Calcium Binding to Calmodulin Mutants Having Domain-Specific Effects on the Regulation of Ion Channels

VanScyoc, Wendy S.; Newman, Rhonda A.; Sorensen, Brenda R.; Shea, Madeline A.

doi:10.1021/bi061134d

Cited by 14 publications

(32 citation statements)

References 53 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…The resulting corrected binding energy of Ca 2+ to CaM was Ϫ 4.9 kcal/mol. This calculated result is very close to the experimentally determined value of ‫ف‬ Ϫ 7 kcal/mol to single EF hands (3 or 4) in CaM ( VanScyoc et al, 2006 ). We also calculated the binding energy with an explicit water added from the crystal structure.…”

Section: R E S U Lt Ssupporting

confidence: 84%

Regulation of Bestrophin Cl Channels by Calcium: Role of the C Terminus

Xiao

Prussia

et al. 2008

The Journal of General Physiology

116

View full text Add to dashboard Cite

Human bestrophin-1 (hBest1), which is genetically linked to several kinds of retinopathy and macular degeneration in both humans and dogs, is the founding member of a family of Cl− ion channels that are activated by intracellular Ca2+. At present, the structures and mechanisms responsible for Ca2+ sensing remain unknown. Here, we have used a combination of molecular modeling, density functional–binding energy calculations, mutagenesis, and patch clamp to identify the regions of hBest1 involved in Ca2+ sensing. We identified a cluster of a five contiguous acidic amino acids in the C terminus immediately after the last transmembrane domain, followed by an EF hand and another regulatory domain that are essential for Ca2+ sensing by hBest1. The cluster of five amino acids (293–308) is crucial for normal channel gating by Ca2+ because all but two of the 35 mutations we made in this region rendered the channel incapable of being activated by Ca2+. Using homology models built on the crystal structure of calmodulin (CaM), an EF hand (EF1) was identified in hBest1. EF1 was predicted to bind Ca2+ with a slightly higher affinity than the third EF hand of CaM and lower affinity than the second EF hand of troponin C. As predicted by the model, the D312G mutation in the putative Ca2+-binding loop (312–323) reduced the apparent Ca2+ affinity by 20-fold. In addition, the D312G and D323N mutations abolished Ca2+-dependent rundown of the current. Furthermore, analysis of truncation mutants of hBest1 identified a domain adjacent to EF1 that is rich in acidic amino acids (350–390) that is required for Ca2+ activation and plays a role in current rundown. These experiments identify a region of hBest1 (312–323) that is involved in the gating of hBest1 by Ca2+ and suggest a model in which Ca2+ binding to EF1 activates the channel in a process that requires the acidic domain (293–308) and another regulatory domain (350–390). Many of the ∼100 disease-causing mutations in hBest1 are located in this region that we have implicated in Ca2+ sensing, suggesting that these mutations disrupt hBest1 channel gating by Ca2+.

show abstract

Section: R E S U Lt Ssupporting

confidence: 84%

Regulation of Bestrophin Cl Channels by Calcium: Role of the C Terminus

Xiao

Prussia

et al. 2008

The Journal of General Physiology

116

View full text Add to dashboard Cite

show abstract

“…Domain cooperativity was previously reported for viable Paramecium CaM mutants in which either the calcium‐binding affinity or the target association properties were determined to be altered 66. One of the mutations in the C‐domain (E104K) changed the calcium‐binding affinity of the N‐domain, whereas four others (D95G, S101F, E104K, and H135R) affected the calcium‐binding affinity of only the mutated C‐domain 66…”

Section: Discussionmentioning

confidence: 83%

Energetics of calmodulin domain interactions with the calmodulin binding domain of CaMKII

Evans

Shea

2008

Proteins

Self Cite

View full text Add to dashboard Cite

Calmodulin (CaM) is an essential eukaryotic calcium receptor that regulates many kinases, including CaMKII. Calcium-depleted CaM does not bind to CaMKII under physiological conditions. However, binding of (Ca 2+ ) 4 -CaM to a basic amphipathic helix in CaMKII releases auto-inhibition of the kinase. The crystal structure of CaM bound to CaMKIIp, a peptide representing the CaM-binding domain (CaMBD) of CaMKII, shows an anti-parallel interface: the C-domain of CaM primarily contacts the N-terminal half of the CaMBD. The two domains of calcium-saturated CaM are believed to play distinct roles in releasing auto-inhibition. To investigate the underlying mechanism of activation, calcium-dependent titrations of isolated domains of CaM binding to CaMKIIp were monitored using fluorescence anisotropy. The binding affinity of CaMKIIp for the domains of CaM increased upon saturation with calcium, with a 35-fold greater increase observed for the C-domain than the N-domain. Because the interdomain linker of CaM regulates calcium-binding affinity and contribute to conformational change, the role of each CaM domain was explored further by investigating effects of CaMKIIp on site-knockout mutants affecting the calcium-binding sites of a single domain. Investigation of the thermodynamic linkage between saturation of individual calcium-binding sites and CaM-domain binding to CaMKIIp showed that calcium binding to sites III and IV was sufficient to recapitulate the behavior of (Ca 2+ ) 4 -CaM. The magnitude of favorable interdomain cooperativity varied depending on which of the four calcium-binding sites were mutated, emphasizing differential regulatory roles for the domains of CaM, despite the high degree of homology among the four EFhands of CaM. KeywordsThermodynamic; anisotropy; fluorescence; cooperativity; mutation; calcium binding Calmodulin (CaM) is a small (148 a.a.), ubiquitous calcium signaling protein that regulates the activities of many cellular proteins. It has four calcium-binding sites (I and II in the Ndomain, and III and IV in the C-domain; Fig. 1A). Favorable calcium binding depends on a glutamate at position 12 in each site, which provides bidentate coordination of the calcium ion 1-3 . Studies of a site IV mutant of CaM lowered the calcium-binding affinity in the Cdomain, but raised the affinity of the N-domain 4 . Mutagenesis and structural studies by Grabarek and coworkers emphasized the key role of the glutamate at position 12 in determining secondary and tertiary structure, as well as coordinating calcium 5 .Upon calcium binding to CaM, methionine-rich hydrophobic patches are exposed to solvent 6,7 , promoting association with the CaM-binding domain (CaMBD) of its target proteins 8- CaM-dependent kinase II (CaMKII) is a multifunctional serine/threonine kinase found in many tissue types with highly homologous α, β, and δ isoforms (for review, see 26 ). Activation of CaMKII contributes to synaptic plasticity and long-term potentiation (LTP) in the brain [26][27][28] , and normal and pathological condit...

show abstract

“…[54] Based on the principle of conservation of energy (shown in the linkage equations below), the difference in energy between apo CaM (ΔG a ) and calcium-saturated CaM (ΔG d ) binding to the Na V 1.2 IQ biosensor (Table 1, ΔΔG +1.93) must be the same as the difference in calcium binding to CaM alone (ΔG c ) and to CaM bound to the Na V 1.2 IQ biosensor (206G b ). …”

Section: Methodsmentioning

confidence: 99%

Calcium triggers reversal of calmodulin on nested anti-parallel sites in the IQ motif of the neuronal voltage-dependent sodium channel Na V 1.2

Hovey

Fowler

Mahling

et al. 2017

Biophysical Chemistry

Self Cite

View full text Add to dashboard Cite

Several members of the voltage-gated sodium channel family are regulated by calmodulin (CaM) and ionic calcium. The neuronal voltage-gated sodium channel NaV1.2 contains binding sites for both apo (calcium-depleted) and calcium-saturated CaM. We have determined equilibrium dissociation constants for rat NaV1.2 IQ motif [IQRAYRRYLLK] binding to apo CaM (~3 nM) and (Ca2+)4-CaM (~85 nM), showing that apo CaM binding is favored by 30-fold. For both apo and (Ca2+)4-CaM, NMR demonstrated that NaV1.2 IQ motif peptide (NaV1.2|Qp) exclusively made contacts with C-domain residues of CaM (CaMC). To understand how calcium triggers conformational change at the CaM-IQ interface, we determined a solution structure (2M5E.pdb) of (Ca2+)2-CaMC bound to NaV1.2IQp. The polarity of (Ca2+)2-CaMC relative to the IQ motif was opposite to that seen in apo CaMC-Nav1.2IQp (2KXW), revealing that CaMC recognizes nested, anti-parallel sites in Nav1.2IQp. Reversal of CaM may require transient release from the IQ motif during calcium binding, and facilitate a re-orientation of CaMN allowing interactions with non-IQ NaV1.2 residues or auxiliary regulatory proteins interacting in the vicinity of the IQ motif.

show abstract

Calcium Binding to Calmodulin Mutants Having Domain-Specific Effects on the Regulation of Ion Channels

Cited by 14 publications

References 53 publications

Regulation of Bestrophin Cl Channels by Calcium: Role of the C Terminus

Regulation of Bestrophin Cl Channels by Calcium: Role of the C Terminus

Energetics of calmodulin domain interactions with the calmodulin binding domain of CaMKII

Calcium triggers reversal of calmodulin on nested anti-parallel sites in the IQ motif of the neuronal voltage-dependent sodium channel Na V 1.2

Contact Info

Product

Resources

About