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, 7 (1), 13425

Calmodulin Confers Calcium Sensitivity to the Stability of the Distal Intracellular Assembly Domain of Kv7.2 Channels

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Calmodulin Confers Calcium Sensitivity to the Stability of the Distal Intracellular Assembly Domain of Kv7.2 Channels

Alessandro Alaimo et al. Sci Rep.

Abstract

Tetrameric coiled-coil structures are present in many ion channels, often adjacent to a calmodulin (CaM) binding site, although the relationship between the two is not completely understood. Here we examine the dynamic properties of the ABCD domain located in the intracellular C-terminus of tetrameric, voltage-dependent, potassium selective Kv7.2 channels. This domain encompasses the CaM binding site formed by helices A and B, followed by helix C, which is linked to the helix D coiled-coil. The data reveals that helix D stabilizes CaM binding, promoting trans-binding (CaM embracing neighboring subunits), and they suggest that the ABCD domain can be exchanged between subunits of the tetramer. Exchange is faster when mutations in AB weaken the CaM interaction. The exchange of ABCD domains is slower in the presence of Ca2+, indicating that CaM stabilization of the tetrameric assembly is enhanced when loaded with this cation. Our observations are consistent with a model that involves a dynamic mechanism of helix D assembly, which supports reciprocal allosteric coupling between the A-B module and the coiled-coil formed by the helix D. Thus, formation of the distal helix D tetramer influences CaM binding and CaM-dependent Kv7.2 properties, whereas reciprocally, CaM and Ca2+ influence the dynamic behavior of the helix D coiled-coil.

Conflict of interest statement

The authors declare that they have no competing interests.

Figures

Figure 1
Figure 1
The presence of a tetramerization domain favors calmodulin binding to a membrane protein. (A and C) Tac-Kv7.2-CFP chimeras extracted from HEK293T cells were immunoprecipitated using anti-GFP antibodies, separated by SDS-PAGE, transferred to membranes (WB), and probed with anti-GFP and anti-CaM antibodies (n ≥ 3, see supplemental Figs 2 and 3 for the full blots). The bottom panel in (C) was obtained after a longer exposure than the top panel (see supplemental Fig. 3). (B) D-CaM (12.5 nM) fluorescence enhancement in the absence of Ca2+ (10 mM EGTA) at the indicated recombinant GST-fusion protein concentrations. The lines are the result of fitting a Hill equation to the data. The EC50 (nM) was 17.8 ± 1.1 and 21.5 ± 0.5, for ABΔ2-CD and ABΔ2-Tet, respectively. The asterisks indicate significantly different maximal fluorescence: **p < 0.01. The data represent the means ± SEM from 3 independent experiments.
Figure 2
Figure 2
Hypothetical cis and trans calmodulin binding in tetramers. (A) A representation of cis-binding, in which CaM embraces helices A and B (boxes) in the same subunit. The N-lobe engages helix B, whereas the C-lobe engages helix A,,. (B) Illustration of CaM in trans-binding mode embracing helices A and B from two different subunits (note that CaM embraces helices of different color). (C) Mutations in either helix A (top) or helix B (bottom) preclude cis- (left) and trans- (right) binding in homomeric tetramers. (D) Trans-binding is allowed between heteromeric helix A and helix B mutants, whereas cis-binding is precluded.
Figure 3
Figure 3
The time-course of trans-binding is affected by the helix D coiled-coil. (A) Time-course of the increase in D-CaM (12.5 nM) fluorescence upon binding to AB and to ABCD (400 nM). Each point represents the average of 4 experiments. The maximal increase in fluorescence was reached in less than 2 min. (B) Comparison of the time-course of the increase in D-CaM fluorescence in the continued presence of the helix A A343D mutant (400 nM), and upon addition of the S511D helix B mutant (400 nM), for proteins devoid of the CD module (AB#, open circles), ABCD# (green upward triangles), and ABCD#-L609R (yellow downward triangles). The hash denotes an equal mixture of helix A and helix B mutants. This set-up was designed to trap trans-binding. Each trace represents the average of 3 experiments. (C) Normalized time-course from the data displayed in (B). (D) Plot of the half-time to reach the maximal D-CaM fluorescence emission vs the apparent affinity for AB#, ABCD# and ABCD#-L609R.
Figure 4
Figure 4
Acquisition of the trans-binding mode depends on the protein concentration and calcium levels. (A) Time course of the increase in D-CaM fluorescence obtained upon binding to ABCD# (50 or 100 nM). Each trace represents the average of 3 experiments. (B) Relationship between the time to reach the half-maximal increase in D-CaM fluorescence and protein concentration in the presence (gray circles) and absence of Ca2+ (white circles). (C) Plot of the apparent binding affinity (EC50) obtained from the concentration-response curves as in Fig. 1B (D-CaM 12.5 nM). The apparent binding affinity was derived from 3 or more experiments. The experiments were performed both in the presence (gray bars) and absence of Ca2+ (white, yellow and green columns). The asterisks indicate significantly different values versus AB: *p < 0.05; ***p < 0.001.
Figure 5
Figure 5
Summary of the maximal D-CaM fluorescence emission. (A) Maximal increases in D-CaM fluorescence emission induced by saturating concentrations of the indicated proteins. The data were collected in the presence (gray bars) or absence of Ca2+ (white, yellow and green bars) (n ≥ 3). Asterisks indicate significantly different values versus AB: *p < 0.05; **p < 0.01; ***p < 0.001. (B) Plot of the time to reach the half-maximal D-CaM fluorescence versus the maximal increase in fluorescence.
Figure 6
Figure 6
The time-course of subunit exchange between FP-ABCD/CaM tetrameric complexes is affected by calcium. Two ABCD/CaM complexes with a fluorescent protein attached to the N-terminus (CFP, donor; YFP, acceptor) were purified and the development of FRET was monitored over time from equimolar mixtures. (A) Cartoon representing the experiment: the CFP-ABCD/CaM complex was mixed with YFP-ABCD/CaM, resulting in an exchange of proteins that led to the development of FRET. Only two subunits of the tetrameric complexes are drawn for clarity. (B) Normalized emission spectra of a mixture of 2.5 µM CFP-ABCD/CaM and 2.5 µM YFP-ABCD/CaM at different times. The yellow traces are the results of subtracting the normalized CFP emission spectra and isolating the emission of the acceptor (YFP). (C) Time course of the increase in the FRET index from a 2.5 µM CFP-ABCD/CaM and 2.5 µM YFP-ABCD/CaM mixture in the presence (gray circles) and absence (white circles) of Ca2+. Each trace represents the average of 3 experiments. (D) Relationship between the time to reach the half-maximal increase in the FRET index and the protein concentration in the presence (gray circles) and absence of Ca2+ (white circles). Each point represents the average of 3 or more experiments.

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References

    1. Lupas A. Coiled coils: new structures and new functions. Trends Biochem. Sci. 1996;21:375–382. doi: 10.1016/S0968-0004(96)10052-9. - DOI - PubMed
    1. Lupas AN, Gruber M. The structure of alpha-helical coiled coils. Adv. Protein Chem. 2005;70:37–78. doi: 10.1016/S0065-3233(05)70003-6. - DOI - PubMed
    1. Woolfson DN. The design of coiled-coil structures and assemblies. Adv. Protein Chem. 2005;70:79–112. doi: 10.1016/S0065-3233(05)70004-8. - DOI - PubMed
    1. Jenke M, et al. C-terminal domains implicated in the functional surface expression of potassium channels. Embo Journal. 2003;22:395–403. doi: 10.1093/emboj/cdg035. - DOI - PMC - PubMed
    1. Wiener R, et al. The KCNQ1 (Kv7.1) COOH terminus, a multitiered scaffold for subunit assembly and protein interaction. J. Biol. Chem. 2008;283:5815–5830. doi: 10.1074/jbc.M707541200. - DOI - PubMed

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