The structural mechanism of KCNH-channel regulation by the eag domain

Abstract

The KCNH voltage-dependent potassium channels (ether-á-go-go, EAG; EAG-related gene, ERG; EAG-like channels, ELK) are important regulators of cellular excitability1-3 and have key roles in diseases such as cardiac long QT syndrome type 2 (LQT2)4, epilepsy5, schizophrenia6 and cancer7. The intracellular domains of KCNH channels are structurally distinct from other voltage-gated channels. The amino-terminal region contains an eag domain, which is comprised of a Per-Arnt-Sim (PAS) domain and a PAS-cap domain8, while the carboxy-terminal region contains a cyclic nucleotide-binding homology domain (CNBHD) which is connected to the pore through a C-linker domain. Many disease-causing mutations localize to these specialized intracellular domains, which underlie the unique gating and regulation of KCNH channels9. It has been suggested that the eag domain may regulate the channel by interacting with either the S4-S5 linker or the CNBHD8,10. Here we present a 2-Å resolution crystal structure of the eag domain-CNBHD complex of the mouse EAG1 (mEAG1) channel. It displays extensive interactions between the eag domain and the CNBHD, indicating that the regulatory mechanism of the eag domain involves primarily the CNBHD. Surprisingly, the structure reveals that a number of LQT2 mutations at homologous positions in hERG, and cancer-associated mutations in EAG channels, localize to the eag domain-CNBHD interface. Furthermore, mutations at the interface produced dramatic effects on channel gating demonstrating the important physiological role of the eag domain-CNBHD interaction. Our structure of the eag domain-CNBHD complex of mEAG1 provides unique insights into the physiological and pathophysiological mechanisms of KCNH channels.

Multiple Interactions between Cytoplasmic Domains Regulate Slow Deactivation of Kv11.1 Channels

“…Although several studies proposed that the N-terminal PAS domains of Kv11.1 channels form interactions with the C-terminal cNBH domains (17,34,35), the exact nature of these interactions, including those of Arg 56 , were hitherto unclear. A recent crystal structure of isolated PAS and cNBH domains from mouse Kv10.1 channels showed that Arg 57 in the PAS domain formed a specific salt bridge interaction with Asp 642 in the cNBH domain (18). Consistent with this, we found a critical salt bridge interaction between Arg 56 and Asp 803 , the corresponding positions in Kv11.1 channels.…”

“…In our tetrameric homology model of the Kv11.1 cNBH domains, which is similar to the one generated by Haitin et al (18), Asp 774 is located within the interface of two subunits and is therefore likely to cause perturbation to the subunit-subunit interaction when mutated to arginine. In contrast, both Glu 788 and Asp 803 are surface-exposed negatively charged residues and could form charge-charge interactions with one or more of the critical N-Cap/PAS positively charged residues (Arg 4 -We performed charge reversal double mutant cycle analysis (see "Experimental Procedures") to identify whether Asp 803 interacts with any of the four positively charged residues from the N-Cap and PAS domains of Kv11.1 channels (Fig.…”

“…Furthermore, this stable interaction is shared between at least two members of the EAG family of channels: Kv11.1 and Kv10.1. However, although in Kv11.1 channels this interaction is important for slowing the kinetics of deactivation (channel closure), in Kv10.1 channels the interaction appears to accelerate the kinetics of channel activation (channel opening) (18). The reason for this difference is unclear, but we postulated that it may reflect differences in the mechanism by which the N-Cap domains interact with the remainder of the channel.…”

“…Interestingly the atomic structures of the PAS domain from the Kv10.1 and Kv11.1 channels are almost identical (19). However, the connection of the N-Cap domain to the PAS domain of these two channels appears to be different (18). Furthermore, in contrast to Kv11.1 channels, Kv10.1 channels do not exhibit slow deactivation kinetics (20,21), suggesting that the functional interaction between N-Cap/PAS and cNBH domains may be different in Kv11.1 compared with other members of the KCNH family.…”

“…For example, the N-Cap/Per-Arnt-Sim (PAS) 2 domain on the N termini plays a critical role in slowing deactivation gating in Kv11.1 channels (12)(13)(14)(15)(16), and this is likely mediated by an interaction with the cyclic nucleotide binding homology (cNBH) domain in the C terminus (17). A crystal structure complex of the PAS and cNBH domains from a closely related murine Kv10.1 channel provides the first atomic detail that these domains form a direct interaction (18). Interestingly the atomic structures of the PAS domain from the Kv10.1 and Kv11.1 channels are almost identical (19).…”