Lörinczi E et al. (2016), Calmodulin Regulates Human Ether à Go-Go 1 (hEA...

XB-ART-53681

J Biol Chem 2016 Aug 19;29134:17907-18. doi: 10.1074/jbc.M116.733576.

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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.

Lörinczi E, Helliwell M, Finch A, Stansfeld PJ, Davies NW, Mahaut-Smith M, Muskett FW, Mitcheson JS.

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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 (Ca(2+) i) through binding of Ca(2+)-calmodulin to three sites adjacent to the eagD and cNBHD. Here, we show that the eagD and cNBHD interact to modulate Ca(2+)-calmodulin as well as voltage-dependent gating. Sustained elevation of Ca(2+) 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 Ca(2+) i However, deletion of just the PAS-cap resulted in a >15-fold potentiation in response to elevated Ca(2+) 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 Ca(2+) i in a manner similar to the ΔPAS-cap mutant. These findings provide the first evidence that eagD and cNBHD interactions are regulating Ca(2+)-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.

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Species referenced: Xenopus laevis
Genes referenced: arnt kcnh1 mia3

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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.
	FIGURE 2. Wild-type hEAG1 channels are profoundly inhibited by raising cytoplasmic calcium. A, representative recordings of WT hEAG1 currents recorded with voltage steps to +60 mV from a holding potential of −90 mV, before (control) and during application of 5 μm of both ionomycin and thapsigargin (I & T). I and T resulted in a profound initial inhibition (Ai, green trace recorded 60 s after I and T application). In most cells, sustained I and T application resulted in the development of a slowly activating current (Aii, red trace) that progressively increased in amplitude and was inhibited by 50 μm astemizole (Aii, magenta trace). Aiii, representative current traces from an oocyte injected with diethyl pyrocarbonate water recorded in control solution (blue) or after 60 s (green) or 300 s (red) of I and T. Small inward tail currents at the time indicated by the dashed box are shown at a higher magnification in the inset. The bottom panel shows the voltage protocol. Vertical and horizontal scales are the same in all current traces (except inset). Dashed horizontal black lines indicate the zero current level. B, current amplitudes (end-pulse minus beginning of pulse current) plotted against time. Each symbol represents the amplitude of current during a single pulse. The time at which I and T and astemizole were applied is indicated by vertical arrows. Current traces and current amplitudes in Ai, Aii, and B were measured in the same oocyte. C, fluorescence imaging of Ca2+i by confocal microscopy. Fluorescence images from a representative oocyte loaded with the Ca2+-sensitive indicator Oregon Green 488 BAPTA-1 were taken before (control) and at indicated times after I and T application. Scale bar on left panel is 200 μm. D, time course of changes of fluorescence following I and T application. The fluorescence signal (F) was normalized to levels prior to I and T application (F0) and plotted against time after compound addition. The time scale is the same as for B. Symbols represent mean levels (n = 9) at 5-s intervals, and dotted lines indicate ± S.E.
	FIGURE 3. With sustained high Ca2+i hEAG1 current inhibition is reduced but activation gating is slowed and shifted to depolarized potentials. A, representative WT hEAG1 current traces before (blue) and >300 s after I and T application (red) elicited by an I-V protocol consisting of 2-s pulses from −50 to +80 mV at 10-mV increments from a holding potential of −90 mV, with a 500-ms step to −60 mV after each test potential. The voltage protocol is illustrated in the bottom left-hand panel (not all voltage steps are shown). B, mean (± S.E., n = 6) conductance-voltage relationships for WT hEAG1 currents before (blue symbols) and during I and T application (red symbols), fitted with Boltzmann functions (solid lines). Conductance is normalized to maximum for control and I and T (I&T) values.
	FIGURE 4. Response of hEAG1 currents to Ca2+i is calmodulin dependent. A–C, left-hand panels, representative current traces with voltage pulses to +60 mV before and at indicated times following I and T (I&T) application. A–C, right-hand panels, mean (± S.E.) conductance-voltage relationships fitted with Boltzmann functions (A and B) or current amplitude relationships (C). Error bars that are too small to extend beyond the symbols are not shown. A, responses in oocytes expressing WT hEAG1 channels and injected with EGTA (estimated final concentration 5 mm, n = 5). B, responses from oocytes expressing F714S/F717S hEAG1, a mutant that reduces the affinity for Ca2+-CaM binding to the BD-C2 domain (n = 7). C, responses in F151N/L154N hEAG1, a mutant with reduced affinity for Ca2+-CaM binding to the BD-N1 domain (n = 7). D, time courses of changes in WT and mutant hEAG1 currents after switching to I and T containing the bath solution. Time-dependent currents with each voltage step to +60 mV were normalized to control current and mean (± S.E.) values plotted against time from switching to I and T. Numbers (n) are indicated in parentheses next to symbol legends.
	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.
	FIGURE 6. PAS-cap is a critical regulatory domain for both voltage- and calcium-dependent hEAG1 channel gating. A, representative ΔPAS-cap hEAG1 current traces elicited by test potentials between −50 and +80 mV. Note the progressive reduction of current amplitudes at potentials positive to +40 mV. Inset shows tail currents recorded at −60 mV following test potentials to +60, +40, and +20 mV. B, representative ΔPAS-cap hEAG1 current traces in control solution (blue trace) and 40 s (green trace) or 300 s (red trace) after applying I and T (I&T). Currents were elicited with voltage steps to +60 mV from a holding potential of −90 mV. I and T caused an initial profound potentiation of ΔPAS-cap hEAG1 current that was in stark contrast to the inhibition of WT hEAG1. The inward tail current observed in the trace after 40 s of I and T is likely to be due to extracellular K+ accumulation following large amplitude test pulse currents. The currents in A and B are from different cells. C, mean (± S.E.) normalized current amplitudes (see Fig. 3D for details) for ΔPAS-cap hEAG1 (n = 9) and ΔPAS-cap/F714S/F717S hEAG1 (n = 14) plotted against time after switching to I and T. The time course of WT hEAG1 is also shown for comparison (n = 21). D, mean (± S.E.) conductance-voltage relationships for ΔPAS-cap hEAG1 (n = 7) before (blue symbols) and during I and T application (red symbols), fitted with Boltzmann functions (solid lines). Black dotted line shows the activation curve for WT hEAG1 for comparison with mutants. E, representative ΔPAS-cap/F714S/F717S hEAG1 currents with voltage pulses to +60 mV before and at indicated times after I and T application.
	FIGURE 7. Molecular model of interactions between cNBHD and PAS-cap of hEAG1. A, homology model of the eagD-cNBHD complex based upon the crystal structure (Protein Data Bank code 4LLO (25)). The PAS-cap is shown in yellow, the PAS domain in green, the cNBHD in blue, and the post-cNBHD region in red. The side chain of Glu-600 is shown in spheres and colored magenta. B, homology model rotated around the horizontal axis by ∼90° with surface rendering to illustrate how the amphipathic α-helix of the PAS-cap sits between the PAS domain and cNBHD. Colors correspond to A.
	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.

References [+] :

Allen, Organization and regulation of small conductance Ca2+-activated K+ channel multiprotein complexes. 2007, Pubmed