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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.
Highlights
Coiled-coils are bundles of intertwined α-helices, a conformation that is one of the most widespread and versatile protein-protein interaction domains found in nature[1,2,3]
The coiled-coil formed by helix D is not essential for Kv7 function and it can be replaced by an unrelated tetrameric coiled-coil domain, channel processing, surface expression and gating are affected by alterations to this helix[23,27,28,29]
We present evidence supporting the notion that the coiled-coil formed by helix D distal to the AB CaM-binding module indirectly favors trans-binding of CaM to Kv7.2, and that CaM binding stabilizes the tetrameric C-terminal assembly
Summary
To study how the assembly/tetramerization domain (CD module) of the Kv7.2 subunit influences CaM binding (see supplemental Fig. 1), we constructed chimeric proteins of the monomeric single transmembrane protein Tac and the Kv7.2 AB CaM-binding domain, and we expressed them in HEK293T cells[33]. The time-course of FRET development was sensitive to Ca2+, taking longer to reach the half-maximal value in the presence of this cation (Fig. 6C,D) This is similar to the response in conditions designed to monitor trans-binding (Fig. 4B), indicating that the ABCD/CaM complexes were more stable in the presence of Ca2+. It might be expected that the dependence on Ca2+ to adopt trans and cis binding might differ substantially in the complete channel relative to the GST-tagged-Kv7.2 C-terminal constructs studied here These cryo-EM images reveal that the helix D region is very flexible[47], an observation in line with the exchange between subunits observed here for the isolated C-terminal domain. Our observations are consistent with a model that involves active reciprocal coupling between the A-B module and the tetramer formed by helices D, CaM and Ca2+ thereby influencing the dynamic behavior of the helix D coiled-coil
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