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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

eagD and cNBHD are both required for Ca2+-CaM-dependent hEAG1 current inhibition.A and C, representative traces of ΔeagD hEAG1 (A) and ΔcNBHD hEAG1 (C) currents elicited by I-V protocols before (left panels) and during I and T (I&T) (right panels) application for >300 s. ΔeagD hEAG1currents exhibited rectification. For clarity, only selected traces, elicited by voltage steps +20 mV apart, are shown. B and D, mean (± S.E.) conductance-voltage relationships for ΔeagD hEAG1 (B, n = 5) and ΔcNBHD hEAG1 (D, n = 7) currents before (blue symbols) and during I and T application (red symbols), fitted with Boltzmann functions (solid lines). The voltage dependence of activation for WT hEAG1 in control solution is shown for comparison (black dashed line). E, mean (± S.E.) normalized current amplitudes (see Fig. 3D for details) for Δ2–135 hEAG1 (n = 6) and ΔcNBHD hEAG1 (n = 7) plotted against time after switching to I and T. The time course of WT hEAG1 (n = 21) is shown for comparison. F, time between 10 and 80% activation (t10–80%) at +60 mV in the presence (red) or absence (blue) of elevated Ca2+i values for WT (n = 8), ΔcNBHD hEAG1 (n = 7), and ΔeagD hEAG1 (n = 5). ****, p < 0.0001. ns, p > 0.05.
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Figure 5: eagD and cNBHD are both required for Ca2+-CaM-dependent hEAG1 current inhibition.A and C, representative traces of ΔeagD hEAG1 (A) and ΔcNBHD hEAG1 (C) currents elicited by I-V protocols before (left panels) and during I and T (I&T) (right panels) application for >300 s. ΔeagD hEAG1currents exhibited rectification. For clarity, only selected traces, elicited by voltage steps +20 mV apart, are shown. B and D, mean (± S.E.) conductance-voltage relationships for ΔeagD hEAG1 (B, n = 5) and ΔcNBHD hEAG1 (D, n = 7) currents before (blue symbols) and during I and T application (red symbols), fitted with Boltzmann functions (solid lines). The voltage dependence of activation for WT hEAG1 in control solution is shown for comparison (black dashed line). E, mean (± S.E.) normalized current amplitudes (see Fig. 3D for details) for Δ2–135 hEAG1 (n = 6) and ΔcNBHD hEAG1 (n = 7) plotted against time after switching to I and T. The time course of WT hEAG1 (n = 21) is shown for comparison. F, time between 10 and 80% activation (t10–80%) at +60 mV in the presence (red) or absence (blue) of elevated Ca2+i values for WT (n = 8), ΔcNBHD hEAG1 (n = 7), and ΔeagD hEAG1 (n = 5). ****, p < 0.0001. ns, p > 0.05.

Mentions: To test whether the hEAG1 channel response to Ca2+-CaM was mediated by interactions between the eag domain and cNBHD, we next tested the effect of deleting each structural domain in turn on Ca2+-CaM-dependent gating. Deleting the eagD (amino acids 2–135) dramatically altered gating and resulted in a slowly activating and slowly deactivating current. At potentials positive to +40 mV, current amplitudes progressively decreased and the tail currents, which also had a smaller peak amplitude, had a 'hooked' appearance (Fig. 5A). This behavior resembles hERG channel gating, in which the rectification is due to rapid onset of inactivation, and the hooked tails are due to rapid recovery from inactivation followed by slow deactivation. Importantly, in ΔeagD hEAG1, the inhibition by elevated Ca2+i was completely abolished; instead, the current was significantly increased by 82 ± 37% (p < 0.005, n = 7, see I-V relationship in Fig. 5A and mean time course data in Fig. 5E). The effect of I and T on the voltage and time dependence of ΔeagD activation was also significantly attenuated compared with WT hEAG1 (p < 0.0001, Fig. 5, B and F).


Calmodulin Regulates Human Ether à Go-Go 1 (hEAG1) Potassium Channels through Interactions of the Eag Domain with the Cyclic Nucleotide Binding Homology Domain *
eagD and cNBHD are both required for Ca2+-CaM-dependent hEAG1 current inhibition.A and C, representative traces of ΔeagD hEAG1 (A) and ΔcNBHD hEAG1 (C) currents elicited by I-V protocols before (left panels) and during I and T (I&T) (right panels) application for >300 s. ΔeagD hEAG1currents exhibited rectification. For clarity, only selected traces, elicited by voltage steps +20 mV apart, are shown. B and D, mean (± S.E.) conductance-voltage relationships for ΔeagD hEAG1 (B, n = 5) and ΔcNBHD hEAG1 (D, n = 7) currents before (blue symbols) and during I and T application (red symbols), fitted with Boltzmann functions (solid lines). The voltage dependence of activation for WT hEAG1 in control solution is shown for comparison (black dashed line). E, mean (± S.E.) normalized current amplitudes (see Fig. 3D for details) for Δ2–135 hEAG1 (n = 6) and ΔcNBHD hEAG1 (n = 7) plotted against time after switching to I and T. The time course of WT hEAG1 (n = 21) is shown for comparison. F, time between 10 and 80% activation (t10–80%) at +60 mV in the presence (red) or absence (blue) of elevated Ca2+i values for WT (n = 8), ΔcNBHD hEAG1 (n = 7), and ΔeagD hEAG1 (n = 5). ****, p < 0.0001. ns, p > 0.05.
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Figure 5: eagD and cNBHD are both required for Ca2+-CaM-dependent hEAG1 current inhibition.A and C, representative traces of ΔeagD hEAG1 (A) and ΔcNBHD hEAG1 (C) currents elicited by I-V protocols before (left panels) and during I and T (I&T) (right panels) application for >300 s. ΔeagD hEAG1currents exhibited rectification. For clarity, only selected traces, elicited by voltage steps +20 mV apart, are shown. B and D, mean (± S.E.) conductance-voltage relationships for ΔeagD hEAG1 (B, n = 5) and ΔcNBHD hEAG1 (D, n = 7) currents before (blue symbols) and during I and T application (red symbols), fitted with Boltzmann functions (solid lines). The voltage dependence of activation for WT hEAG1 in control solution is shown for comparison (black dashed line). E, mean (± S.E.) normalized current amplitudes (see Fig. 3D for details) for Δ2–135 hEAG1 (n = 6) and ΔcNBHD hEAG1 (n = 7) plotted against time after switching to I and T. The time course of WT hEAG1 (n = 21) is shown for comparison. F, time between 10 and 80% activation (t10–80%) at +60 mV in the presence (red) or absence (blue) of elevated Ca2+i values for WT (n = 8), ΔcNBHD hEAG1 (n = 7), and ΔeagD hEAG1 (n = 5). ****, p < 0.0001. ns, p > 0.05.
Mentions: To test whether the hEAG1 channel response to Ca2+-CaM was mediated by interactions between the eag domain and cNBHD, we next tested the effect of deleting each structural domain in turn on Ca2+-CaM-dependent gating. Deleting the eagD (amino acids 2–135) dramatically altered gating and resulted in a slowly activating and slowly deactivating current. At potentials positive to +40 mV, current amplitudes progressively decreased and the tail currents, which also had a smaller peak amplitude, had a 'hooked' appearance (Fig. 5A). This behavior resembles hERG channel gating, in which the rectification is due to rapid onset of inactivation, and the hooked tails are due to rapid recovery from inactivation followed by slow deactivation. Importantly, in ΔeagD hEAG1, the inhibition by elevated Ca2+i was completely abolished; instead, the current was significantly increased by 82 ± 37% (p < 0.005, n = 7, see I-V relationship in Fig. 5A and mean time course data in Fig. 5E). The effect of I and T on the voltage and time dependence of ΔeagD activation was also significantly attenuated compared with WT hEAG1 (p < 0.0001, Fig. 5, B and F).

View Article: PubMed Central - PubMed

ABSTRACT

The ether &agrave; 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&ndash;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 &gt;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 &Delta;PAS-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