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Calmodulin Regulates Human Ether à Go-Go 1 (hEAG1) Potassium Channels through Interactions of the Eag Domain with the Cyclic Nucleotide Binding Homology Domain *

View Article: PubMed Central - PubMed

ABSTRACT

The ether à go-go family of voltage-gated potassium channels is structurally distinct. The N terminus contains an eag domain (eagD) that contains a Per-Arnt-Sim (PAS) domain that is preceded by a conserved sequence of 25–27 amino acids known as the PAS-cap. The C terminus contains a region with homology to cyclic nucleotide binding domains (cNBHD), which is directly linked to the channel pore. The human EAG1 (hEAG1) channel is remarkably sensitive to inhibition by intracellular calcium (Ca2+i) through binding of Ca2+-calmodulin to three sites adjacent to the eagD and cNBHD. Here, we show that the eagD and cNBHD interact to modulate Ca2+-calmodulin as well as voltage-dependent gating. Sustained elevation of Ca2+i resulted in an initial profound inhibition of hEAG1 currents, which was followed by a phase when current amplitudes partially recovered, but activation gating was slowed and shifted to depolarized potentials. Deletion of either the eagD or cNBHD abolished the inhibition by Ca2+i. However, deletion of just the PAS-cap resulted in a >15-fold potentiation in response to elevated Ca2+i. Mutations of residues at the interface between the eagD and cNBHD have been linked to human cancer. Glu-600 on the cNBHD, when substituted with residues with a larger volume, resulted in hEAG1 currents that were profoundly potentiated by Ca2+i in a manner similar to the ΔPAS-cap mutant. These findings provide the first evidence that eagD and cNBHD interactions are regulating Ca2+-dependent gating and indicate that the binding of the PAS-cap with the cNBHD is required for the closure of the channels upon CaM binding.

No MeSH data available.


hEAG1 secondary structure and sequence alignments.A, schematic representation of the secondary structure of hEAG1 K+ channels showing the location of three CaM binding domains (magenta circles) per subunit. CaM binding domain N (BD-N) is on the N terminus, close to the PAS domain. CaM binding domains C1 and C2 (BD-C1 and BD-C2) are located on the C terminus and adjacent to the cNBHD. B and C, protein sequence alignments of hEAG1 eagD (B) and cNBHD (C) with other KCNH family members. Numbering refers to hEAG1 sequences. White text on a red background indicates identical sequence, and red text indicates a semi-conserved sequence. Black boxes indicate positions of BD-C1 and BD-C2 in the post-cNBHD sequence of hEAG1.
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Figure 1: hEAG1 secondary structure and sequence alignments.A, schematic representation of the secondary structure of hEAG1 K+ channels showing the location of three CaM binding domains (magenta circles) per subunit. CaM binding domain N (BD-N) is on the N terminus, close to the PAS domain. CaM binding domains C1 and C2 (BD-C1 and BD-C2) are located on the C terminus and adjacent to the cNBHD. B and C, protein sequence alignments of hEAG1 eagD (B) and cNBHD (C) with other KCNH family members. Numbering refers to hEAG1 sequences. White text on a red background indicates identical sequence, and red text indicates a semi-conserved sequence. Black boxes indicate positions of BD-C1 and BD-C2 in the post-cNBHD sequence of hEAG1.

Mentions: Like other voltage-gated K+ channels, the central pore of KCNH channels is formed by the tetrameric assembly of S5–S6 helices and is surrounded by voltage sensor domains formed by S1–S4. The N terminus of hEAG1 contains an eag domain (eagD), which is unique to the KCNH channel family and contains a Per-Arnt-Sim (PAS) homology domain. PAS domains are structural folds that mediate protein-protein interactions in a variety of signaling proteins (15). In KCNH channels, the PAS domain is preceded by a highly conserved sequence of 25–27 amino acids that has become known as the PAS-cap (see Fig. 1) (16). NMR studies reveal that the first part of the PAS-cap is disordered, whereas the second half contains a stable amphipathic α-helix (17–19). Both segments have been shown to be important for gating of hEAG1 and hERG1 channels (17, 18, 20–22). The C terminus of the KCNH channel family contains a cyclic nucleotide binding homology domain (cNBHD) that is structurally similar to the cyclic nucleotide binding domains of CNG and HCN channels (23–26). However, KCNH channels lack key residues for cyclic nucleotide binding and are not directly regulated by cAMP or cGMP (27). Instead, the functional role of the KCNH cNBHD appears to be to regulate channel gating through interactions with the eagD (17, 28–30). The cNBHD is connected to the S6 inner helix of the pore by a region of ∼60 amino acids known as the C-linker, providing a mechanism for coupling conformational changes in the cNBHD to changes in gating of the pore (Fig. 1).


Calmodulin Regulates Human Ether à Go-Go 1 (hEAG1) Potassium Channels through Interactions of the Eag Domain with the Cyclic Nucleotide Binding Homology Domain *
hEAG1 secondary structure and sequence alignments.A, schematic representation of the secondary structure of hEAG1 K+ channels showing the location of three CaM binding domains (magenta circles) per subunit. CaM binding domain N (BD-N) is on the N terminus, close to the PAS domain. CaM binding domains C1 and C2 (BD-C1 and BD-C2) are located on the C terminus and adjacent to the cNBHD. B and C, protein sequence alignments of hEAG1 eagD (B) and cNBHD (C) with other KCNH family members. Numbering refers to hEAG1 sequences. White text on a red background indicates identical sequence, and red text indicates a semi-conserved sequence. Black boxes indicate positions of BD-C1 and BD-C2 in the post-cNBHD sequence of hEAG1.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC5016179&req=5

Figure 1: hEAG1 secondary structure and sequence alignments.A, schematic representation of the secondary structure of hEAG1 K+ channels showing the location of three CaM binding domains (magenta circles) per subunit. CaM binding domain N (BD-N) is on the N terminus, close to the PAS domain. CaM binding domains C1 and C2 (BD-C1 and BD-C2) are located on the C terminus and adjacent to the cNBHD. B and C, protein sequence alignments of hEAG1 eagD (B) and cNBHD (C) with other KCNH family members. Numbering refers to hEAG1 sequences. White text on a red background indicates identical sequence, and red text indicates a semi-conserved sequence. Black boxes indicate positions of BD-C1 and BD-C2 in the post-cNBHD sequence of hEAG1.
Mentions: Like other voltage-gated K+ channels, the central pore of KCNH channels is formed by the tetrameric assembly of S5–S6 helices and is surrounded by voltage sensor domains formed by S1–S4. The N terminus of hEAG1 contains an eag domain (eagD), which is unique to the KCNH channel family and contains a Per-Arnt-Sim (PAS) homology domain. PAS domains are structural folds that mediate protein-protein interactions in a variety of signaling proteins (15). In KCNH channels, the PAS domain is preceded by a highly conserved sequence of 25–27 amino acids that has become known as the PAS-cap (see Fig. 1) (16). NMR studies reveal that the first part of the PAS-cap is disordered, whereas the second half contains a stable amphipathic α-helix (17–19). Both segments have been shown to be important for gating of hEAG1 and hERG1 channels (17, 18, 20–22). The C terminus of the KCNH channel family contains a cyclic nucleotide binding homology domain (cNBHD) that is structurally similar to the cyclic nucleotide binding domains of CNG and HCN channels (23–26). However, KCNH channels lack key residues for cyclic nucleotide binding and are not directly regulated by cAMP or cGMP (27). Instead, the functional role of the KCNH cNBHD appears to be to regulate channel gating through interactions with the eagD (17, 28–30). The cNBHD is connected to the S6 inner helix of the pore by a region of ∼60 amino acids known as the C-linker, providing a mechanism for coupling conformational changes in the cNBHD to changes in gating of the pore (Fig. 1).

View Article: PubMed Central - PubMed

ABSTRACT

The ether à go-go family of voltage-gated potassium channels is structurally distinct. The N terminus contains an eag domain (eagD) that contains a Per-Arnt-Sim (PAS) domain that is preceded by a conserved sequence of 25–27 amino acids known as the PAS-cap. The C terminus contains a region with homology to cyclic nucleotide binding domains (cNBHD), which is directly linked to the channel pore. The human EAG1 (hEAG1) channel is remarkably sensitive to inhibition by intracellular calcium (Ca2+i) through binding of Ca2+-calmodulin to three sites adjacent to the eagD and cNBHD. Here, we show that the eagD and cNBHD interact to modulate Ca2+-calmodulin as well as voltage-dependent gating. Sustained elevation of Ca2+i resulted in an initial profound inhibition of hEAG1 currents, which was followed by a phase when current amplitudes partially recovered, but activation gating was slowed and shifted to depolarized potentials. Deletion of either the eagD or cNBHD abolished the inhibition by Ca2+i. However, deletion of just the PAS-cap resulted in a >15-fold potentiation in response to elevated Ca2+i. Mutations of residues at the interface between the eagD and cNBHD have been linked to human cancer. Glu-600 on the cNBHD, when substituted with residues with a larger volume, resulted in hEAG1 currents that were profoundly potentiated by Ca2+i in a manner similar to the ΔPAS-cap mutant. These findings provide the first evidence that eagD and cNBHD interactions are regulating Ca2+-dependent gating and indicate that the binding of the PAS-cap with the cNBHD is required for the closure of the channels upon CaM binding.

No MeSH data available.