A mutually induced conformational fit underlies Ca2+-directed interactions between calmodulin and the proximal C terminus of KCNQ4 K+ channels

Crystal R Archer,Benjamin T Enslow,Alexander B Taylor,Victor De La Rosa,Akash Bhattacharya,Mark S Shapiro

doi:10.1074/jbc.ra118.006857

Abstract

Calmodulin (CaM) conveys intracellular Ca2+ signals to KCNQ (Kv7, "M-type") K+ channels and many other ion channels. Whether this "calmodulation" involves a dramatic structural rearrangement or only slight perturbations of the CaM/KCNQ complex is as yet unclear. A consensus structural model of conformational shifts occurring between low nanomolar and physiologically high intracellular [Ca2+] is still under debate. Here, we used various techniques of biophysical chemical analyses to investigate the interactions between CaM and synthetic peptides corresponding to the A and B domains of the KCNQ4 subtype. We found that in the absence of CaM, the peptides are disordered, whereas Ca2+/CaM imposed helical structure on both KCNQ A and B domains. Isothermal titration calorimetry revealed that Ca2+/CaM has higher affinity for the B domain than for the A domain of KCNQ2-4 and much higher affinity for the B domain when prebound with the A domain. X-ray crystallography confirmed that these discrete peptides spontaneously form a complex with Ca2+/CaM, similar to previous reports of CaM binding KCNQ-AB domains that are linked together. Microscale thermophoresis and heteronuclear single-quantum coherence NMR spectroscopy indicated the C-lobe of Ca2+-free CaM to interact with the KCNQ4 B domain (Kd ∼10-20 μm), with increasing Ca2+ molar ratios shifting the CaM-B domain interactions via only the CaM C-lobe to also include the N-lobe. Our findings suggest that in response to increased Ca2+, CaM undergoes lobe switching that imposes a dramatic mutually induced conformational fit to both the proximal C terminus of KCNQ4 channels and CaM, likely underlying Ca2+-dependent regulation of KCNQ gating.

Highlights

Calmodulin (CaM) conveys intracellular Ca2؉ signals to KCNQ (Kv7, “M-type”) K؉ channels and many other ion channels
We demonstrated that apoCaM does bind KCNQ4 subunits with moderate affinity with only the C-lobe of apoCaM interacting with the B domain of KCNQ4
The major advance of this work is our finding that a dramatic lobe switching of CaM interaction with the KCNQ4 A and B domain occurs from low to high [Ca2ϩ]

Summary

The abbreviations used are

CaM, calmodulin; HSQC, heteronuclear singlequantum coherence; TROSY, transverse relaxation optimized spectroscopy; M⍀, megaohms; apoCaM, non-Ca2ϩ-loaded CaM; VGCC, voltagegated Ca2ϩ channel; RD, regulatory domain; PIP2, phosphatidylinositol 4,5-bisposphate; PDB, Protein Data Bank; HBS, Hepes-buffered saline; RMSD, root mean square deviation; ITC, isothermal titration calorimetry; MST, microscale thermophoresis; CHO, Chinese hamster ovary; LB, Luria broth A recent solution NMR study of a similar complex of the A and B domains of KCNQ2 and CaM suggested only minor changes in the structure of the complex between low and high [Ca2ϩ], arguing against a dramatic structural change in KCNQ channels in response to intracellular rises in [Ca2ϩ] [50] We investigated this issue for KCNQ4, as this isoform is expressed in cells and tissues mainly as homomeric channels, simplifying our interpretations. The goal of this inquiry was to build a stepwise model of the mechanism of CaM binding to the KCNQ4 C terminus from Ͻ10 nM to physiologically high [Ca2ϩ]

Results

Discussion

Experimental procedures