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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.


Related in: MedlinePlus

Point mutations to the cNBHD mimic the effect of deleting the PAS-cap on voltage- and calcium-dependent properties.A and C, representative current traces elicited by the I-V protocol for E600R (A) and E600A (C) hEAG1 before (control, blue traces) and during I and T (I&T) application (red traces). Traces at +20-mV increments are shown for clarity. B and D, mean (± S.E.) normalized current-voltage relationships for E600R (B, n = 7) and E600A (D, n = 8) hEAG1. Time-dependent currents at each potential were normalized to the maximum current in control conditions to illustrate the fold-change of current amplitude in response to I and T. The dotted lines in D show the mean normalized relationships for WT hEAG1 currents for comparison. E, mean (± S.E.) normalized to control current amplitudes against time in I and T for cNBHD point mutants E600R (n = 5), E600A (n = 7), E600L (n = 13), E600I (n = 11), and WT hEAG1 (n = 21). Note the split current axis to accommodate the substantial potentiation of currents exhibited by E600R, E600I, and E600L hEAG1. Dotted lines indicate ± S.E. F, mean maximum changes of current for Glu-600 mutants and WT hEAG1 in response to I and T, normalized to control current amplitudes. n = 7 and 14 for E600A and E600Q, respectively, and the other numbers (n) are the same as in E. The normalized current axis has been split for the same reason as given in E.
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Figure 8: Point mutations to the cNBHD mimic the effect of deleting the PAS-cap on voltage- and calcium-dependent properties.A and C, representative current traces elicited by the I-V protocol for E600R (A) and E600A (C) hEAG1 before (control, blue traces) and during I and T (I&T) application (red traces). Traces at +20-mV increments are shown for clarity. B and D, mean (± S.E.) normalized current-voltage relationships for E600R (B, n = 7) and E600A (D, n = 8) hEAG1. Time-dependent currents at each potential were normalized to the maximum current in control conditions to illustrate the fold-change of current amplitude in response to I and T. The dotted lines in D show the mean normalized relationships for WT hEAG1 currents for comparison. E, mean (± S.E.) normalized to control current amplitudes against time in I and T for cNBHD point mutants E600R (n = 5), E600A (n = 7), E600L (n = 13), E600I (n = 11), and WT hEAG1 (n = 21). Note the split current axis to accommodate the substantial potentiation of currents exhibited by E600R, E600I, and E600L hEAG1. Dotted lines indicate ± S.E. F, mean maximum changes of current for Glu-600 mutants and WT hEAG1 in response to I and T, normalized to control current amplitudes. n = 7 and 14 for E600A and E600Q, respectively, and the other numbers (n) are the same as in E. The normalized current axis has been split for the same reason as given in E.

Mentions: Recently, an x-ray crystal structure of the eag domain in complex with the cNBHD of mEAG has been solved (25). There are a number of sites of contact between the two domains, including contact of the PAS domain with the intrinsic ligand and post-cNBHD motifs of the cNBHD, which are adjacent to the BD-C1 and BD-C2 CaM-binding sites of the channel. The other important region of contact is between the PAS-cap and the cNBHD, although it should be noted that the first 16 amino acids of the PAS-cap were not resolved. Interestingly, there are relatively few structural changes between the domains in the complex and those of the isolated domains when they were solved independently of one another. This suggests that the contacts are quite weak and consistent with the low binding affinity (13.2 μm) measured using fluorescence anisotropy (25). Other regions of the channel may both stabilize and regulate the interaction of these crucial regions. Of particular interest was that a number of disease-associated mutations (long QT causing mutations in hERG1 and cancer-linked mutations in hEAG1) mapped to the interface between the eag domain and cNBHD. We modeled the interaction of the hEAG1 eag domain in complex with the cNBHD, including the section of the PAS-cap missing in the crystal structure (Fig. 7). The PAS-cap folds back, and the amphipathic helix sits in a groove between the PAS domain and cNBHD, making contacts with both domains. To test whether the Ca2+-CaM-mediated inhibition of hEAG1 could be due to interactions of the PAS-cap with the cNBHD, we mutated Glu-600 (equivalent to Glu-627 in mEAG and Glu-788 in hERG1) to Ala, Arg, Gln, Ile, or Leu and tested the response of these point mutants to I and T. This position is highly conserved in the EAG channel family and mutations in the homologous hERG1 channel position are associated with long QT syndrome (34). In mEAG, Ala and Arg mutations have been reported to cause robust depolarizing shifts in the voltage dependence of activation of >100 mV (25). In hEAG1, the effects were not as marked but, remarkably, the E600R mutation reproduced much of the change in gating properties (slowed activation and deactivation, and rectification at positive potentials) seen in ΔPAS-cap hEAG1 (Fig. 8, A and B). The substantial Ca2+i-dependent potentiation was also seen with the cNBHD E600R mutant. I and T resulted in a 12.3 ± 2.6-fold increase in E600R hEAG1 current measured at +40 mV relative to control (Table 1). However, unlike the ΔPAS-cap mutant, the increase of current in I and T was sustained (Fig. 8E). The response of Glu-600 mutants to elevated Ca2+i was highly dependent on residue volume and not just the charge of the substituted residue. The E600A and E600Q mutants exhibited WT hEAG1 behavior and were inhibited by I and T (Fig. 8F and Table 1). Ala and Gln have van der Waals residue volumes of 67 and 96 Å3, respectively, which are smaller than Glu (109 Å3) (35). Substituting Glu-600 for Leu or Ile (each 124 Å3) resulted in potentiation of current by I and T of 3.16 ± 0.6 (n = 13) and 6.43 ± 0.72 (n = 11)-fold, respectively. The volume of Arg is largest of all (196 Å3) and gave the greatest potentiation. Collectively, these results suggest that Glu-600 is an important site of contact with the PAS-cap. They also suggest that the charge of the residue is not the critical factor. The functional data support a molecular model in which the PAS-cap packs against the cNBHD. Substitution of large residues at position 600 reduces PAS-cap binding affinity resulting in similar functional effects to deleting the PAS-cap entirely.


Calmodulin Regulates Human Ether à Go-Go 1 (hEAG1) Potassium Channels through Interactions of the Eag Domain with the Cyclic Nucleotide Binding Homology Domain *
Point mutations to the cNBHD mimic the effect of deleting the PAS-cap on voltage- and calcium-dependent properties.A and C, representative current traces elicited by the I-V protocol for E600R (A) and E600A (C) hEAG1 before (control, blue traces) and during I and T (I&T) application (red traces). Traces at +20-mV increments are shown for clarity. B and D, mean (± S.E.) normalized current-voltage relationships for E600R (B, n = 7) and E600A (D, n = 8) hEAG1. Time-dependent currents at each potential were normalized to the maximum current in control conditions to illustrate the fold-change of current amplitude in response to I and T. The dotted lines in D show the mean normalized relationships for WT hEAG1 currents for comparison. E, mean (± S.E.) normalized to control current amplitudes against time in I and T for cNBHD point mutants E600R (n = 5), E600A (n = 7), E600L (n = 13), E600I (n = 11), and WT hEAG1 (n = 21). Note the split current axis to accommodate the substantial potentiation of currents exhibited by E600R, E600I, and E600L hEAG1. Dotted lines indicate ± S.E. F, mean maximum changes of current for Glu-600 mutants and WT hEAG1 in response to I and T, normalized to control current amplitudes. n = 7 and 14 for E600A and E600Q, respectively, and the other numbers (n) are the same as in E. The normalized current axis has been split for the same reason as given in E.
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Figure 8: Point mutations to the cNBHD mimic the effect of deleting the PAS-cap on voltage- and calcium-dependent properties.A and C, representative current traces elicited by the I-V protocol for E600R (A) and E600A (C) hEAG1 before (control, blue traces) and during I and T (I&T) application (red traces). Traces at +20-mV increments are shown for clarity. B and D, mean (± S.E.) normalized current-voltage relationships for E600R (B, n = 7) and E600A (D, n = 8) hEAG1. Time-dependent currents at each potential were normalized to the maximum current in control conditions to illustrate the fold-change of current amplitude in response to I and T. The dotted lines in D show the mean normalized relationships for WT hEAG1 currents for comparison. E, mean (± S.E.) normalized to control current amplitudes against time in I and T for cNBHD point mutants E600R (n = 5), E600A (n = 7), E600L (n = 13), E600I (n = 11), and WT hEAG1 (n = 21). Note the split current axis to accommodate the substantial potentiation of currents exhibited by E600R, E600I, and E600L hEAG1. Dotted lines indicate ± S.E. F, mean maximum changes of current for Glu-600 mutants and WT hEAG1 in response to I and T, normalized to control current amplitudes. n = 7 and 14 for E600A and E600Q, respectively, and the other numbers (n) are the same as in E. The normalized current axis has been split for the same reason as given in E.
Mentions: Recently, an x-ray crystal structure of the eag domain in complex with the cNBHD of mEAG has been solved (25). There are a number of sites of contact between the two domains, including contact of the PAS domain with the intrinsic ligand and post-cNBHD motifs of the cNBHD, which are adjacent to the BD-C1 and BD-C2 CaM-binding sites of the channel. The other important region of contact is between the PAS-cap and the cNBHD, although it should be noted that the first 16 amino acids of the PAS-cap were not resolved. Interestingly, there are relatively few structural changes between the domains in the complex and those of the isolated domains when they were solved independently of one another. This suggests that the contacts are quite weak and consistent with the low binding affinity (13.2 μm) measured using fluorescence anisotropy (25). Other regions of the channel may both stabilize and regulate the interaction of these crucial regions. Of particular interest was that a number of disease-associated mutations (long QT causing mutations in hERG1 and cancer-linked mutations in hEAG1) mapped to the interface between the eag domain and cNBHD. We modeled the interaction of the hEAG1 eag domain in complex with the cNBHD, including the section of the PAS-cap missing in the crystal structure (Fig. 7). The PAS-cap folds back, and the amphipathic helix sits in a groove between the PAS domain and cNBHD, making contacts with both domains. To test whether the Ca2+-CaM-mediated inhibition of hEAG1 could be due to interactions of the PAS-cap with the cNBHD, we mutated Glu-600 (equivalent to Glu-627 in mEAG and Glu-788 in hERG1) to Ala, Arg, Gln, Ile, or Leu and tested the response of these point mutants to I and T. This position is highly conserved in the EAG channel family and mutations in the homologous hERG1 channel position are associated with long QT syndrome (34). In mEAG, Ala and Arg mutations have been reported to cause robust depolarizing shifts in the voltage dependence of activation of >100 mV (25). In hEAG1, the effects were not as marked but, remarkably, the E600R mutation reproduced much of the change in gating properties (slowed activation and deactivation, and rectification at positive potentials) seen in ΔPAS-cap hEAG1 (Fig. 8, A and B). The substantial Ca2+i-dependent potentiation was also seen with the cNBHD E600R mutant. I and T resulted in a 12.3 ± 2.6-fold increase in E600R hEAG1 current measured at +40 mV relative to control (Table 1). However, unlike the ΔPAS-cap mutant, the increase of current in I and T was sustained (Fig. 8E). The response of Glu-600 mutants to elevated Ca2+i was highly dependent on residue volume and not just the charge of the substituted residue. The E600A and E600Q mutants exhibited WT hEAG1 behavior and were inhibited by I and T (Fig. 8F and Table 1). Ala and Gln have van der Waals residue volumes of 67 and 96 Å3, respectively, which are smaller than Glu (109 Å3) (35). Substituting Glu-600 for Leu or Ile (each 124 Å3) resulted in potentiation of current by I and T of 3.16 ± 0.6 (n = 13) and 6.43 ± 0.72 (n = 11)-fold, respectively. The volume of Arg is largest of all (196 Å3) and gave the greatest potentiation. Collectively, these results suggest that Glu-600 is an important site of contact with the PAS-cap. They also suggest that the charge of the residue is not the critical factor. The functional data support a molecular model in which the PAS-cap packs against the cNBHD. Substitution of large residues at position 600 reduces PAS-cap binding affinity resulting in similar functional effects to deleting the PAS-cap entirely.

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.


Related in: MedlinePlus