Calmodulin as a universal regulator of voltage gated calcium channels

Calmodulin (CaM) is a ubiquitous calcium-binding protein responsible for the binding and activation of a vast number of enzymes and signaling pathways. It contains two lobes that bind two calcium ions each, separated by a flexible central linker. This structural flexibility allows CaM to bind and regulate a large number of diverse protein targets within the cell in response to Ca2+ gradients. Voltage gated calcium channels (CaVs), as main sources of extracellular Ca2+, are crucial for a number of physiological processes, from muscle contraction to neurotransmission and endocrine function. These large transmembrane proteins open in response to membrane depolarization and allow gated entry of Ca2+ ions into the cytoplasm. Their regulation is currently the subject of intense investigation due to its pharmacological and scientific importance. CaM has been previously shown to pre-associate and act as a potent inhibitor of one class of high-voltage activated (HVA) channels called L-type channels via its interaction with their C-terminal cytoplasmic region. This interaction is primarily mediated by a conserved CaM-binding motif called the ‘IQ’ motif (for conserved isoleucine and glutamine residues), although the exact molecular details of its involvement in inactivation are currently unclear. Elucidation of these details was the primary objective of this dissertation. Recently, a novel sequence motif within this channel called ‘NSCaTE’ (N-terminal spatial calcium transforming element) has been described as an important contributor to calcium-dependent inactivation (CDI) of L-type channels. It was presumed to be unique to vertebrates, but we also show its conservation in a distantly related L-type channel homolog of Lymnaea stagnalis (pond snail). The interaction of CaM with a number of peptides representing the different regulatory motifs (IQ and NSCaTE) for both mammalian and snail isoforms was characterized in an attempt to better understand their role in CDI. Biophysical work with peptides as well as electrophysiology recordings with an N-terminal truncation mutant of Lymnaea CaV1 homolog were performed to expand our understanding of how the interplay between these channel elements might occur. In brief, the most striking feature of the interaction concerns the strong evidence for a CaM-mediated bridge between the N- and C-terminal elements of L-type channels. Further investigation of the CaM interaction with both IQ and NSCaTE peptides using Ca2+-deficient CaM mutants reveals a preference of both peptides for the Ca2+-C-lobe of CaM, and a much higher affinity of CaM for the IQ peptide, suggesting that the N-lobe of CaM is the main interaction responsible for the physiological effects of NSCaTE. These results are consistent with our electrophysiology findings that reveal a distinct buffer-sensitive CDI in wild type LCaV1 that can be abolished by the N-terminal truncation spanning the NSCaTE region. In addition to L-type channels, CaM has also been shown to have an indirect role in the regulation of low-voltage activated (LVA) or T-type channels (CaV3.x), via their phosphorylation by CaM-dependent protein kinase II (CaMKII). Using a primary sequence scanning algorithm, a CaM-binding site was predicted in a cytoplasmic region of these channels that was also previously shown to be important in channel gating. Biophysical experiments with synthetic peptides spanning this gating brake region from the three human and the single Lymnaea isoform strongly suggest that there is a novel, bona fide CaM interaction in this channel region, and also hint that this interaction may be a Ca2+-dependent switch of some sort. The results confirm a possible new role for CaM in the direct regulation of these channels, although the exact mechanism remains to be elucidated.1 yea

Structural Consequences of Calmodulin EF Hand Mutations

This document is the Accepted Manuscript version of a Published Work that appeared in final form in Biochemistry, copyright © American Chemical Society after peer review and technical editing by publisher. To access the final edited and published work see http://dx.doi.org/10.1021/acs.biochem.6b01296Calmodulin (CaM) is a cytosolic Ca2+-binding protein that serves as a control element for many enzymes. It consists of two globular domains, each containing two EF hand pairs capable of binding Ca2+, joined by a flexible central linker region. CaM is able to bind and activate its target proteins in the Ca2+-replete and Ca2+-deplete forms. To study the Ca2+-dependent/independent properties of binding and activation of target proteins by CaM, CaM constructs with Ca2+-binding disrupting mutations of Asp to Ala at position one of each EF hand have been used. These CaM mutant proteins are deficient in binding Ca2+ in either the N-lobe EF hands (CaM12), C-lobe EF hands (CaM34), or all four EF hands (CaM1234). To investigate potential structural changes these mutations may cause, we performed detailed NMR studies of CaM12, CaM34, and CaM1234 including determining the solution structure of CaM1234. We then investigated if these CaM mutants affected the interaction of CaM with a target protein known to interact with apoCaM by determining the solution structure of CaM34 bound to the iNOS CaM binding domain peptide. The structures provide direct structural evidence of changes that are present in these Ca2+-deficient CaM mutants and show these mutations increase the hydrophobic exposed surface and decrease the electronegative surface potential throughout each lobe of CaM. These Ca2+-deficient CaM mutants may not be a true representation of apoCaM and may not allow for native-like interactions of apoCaM with its target proteins.Natural Sciences and Engineering Research Council of Canada (NSERC) [326911, 183521

Solution Structure of Calmodulin Bound to the Target Peptide of Endothelial Nitric Oxide Synthase Phosphorylated at Thr495

This document is the Accepted Manuscript version of a Published Work that appeared in final form in Biochemistry, copyright © American Chemical Society after peer review and technical editing by publisher. To access the final edited and published work see http://dx.doi.org/10.1021/bi401466sNitric oxide synthase (NOS) plays a major role in a number of key physiological and pathological processes, and it is important to understand how this enzyme is regulated. The small acidic calcium binding protein, calmodulin (CaM), is required to fully activate the enzyme. The exact mechanism of how CaM activates NOS is not fully understood at this time. Studies have shown CaM to act like a switch that causes a conformational change in NOS to allow for the transfer of an electron between the reductase and oxygenase domains through a process that is thought to be highly dynamic and at least in part controlled by several possible phosphorylation sites. We have determined the solution structure of CaM bound to a peptide that contains a phosphorylated threonine corresponding to Thr495 in full size endothelial NOS (eNOS) to investigate the structural and functional effects that the phosphorylation of this residue may have on nitric oxide production. Our biophysical studies show that phosphorylation of Thr495 introduces electrostatic repulsions between the target sequence and CaM as well as a diminished propensity for the peptide to form an α-helix. The calcium affinity of the CaM–target peptide complex is reduced because of phosphorylation, and this leads to weaker binding at low physiological calcium concentrations. This study provides an explanation for the reduced level of NO production by eNOS carrying a phosphorylated Thr495 residue.National Science and Engineering Research Council (NSERC) [326911-2009, 183521

Correction: The Calmodulin-Binding, Short Linear Motif, NSCaTE Is Conserved in L-Type Channel Ancestors of Vertebrate Cav1.2 and Cav1.3 Channels.

NSCaTE is a short linear motif of (xWxxx(I or L)xxxx), composed of residues with a high helix-forming propensity within a mostly disordered N-terminus that is conserved in L-type calcium channels from protostome invertebrates to humans. NSCaTE is an optional, lower affinity and calcium-sensitive binding site for calmodulin (CaM) which competes for CaM binding with a more ancient, C-terminal IQ domain on L-type channels. CaM bound to N- and C- terminal tails serve as dual detectors to changing intracellular Ca(2+) concentrations, promoting calcium-dependent inactivation of L-type calcium channels. NSCaTE is absent in some arthropod species, and is also lacking in vertebrate L-type isoforms, Cav1.1 and Cav1.4 channels. The pervasiveness of a methionine just downstream from NSCaTE suggests that L-type channels could generate alternative N-termini lacking NSCaTE through the choice of translational start sites. Long N-terminus with an NSCaTE motif in L-type calcium channel homolog LCav1 from pond snail Lymnaea stagnalis has a faster calcium-dependent inactivation than a shortened N-termini lacking NSCaTE. NSCaTE effects are present in low concentrations of internal buffer (0.5 mM EGTA), but disappears in high buffer conditions (10 mM EGTA). Snail and mammalian NSCaTE have an alpha-helical propensity upon binding Ca(2+)-CaM and can saturate both CaM N-terminal and C-terminal domains in the absence of a competing IQ motif. NSCaTE evolved in ancestors of the first animals with internal organs for promoting a more rapid, calcium-sensitive inactivation of L-type channels

Isothermal Calorimetry (ITC) analysis indicates a 2∶1 stoichiometry of NSCaTE:CaM in the absence of IQ, and a 1∶1 NSCaTE:CaM stoichiometry when CaM is first pre-bound to IQ.

<p>(A) Representative raw sample data for several CaM-peptide titrations. (B) Summary Table of ITC data. NSCaTE or IQ peptides were titrated into Ca²⁺-CaM alone or Ca²⁺-CaM pre-bound to a competing peptide at a 1∶1 ratio. All binding reactions exhibited negative enthalpy under the experimental conditions used (exothermic, ΔH <0). Despite the lack of mobility shift of Ca²⁺-CaM (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0061765#pone-0061765-g007" target="_blank">Figure 7</a>), NSCaTE does bind Ca²⁺-CaM alone. LCa_v1 and Ca_v1.2 IQ peptides have a higher affinity (Kd = 80 nM and 130 nM, respectively) for Ca²⁺-CaM than do either of the NSCaTE peptides (Kd = 0.83 µM and 3.24 µM for Ca_v1.2 and LCa_v1 NSCaTEs). Both mammalian NSCaTE_Cav1.2 and snail NSCaTE_LCav1 is able to bind to Ca²⁺-CaM pre-bound to IQ motifs, and both IQ peptides are able to bind to CaM when it is first bound to NSCaTE, which is consistent with the possibility of a NSCaTE-CaM-IQ complex <i>in vivo</i>. N values indicate that NSCaTE_LCav1 and NSCaTE_Cav1.2 can bind to Ca²⁺-CaM in a 2∶1 stoichiometry, consistent with NSCaTE possibly binding simultaneously to both the N- and C-lobes of CaM, as previously reported <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0061765#pone.0061765-Liu1" target="_blank">[30]</a>.</p

No major differences in biophysical properties between snail LCa_v1 channel containing a full-length N-terminus with NSCaTE translated from upstream methionine Met₁ or truncated N-terminus missing NSCaTE generated from the downstream methionine Met₂.

<p>LCa_v1-Met₁ and LCa_v2-Met₂ were transfected in HEK-293T cells alongside mammalian α₂δ₁ and β1b accessory subunits and recorded in 10 mM Barium (Ba) or Calcium (Ca) containing extracellular solution using patch clamp electrophysiology. Intracellular solution contained 9 mM EGTA. (A) Representative current traces generated from voltage steps (−40 mV to 60 mV in 10 mV steps) from a holding potential of −60 mV) illustrating the typical buffer resistant (9 mM EGTA) calcium-dependent inactivation when calcium is the charge carrier, leaving residual voltage-dependent inactivation when barium replaces calcium in the extracellular solution. (B) Normalized current-voltage relationships (n = 10), transformed and Boltzmann-fitted as activation curves in (C). (D) Steady-state availability curves (Ba_ex: n = 10, Ca_ex: n = 6), generated by measuring the fraction of maximal current generated after a 10 s sustained prepulse voltage from −100 to +50 mV in 10 mV steps). (E) Time of recovery from inactivation (n = 4) measured as the fraction of maximal current recovery after time delays, plotted on a log scale.</p

Gel Mobility Shift Assays illustrate that snail NSCaTE of LCav1 and mammalian NSCaTE of Cav1.2 can displace calcium-calmodulin (Ca²⁺-CaM) prebound to either snail IQ or mammalian IQ motifs.

<p>Gel shift mobility assays of CaM and individual peptides (A) or CaM pre-bound to an IQ peptide competing with increasing amounts of NSCaTE (B). Each lane contains 300pmol wild-type Ca²⁺-CaM; first lane is a CaM-only control for reference (Positions #2,#3,#5). (A) Each subsequent lane has increasing ratios of C-terminal (IQ) and/or N-terminal NSCaTE peptides at the ratios indicated. The changing conformation of CaM by IQ peptide causes a mobility shift of the CaM band (from Positions #2 → #1, top panels). Neither snail NSCaTE_LCav1 or mammalian NSCaTE_Cav1.2 changed CaM mobility alone, at any ratio (Position #3, bottom panels). (B) First lane is again Ca²⁺-CaM-only control (Position #5). Second lane is Ca²⁺-CaM with 1.5×IQ peptide alone control (for the maximum shift reference, Position #4). In subsequent lanes, increasing the amount of added NSCaTE peptide eventually reversed the slow mobility of CaM-IQ peptide back to the faster mobility of CaM without IQ peptide (Position #4 → #5), but not at a 100% effectiveness.</p

Full-length LCa_v1-Met₁ channels containing NSCaTE have a buffer-sensitive form of calcium dependent inactivation not found in truncated LCa_v1-Met₂ channels lacking NSCaTE.

<p>LCa_v1-Met₁ and LCa_v2-Met₂ were transfected in HEK-293T cells alongside mammalian α₂δ₁ and β2a accessory subunits and recorded using patch clamp electrophysiology. (A) Overlapping sample traces illustrating the ultra-fast calcium-dependent inactivation (red trace) of LCav₁-Met₁ channel currents in low (0.5 mM) EGTA buffering conditions. (B) Graph of fraction of peak current size at 300 ms time point of inactivation decay (R300). * represents a statistically significantly smaller R300 value (p<0.001, ANOVA) for LCav₁-Met₁ calcium currents (red trace), reflecting the buffer-sensitive calcium dependent inactivation uniquely in LCav₁ channels with a full N-terminus, containing NSCaTE.</p

Summary of biophysical parameters of LCav3 channel variants containing exons 8b and 25c expressed in HEK-293T cells, with one-way analysis of variance to assess statistical significance.

<p>Summary of biophysical parameters of LCav3 channel variants containing exons 8b and 25c expressed in HEK-293T cells, with one-way analysis of variance to assess statistical significance.</p

Amino acid sequence conservation of calmodulin binding, amino terminal (NSCaTE) and carboxyl terminal (Pre-IQ/IQ) motifs in L-type Cav1 channels.

<p>(A) Running window of amino acid similarity of aligned Cav1.2, Cav1.3 and four representative invertebrate L-type channels (red asterisk in B). DI, DII, DIII, DIV are the location of the four major domains, each domain consisting of six transmembrane helices. (B) Multiple alignment of N-terminal sequences illustrating the conservation of NSCaTE and a downstream methionine (Met₂) in L-type channels of coelomate animals. (C) C-terminal sequence alignments illustrating the nearly invariant Pre-IQ/IQ motifs in coelomate animals, with a recognizable “IQ” even in single-celled, <i>Paramecium</i>.</p