁺ and Ca²⁺ currents from wild-type (WT) and TripleA channels during a 600-ms depolarizing step from −90 mV to +20 mV. Traces are normalized to the peak current to facilitate comparison. Tail currents are not shown. (b) r₃₀₀ values (current fraction 300 ms after depolarization) in Ba²⁺ (black) and Ca²⁺ (grey). Error bars show s.d. (c) I1624A and I1624A TripleA Ca²⁺ currents in a 3-Hz 40-pulse train (50-ms steps to +20-mV from −90-mV holding potential). The first pulse currents are scaled to the same level for comparison. (d) Relative current increase between last and first pulses for I1624A and I1624A TripleA. Error bars show s.d.

Figure 5

Schematic of Ca²⁺/CaM lobe multiple binding modes on the Ca_V1.2 IQ domain. (a) Ca²⁺/N lobe (green) has medium-affinity and low-affinity binding sites on the IQ domain. (b) Ca²⁺/C lobe (blue) has a high-affinity binding site on the IQ domain. (c) Binding of Ca²⁺/C lobe to the IQ domain blocks Ca²⁺/N lobe access to the Ca²⁺/N lobe medium-affinity site (black X) and reduces Ca²⁺/N lobe binding to the Ca²⁺/N lobe low-affinity site (grey X). (d) Representation of how Ca²⁺/C lobe binding to the IQ domain tethers Ca²⁺/N lobe near the Ca²⁺/N lobe low-affinity site. Yellow line indicates the CaM interlobe linker. In all panels, the approximate position of aromatic anchor Phe1628, which is shared by the Ca²⁺/N lobe medium-affinity site and the Ca²⁺/C lobe high-affinity site, is indicated by the white hexagon.