Three-dimensional structure of the KChIP1–Kv4.3 T1 complex reveals a cross-shaped octamer

Abstract

Brain I A and cardiac I to currents arise from complexes containing Kv4 voltage-gated potassium channels and cytoplasmic calcium-sensor proteins (KChIPs). Here, we present X-ray crystallographic and small-angle X-ray scattering data that show that the KChIP1-Kv4.3 Nterminal cytoplasmic domain complex is a cross-shaped octamer bearing two principal interaction sites. Site 1 comprises interactions between a unique Kv4 channel N-terminal hydrophobic segment and a hydrophobic pocket formed by displacement of the KChIP H10 helix. Site 2 comprises interactions between a T1 assembly domain loop and the KChIP H2 helix. Functional and biochemical studies indicate that site 1 influences channel trafficking, whereas site 2 affects channel gating, and that calcium binding is intimately linked to KChIP folding and complex formation. Together, the data resolve how Kv4 channels and KChIPs interact and provide a framework for understanding how KChIPs modulate Kv4 function.In biological systems, electrical information is encoded and processed by changes in actionpotential timing, duration, frequency, waveform and number 1 . Modulation of voltage-gated potassium channels is central to these events and affects heart rate, sensory transduction and cognition. The ion transport 2 and voltage-dependent gating properties 3 of the potassium channel α subunits that form the ion conduction pathway are well characterized. Although α subunits form the pore, many channels function as complexes that require cytoplasmic and transmembrane auxiliary subunits 4 . To date, little is known regarding the way in which such components bind α subunits and modulate channel action. Understanding the interplay between regulatory components and pore-forming domains is crucial for unraveling how modulatory signals 4 and homeostatic mechanisms 5 allow excitable cells to sense and respond to environmental cues. Two potassium currents, I A (ref. 6 ) and I to (ref. 7 ), exert strong control over neuronal and cardiac excitability, respectively, and provide clear examples of the importance of auxiliary subunits for tuning properties of α subunits. Both currents arise from complexes of Kv4 The structure of the KChIP1-Kv4.3 T1 complex shows that the assembly forms a crossshaped octamer having the T1 tetramer at the center (Fig. 1a, chains A-D) and individual KChIPs extending radially (Fig. 1a, chains E-H). The asymmetric unit has two octameric complexes that are apposed on the cytoplasmic T1 faces (Fig. 1b). T1 shows few differences from the isolated Kv4.3 T1 structure 17 (r.m.s. deviation = 0.64 Å 2 over 408 Cα positions). In contrast, KChIP1 shows substantial differences (r.m.s. deviation = 4.03 Å 2 over 179 Cα positions) from isolated KChIP1 (ref. 17 ).The structure reveals two main interaction sites. Site 1 buries ~2,100 Å 2 total surface area between residues 3 and 21 of a conserved hydrophobic segment that is on the N-terminal side of T1 (called T1N; Fig. 2a) and a large hydrophobic pocket (28 Å long, 12 Å deep, 10 Å wide) formed by KChIP1 (Fig. 2b,c...

Kv channel-interacting proteins as neuronal and non-neuronal calcium sensors

“…Taken together, these findings suggest that in KChIPs EF2 mediates low affinity Ca 2+ binding and is occupied by Mg 2+ under physiological conditions, whereas EF3 and EF4 mediate high affinity Ca 2+ binding [34–37,39]. The data are in accordance with crystal structure and NMR analysis, which showed only two bound Ca 2+ ions per KChIP subunit residing in EF3 and EF4 (Figure 2) [30–33]. Obviously, EF3 and EF4 are critical sites for a putative calcium sensor function of the KChIPs.…”

“…The conserved NCS core domain contains four EF-hand motifs (EF1, EF2, EF3 and EF4), a feature known from calmodulin, but in all NCS proteins the most N-terminal EF-hand motif (EF1) is degenerated and cannot bind Ca 2+ [14]. Recoverin [26], KChIP1 [30–32] and KChIP3 [33] were even isolated with only two Ca 2+ ions bound (see Figure 2 and next section). The Ca 2+ sensitivity of the NCS proteins is very high.…”

“…Lower picture: Rotating the KChIP1 structure by about 180° around the vertical axis (N-terminus on the right) offers a better view of the H10 α-helix (green), which lines the hydrophobic groove. (B) Crystal structure of a KChIP1 molecule when bound to Kv4.3 (PDB 2I2R) [30]. Positioning according to the N-terminal H1 and H2 α-helices of the lower picture in A suggests conformational changes, especially of the C-terminal H10 α-helix which is moved aside for target molecule binding.…”

“…Ca 2+ withdrawal from KChIP1 [30], KChIP2 [38] and KChIP3 [39] has been shown to cause a reduced helical content and increased flexibility of these proteins. Even an EF3,EF4 fragment (amino acid 161 – 256) of KChIP3 showed a reduced spectral resolution in the absence of Ca 2+ , corroborating the central role of EF3 and EF4 in Ca 2+ binding and associated conformational changes [40].…”

“…As described above, Ca 2+ -dependent complex formation undoubtedly underlies the function of recoverin in retinal photoreceptors and, in a reverse manner, the DNA/KChIP-mediated control of gene expression, but probably also the presenilin/KChIP-mediated control of APP γ-cleavage and ER Ca 2+ release. The reports on the role of EF-hand-mediated Ca 2+ binding in Kv4/KChIP complex formation are conflicting: Pioletti and coworkers, who studied the interaction between Kv4.3 and KChIP1 in great detail, found that neither a KChIP1ΔEF2,3,4 nor a KChIP1ΔEF3,4 mutant was able to modulate Kv4.3 channel inactivation gating when coexpressed [30]. These results correlated well with their pull-down experiments showing that for both KChIP1ΔEF2,3,4 and KChIP1ΔEF3,4 binding to a 143 amino acid N-terminal fragment (NTF) of Kv4.3 was impaired, and the authors concluded that Ca 2+ binding to KChIP1 EF-hands is required for Kv4.3/KChIP1 complex formation [30].…”