Thermodynamic linkage between calmodulin domains binding calcium and contiguous sites in the C-terminal tail of Ca(V)1.2

Calmodulin (CaM) binding to the intracellular C-terminal tail (CTT) of the cardiac L-type Ca²⁺ channel (Ca_V1.2) regulates Ca²⁺ entry by recognizing sites that contribute to negative feedback mechanisms for channel closing. CaM associates with Ca_V1.2 under low resting [Ca²⁺], but is poised to change conformation and position when intracellular [Ca²⁺] rises. CaM binding Ca²⁺, and the domains of CaM binding the CTT are linked thermodynamic functions. To better understand regulation, we determined the energetics of CaM domains binding to peptides representing pre-IQ sites A₁₅₈₈, and C₁₆₁₄ and the IQ motif studied as overlapping peptides IQ₁₆₄₄ and IQ′₁₆₅₀ as well as their effect on calcium binding. (Ca²⁺)₄-CaM bound to all four peptides very favorably (K_d ≤ 2 nM). Linkage analysis showed that IQ_1644-1670 bound with a K_d ~ 1 pM. In the pre-IQ region, (Ca²⁺)₂-N-domain bound preferentially to A₁₅₈₈, while (Ca²⁺)₂-C-domain preferred C₁₆₁₄. When bound to C₁₆₁₄, calcium binding in the N-domain affected the tertiary conformation of the C-domain. Based on the thermodynamics, we propose a structural mechanism for calcium-dependent conformational change in which the linker between CTT sites A and C buckles to form an A-C hairpin that is bridged by calcium-saturated CaM.     

Backbone resonance assignments of complexes of apo human calmodulin bound to IQ motif peptides of voltage-dependent sodium channels Na<Subscript>V</Subscript>1.1, Na<Subscript>V</Subscript>1.4 and Na<Subscript>V</Subscript>1.7

Human voltage-gated sodium (Na_V) channels are critical for initiating and propagating action potentials in excitable cells. Nine isoforms have different roles but similar topologies, with a pore-forming α-subunit and auxiliary transmembrane β-subunits. Na_V pathologies lead to debilitating conditions including epilepsy, chronic pain, cardiac arrhythmias, and skeletal muscle paralysis. The ubiquitous calcium sensor calmodulin (CaM) binds to an IQ motif in the C-terminal tail of the α-subunit of all Na_V isoforms, and contributes to calcium-dependent pore-gating in some channels. Previous structural studies of calcium-free (apo) CaM bound to the IQ motifs of Na_V1.2, Na_V1.5, and Na_V1.6 showed that CaM binding was mediated by the C-domain of CaM (CaM_C), while the N-domain (CaM_N) made no detectable contacts. To determine whether this domain-specific recognition mechanism is conserved in other Na_V isoforms, we used solution NMR spectroscopy to assign the backbone resonances of complexes of apo CaM bound to peptides of IQ motifs of Na_V1.1, Na_V1.4, and Na_V1.7. Analysis of chemical shift differences showed that peptide binding only perturbed resonances in CaM_C; resonances of CaM_N were identical to free CaM. Thus, CaM_C residues contribute to the interface with the IQ motif, while CaM_N is available to interact elsewhere on the channel.     

Determinants in CaV1 Channels That Regulate the Ca2+ Sensitivity of Bound Calmodulin

Calmodulin binds to IQ motifs in the α₁ subunit of Ca_V1.1 and Ca_V1.2, but the affinities of calmodulin for the motif and for Ca²⁺ are higher when bound to Ca_V1.2 IQ. The Ca_V1.1 IQ and Ca_V1.2 IQ sequences differ by four amino acids. We determined the structure of calmodulin bound to Ca_V1.1 IQ and compared it with that of calmodulin bound to Ca_V1.2 IQ. Four methionines in Ca²⁺-calmodulin form a hydrophobic binding pocket for the peptide, but only one of the four nonconserved amino acids (His-1532 of Ca_V1.1 and Tyr-1675 of Ca_V1.2) contacts this calmodulin pocket. However, Tyr-1675 in Ca_V1.2 contributes only modestly to the higher affinity of this peptide for calmodulin; the other three amino acids in Ca_V1.2 contribute significantly to the difference in the Ca²⁺ affinity of the bound calmodulin despite having no direct contact with calmodulin. Those residues appear to allow an interaction with calmodulin with one lobe Ca²⁺-bound and one lobe Ca²⁺-free. Our data also provide evidence for lobe-lobe interactions in calmodulin bound to Ca_V1.2.The complexity of eukaryotic Ca²⁺ signaling arises from the ability of cells to respond differently to Ca²⁺ signals that vary in amplitude, duration, and location. A variety of mechanisms decode these signals to drive the appropriate physiological responses. The Ca²⁺ sensor for many of these physiological responses is the Ca²⁺-binding protein calmodulin (CaM).² The primary sequence of CaM is tightly conserved in all eukaryotes, yet it binds and regulates a broad set of target proteins in response to Ca²⁺ binding. CaM has two domains that bind Ca²⁺ as follows: an amino-terminal domain (N-lobe) and a carboxyl-terminal domain (C-lobe) joined via a flexible α-helix. Each lobe of CaM binds two Ca²⁺ ions, and binding within each lobe is highly cooperative. The two lobes of CaM, however, have distinct Ca²⁺ binding properties; the C-lobe has higher Ca²⁺ affinity because of a slower rate of dissociation, whereas the N-lobe has weaker Ca²⁺ affinity and faster kinetics (1). CaM can also bind to some target proteins in both the presence and absence of Ca²⁺, and the preassociation of CaM in low Ca²⁺ modulates the apparent Ca²⁺ affinity of both the amino-terminal and carboxyl-terminal lobes. Differences in the Ca²⁺ binding properties of the lobes and in the interaction sites of the amino- and carboxyl-terminal lobes enable CaM to decode local versus global Ca²⁺ signals (2).Even though CaM is highly conserved, CaM target (or recognition) sites are quite heterogeneous. The ability of CaM to bind to very different targets is at least partially due to its flexibility, which allows it to assume different conformations when bound to different targets. CaM also binds to various targets in distinct Ca²⁺ saturation states as follows: Ca²⁺-free (3), Ca²⁺ bound to only one of the two lobes, or fully Ca²⁺-bound (4–7). In addition, CaM may bind with both lobes bound to a target (5, 6) or with only a single lobe engaged (8). If a target site can bind multiple conformers of CaM, CaM may undergo several transitions that depend on Ca²⁺ concentration, thereby tuning the functional response. Identification of stable intermediate states of CaM bound to individual targets will help to elucidate the steps involved in this fine-tuned control.Both Ca_V1.1 and Ca_V1.2 belong to the L-type family of voltage-dependent Ca²⁺ channels, which bind apoCaM and Ca²⁺-CaM at carboxyl-terminal recognition sites in their α₁ subunits (9–14). Ca²⁺ binding to CaM, bound to Ca_V1.2 produces Ca²⁺-dependent facilitation (CDF) (14). Whether Ca_V1.1 undergoes CDF is not known. However, both Ca_V1.2 and Ca_V1.1 undergo Ca²⁺- and CaM-dependent inactivation (CDI) (14, 15). Ca_V1.1 CDI is slower and more sensitive to buffering by 1,2-bis(o-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid than Ca_V1.2 CDI (15). Ca²⁺ buffers are thought to influence CDI and/or CDF in voltage-dependent Ca²⁺ channels by competing with CaM for Ca²⁺ (16).The conformation of the carboxyl terminus of the α₁ subunit is critical for channel function and has been proposed to regulate the gating machinery of the channel (17, 18). Several interactions of this region include intramolecular contacts with the pore inactivation machinery and intermolecular contacts with CaM kinase II and ryanodine receptors (17, 19–22). Ca²⁺ regulation of Ca_V1.2 may involve several motifs within this highly conserved region, including an EF hand motif and three contiguous CaM-binding sequences (10, 12). ApoCaM and Ca²⁺-CaM-binding sites appear to overlap at the site designated as the “IQ motif” (9, 12, 13), which are critical for channel function at the molecular and cellular level (14, 23).Differences in the rate at which 1,2-bis(o-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid affects CDI of Ca_V1.1 and Ca_V1.2 could reflect differences in their interactions with CaM. In this study we describe the differences in CaM interactions with the IQ motifs of the Ca_V1.1 and the Ca_V1.2 channels in terms of crystal structure, CaM affinity, and Ca²⁺ binding to CaM. We find the structures of Ca²⁺-CaM-IQ complexes are similar except for a single amino acid change in the peptide that contributes to its affinity for CaM. We also find that the other three amino acids that differ in Ca_V1.2 and Ca_V1.1 contribute to the ability of Ca_V1.2 to bind a partially Ca²⁺-saturated form of CaM.     

Neurogranin Alters the Structure and Calcium Binding Properties of Calmodulin

Neurogranin (Ng) is a member of the IQ motif class of calmodulin (CaM)-binding proteins, and interactions with CaM are its only known biological function. In this report we demonstrate that the binding affinity of Ng for CaM is weakened by Ca²⁺ but to a lesser extent (2–3-fold) than that previously suggested from qualitative observations. We also show that Ng induced a >10-fold decrease in the affinity of Ca²⁺ binding to the C-terminal domain of CaM with an associated increase in the Ca²⁺ dissociation rate. We also discovered a modest, but potentially important, increase in the cooperativity in Ca²⁺ binding to the C-lobe of CaM in the presence of Ng, thus sharpening the threshold for the C-domain to become Ca²⁺-saturated. Domain mapping using synthetic peptides indicated that the IQ motif of Ng is a poor mimetic of the intact protein and that the acidic sequence just N-terminal to the IQ motif plays an important role in reproducing Ng-mediated decreases in the Ca²⁺ binding affinity of CaM. Using NMR, full-length Ng was shown to make contacts largely with residues in the C-domain of CaM, although contacts were also detected in residues in the N-terminal domain. Together, our results can be consolidated into a model where Ng contacts residues in the N- and C-lobes of both apo- and Ca²⁺-bound CaM and that although Ca²⁺ binding weakens Ng interactions with CaM, the most dramatic biochemical effect is the impact of Ng on Ca²⁺ binding to the C-terminal lobe of CaM.     

Mechanisms of a Human Skeletal Myotonia Produced by Mutation in the C-Terminus of NaV1.4: Is Ca2+ Regulation Defective?

Mutations in the cytoplasmic tail (CT) of voltage gated sodium channels cause a spectrum of inherited diseases of cellular excitability, yet to date only one mutation in the CT of the human skeletal muscle voltage gated sodium channel (hNa_V1.4_F1705I) has been linked to cold aggravated myotonia. The functional effects of altered regulation of hNa_V1.4_F1705I are incompletely understood. The location of the hNa_V1.4_F1705I in the CT prompted us to examine the role of Ca²⁺ and calmodulin (CaM) regulation in the manifestations of myotonia. To study Na channel related mechanisms of myotonia we exploited the differences in rat and human Na_V1.4 channel regulation by Ca²⁺ and CaM. hNa_V1.4_F1705I inactivation gating is Ca²⁺-sensitive compared to wild type hNa_V1.4 which is Ca²⁺ insensitive and the mutant channel exhibits a depolarizing shift of the V_1/2 of inactivation with CaM over expression. In contrast the same mutation in the rNa_V1.4 channel background (rNa_V1.4_F1698I) eliminates Ca²⁺ sensitivity of gating without affecting the CaM over expression induced hyperpolarizing shift in steady-state inactivation. The differences in the Ca²⁺ sensitivity of gating between wild type and mutant human and rat Na_V1.4 channels are in part mediated by a divergence in the amino acid sequence in the EF hand like (EFL) region of the CT. Thus the composition of the EFL region contributes to the species differences in Ca²⁺/CaM regulation of the mutant channels that produce myotonia. The myotonia mutation F1705I slows I_Na decay in a Ca²⁺-sensitive fashion. The combination of the altered voltage dependence and kinetics of I_Na decay contribute to the myotonic phenotype and may involve the Ca²⁺-sensing apparatus in the CT of Na_V1.4.     

Current view on regulation of voltage‐gated sodium channels by calcium and auxiliary proteins

In cardiac and skeletal myocytes, and in most neurons, the opening of voltage‐gated Na⁺ channels (Na_V channels) triggers action potentials, a process that is regulated via the interactions of the channels’ intercellular C‐termini with auxiliary proteins and/or Ca²⁺. The molecular and structural details for how Ca²⁺ and/or auxiliary proteins modulate Na_V channel function, however, have eluded a concise mechanistic explanation and details have been shrouded for the last decade behind controversy about whether Ca²⁺ acts directly upon the Na_V channel or through interacting proteins, such as the Ca²⁺ binding protein calmodulin (CaM). Here, we review recent advances in defining the structure of Na_V intracellular C‐termini and associated proteins such as CaM or fibroblast growth factor homologous factors (FHFs) to reveal new insights into how Ca²⁺ affects Na_V function, and how altered Ca²⁺‐dependent or FHF‐mediated regulation of Na_V channels is perturbed in various disease states through mutations that disrupt CaM or FHF interaction.     

Rem GTPase interacts with the proximal CaV1.2 C-terminus and modulates calcium-dependent channel inactivation

The Rem, Rem2, Rad, and Gem/Kir (RGK) GTPases, comprise a subfamily of small Ras-related GTP-binding proteins, and have been shown to potently inhibit high voltage-activated Ca²⁺ channel current following overexpression. Although the molecular mechanisms underlying RGK-mediated Ca²⁺ channel regulation remains controversial, recent studies suggest that RGK proteins inhibit Ca²⁺ channel currents at the plasma membrane in part by interactions with accessory channel β subunits. In this paper, we extend our understanding of the molecular determinants required for RGK-mediated channel regulation by demonstrating a direct interaction between Rem and the proximal C-terminus of Ca_V1.2 (PCT), including the CB/IQ domain known to contribute to Ca²⁺/calmodulin (CaM)-mediated channel regulation. The Rem2 and Rad GTPases display similar patterns of PCT binding, suggesting that the Ca_V1.2 C-terminus represents a common binding partner for all RGK proteins. In vitro Rem:PCT binding is disrupted by Ca²⁺/CaM, and this effect is not due to Ca²⁺/CaM binding to the Rem C-terminus. In addition, co-overexpression of CaM partially relieves Rem-mediated L-type Ca²⁺ channel inhibition and slows the kinetics of Ca²⁺-dependent channel inactivation. Taken together, these results suggest that the association of Rem with the PCT represents a crucial molecular determinant in RGK-mediated Ca²⁺ channel regulation and that the physiological function of the RGK GTPases must be re-evaluated. Rather than serving as endogenous inhibitors of Ca²⁺ channel activity, these studies indicate that RGK proteins may play a more nuanced role, regulating Ca²⁺ currents via modulation of Ca²⁺/CaM-mediated channel inactivation kinetics.     

The Calmodulin Regulator Protein,PEP-19, Sensitizes ATP-induced Ca2+ Release

PEP-19 is a small, intrinsically disordered protein that binds to the C-domain of calmodulin (CaM) via an IQ motif and tunes its Ca²⁺ binding properties via an acidic sequence. We show here that the acidic sequence of PEP-19 has intrinsic Ca²⁺ binding activity, which may modulate Ca²⁺ binding to CaM by stabilizing an initial Ca²⁺-CaM complex or by electrostatically steering Ca²⁺ to and from CaM. Because PEP-19 is expressed in cells that exhibit highly active Ca²⁺ dynamics, we tested the hypothesis that it influences ligand-dependent Ca²⁺ release. We show that PEP-19 increases the sensitivity of HeLa cells to ATP-induced Ca²⁺ release to greatly increase the percentage of cells responding to sub-saturating doses of ATP and increases the frequency of Ca²⁺ oscillations. Mutations in the acidic sequence of PEP-19 that inhibit or prevent it from modulating Ca²⁺ binding to CaM greatly inhibit its effect on ATP-induced Ca²⁺ release. Thus, this cellular effect of PEP-19 does not depend simply on binding to CaM via the IQ motif but requires its acidic metal binding domain. Tuning the activities of Ca²⁺ mobilization pathways places PEP-19 at the top of CaM signaling cascades, with great potential to exert broad effects on downstream CaM targets, thus expanding the biological significance of this small regulator of CaM signaling.     

Solution NMR structure of Apo-calmodulin in complex with the IQ motif of human cardiac sodium channel NaV1.5

The function of the human voltage-gated sodium channel Na_V1.5 is regulated in part by intracellular calcium signals. The ubiquitous calcium sensor protein calmodulin (CaM) is an important part of the complex calcium-sensing apparatus in Na_V1.5. CaM interacts with an IQ (isoleucine-glutamine) motif in the large intracellular C-terminal domain of the channel. Using co-expression and co-purification, we have been able to isolate a CaM-IQ motif complex and to determine its high-resolution structure in absence of calcium using multi-dimensional solution NMR. Under these conditions, the Na_V1.5 IQ motif interacts with the C-terminal domain (C-lobe) of CaM, with the N-terminal domain remaining free in solution. The structure reveals that the C-lobe adopts a semi-open conformation with the IQ motif bound in a narrow hydrophobic groove. Sequence similarities between voltage-gated sodium channels and voltage-gated calcium channels suggest that the structure of the CaM-Na_V1.5 IQ motif complex can serve as a general model for the interaction between CaM and ion channel IQ motifs under low-calcium conditions. The structure also provides insight into the biochemical basis for disease-associated mutations that map to the IQ motif in Na_V1.5.     

Seeing the Forest through the Trees: towards a Unified View on Physiological Calcium Regulation of Voltage-Gated Sodium Channels

Voltage-gated sodium channels (Na_Vs) underlie the upstroke of the action potential in the excitable tissues of nerve and muscle. After opening, Na_Vs rapidly undergo inactivation, a crucial process through which sodium conductance is negatively regulated. Disruption of inactivation by inherited mutations is an established cause of lethal cardiac arrhythmia, epilepsy, or painful syndromes. Intracellular calcium ions (Ca²⁺) modulate sodium channel inactivation, and multiple players have been suggested in this process, including the cytoplasmic Na_V C-terminal region including two EF-hands and an IQ motif, the Na_V domain III-IV linker, and calmodulin. Calmodulin can bind to the IQ domain in both Ca²⁺-bound and Ca²⁺-free conditions, but only to the DIII-IV linker in a Ca²⁺-loaded state. The mechanism of Ca²⁺ regulation, and its composite effect(s) on channel gating, has been shrouded in much controversy owing to numerous apparent experimental inconsistencies. Herein, we attempt to summarize these disparate data and propose a novel, to our knowledge, physiological mechanism whereby calcium ions promote sodium current facilitation due to Ca²⁺ memory at high-action-potential frequencies where Ca²⁺ levels may accumulate. The available data suggest that this phenomenon may be disrupted in diseases where cytoplasmic calcium ion levels are chronically high and where targeted phosphorylation may decouple the Ca²⁺ regulatory machinery. Many Na_V disease mutations associated with electrical dysfunction are located in the Ca²⁺-sensing machinery and misregulation of Ca²⁺-dependent channel modulation is likely to contribute to disease phenotypes.     

Both N- and C-lobes of calmodulin are required for Ca-dependent regulations of CaV1.2 Ca channels

We investigated the concentration- and Ca²⁺-dependent effects of CaM mutants, CaM₁₂ and CaM₃₄, in which Ca²⁺-binding to its N- and C-lobes was eliminated, respectively, on the Ca_V1.2 Ca²⁺ channel by inside-out patch clamp in guinea-pig cardiomyocytes. Both CaM₁₂ and CaM₃₄ (0.7-10 μM) applied with 3 mM ATP produced channel activity after “rundown”. Concentration-response curves were bell-shaped, similar to that for wild-type CaM. However, there was no obvious leftward shift of the curves by increasing [Ca²⁺], suggesting that both functional lobes of CaM were necessary for the Ca²⁺-dependent shift. However, channel activity induced by the CaM mutants showed Ca²⁺-dependent decrease, implying a Ca²⁺ sensor existing besides CaM. These results suggest that both N- and C-lobes of CaM are required for the Ca²⁺-dependent regulations of Ca_V1.2 Ca²⁺ channels.     

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 Ca²⁺ 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, Ca_v1.1 and Ca_v1.4 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 LCa_v1 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 Ca²⁺-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.     

The Ca-dependent interaction of calpastatin domain L with the C-terminal tail of the Cav1.2 channel

To demonstrate the interaction of calpastatin (CS) domain L (CS_L) with Cav1.2 channel, we investigated the binding of CS_L with various C-terminus-derived peptides at ≈ free, 100 nM, 10 μM, and 1 mM Ca²⁺ by using the GST pull-down assay method. Besides binding with the IQ motif, CS_L was also found to bind with the PreIQ motif. With increasing [Ca²⁺], the affinity of the CS_L–IQ interaction gradually decreased, and the affinity of the CS_L–PreIQ binding gradually increased. The results suggest that CS_L may bind with both the IQ and PreIQ motifs of the Cav1.2 channel in different Ca²⁺-dependent manners.     

The individual N- and C-lobes of calmodulin tether to the Cav1.2 channel and rescue the channel activity from run-down in ventricular myocytes of guinea-pig heart

The present study examined the binding of the individual N- and C-lobes of calmodulin (CaM) to Cav1.2 at different Ca²⁺ concentration ([Ca²⁺]) from ≈ free to 2 mM, and found that they may bind to Cav1.2 Ca²⁺-dependently. In particular, using the patch-clamp technique, we confirmed that the N- or C-lobes can rescue the basal activity of Cav1.2 from run-down, demonstrating the functional relevance of the individual lobes. The data imply that at resting [Ca²⁺], CaM may tether to the channel with its single lobe, leading to multiple CaM molecule binding to increase the grade of Ca²⁺-dependent regulation of Cav1.2.     

Role of calcium and calmodulin in the regulation of the rabbit ileal brush-border membrane Na+/H+ antiporter

Summary In rabbit ileum, Ca²⁺/calmodulin (CaM) appears to be involved in physiologically inhibiting the linked NaCl absorptive process, since inhibitors of Ca²⁺/CaM stimulate linked Na⁺ and Cl^– absorption. The role of Ca²⁺/CaM-dependent phosphorylation in regulation of the brush-border Na⁺/H⁺ antiporter, which is believed to be part of the neutral linked NaCl absorptive process, was studied using purified brush-border membrane vesicles, which contain both the Na⁺/H⁺ antiporter and Ca²⁺/CaM-dependent protein kinase(s) and its phosphoprotein substrates. Rabbit ileal villus cell brush-border membrane vesicles were prepared by Mg precipitation and depleted of ATP. Using a freezethaw technique, the ATP-depleted vesicles were loaded with Ca²⁺, CaM, ATP and an ATP-regenerating system consisting of creatine kinase and creatine phosphate. The combination of Ca²⁺/CaM and ATP inhibited Na⁺/H⁺ exchange by 45±13%. This effect was specific since Ca²⁺/CaM and ATP did not alter diffusive Na⁺ uptake, Na⁺-dependent glucose entry, or Na⁺ or glucose equilibrium volumes. The inhibition of the Na⁺/H⁺ exchanger by Ca²⁺/CaM/ATP was due to an effect on theV _max and not on theK _m for Na⁺. In the presence of CaM and ATP, Ca²⁺ caused a concentration-dependent inhibition of Na⁺ uptake, with an effect 50% of maximum occurring at 120nm. This Ca²⁺ concentration dependence was similar to the Ca²⁺ concentration dependence of Ca²⁺/CaM-dependent phosphorylation of specific proteins in the vesicles. The Ca²⁺/CaM/ATP-inhibition of Na⁺/H⁺ exchange was reversed by W₁₃, a Ca²⁺/CaM antagonist, but not by a hydrophobic control, W₁₂, or by H-7, a protein kinase C antagonist. we conclude that Ca²⁺, acting through CaM, regulates ileal brush-border Na⁺/H⁺ exchange, and that this may be involved in the regulation of neutral linked NaCl absorption.     

Characterization of novel calmodulin binding domains within IQ motifs of IQGAP1

IQ motif-containing GTPase-activating protein 1 (IQGAP1), which is a well-known calmodulin (CaM) binding protein, is involved in a wide range of cellular processes including cell proliferation, tumorigenesis, adhesion, and migration. Interaction of IQGAP1 with CaM is important for its cellular functions. Although each IQ domain of IQGAP1 for CaM binding has been characterized in a Ca²⁺-dependent or -independent manner, it was not clear which IQ motifs are physiologically relevant for CaM binding in the cells. In this study, we performed immunoprecipitation using 3xFLAGhCaM in mammalian cell lines to characterize the domains of IQGAP1 that are key for CaM binding under physiological conditions. Interestingly, using this method, we identified two novel domains, IQ(2.7–3) and IQ(3.5–4.4), within IQGAP1 that were involved in Ca²⁺-independent or -dependent CaM binding, respectively. Mutant analysis clearly showed that the hydrophobic regions within IQ(2.7–3) were mainly involved in apoCaM binding, while the basic amino acids and hydrophobic region of IQ(3.5–4.4) were required for Ca²⁺/CaM binding. Finally, we showed that IQ(2.7–3) was the main apoCaM binding domain and both IQ(2.7–3) and IQ(3.5–4.4) were required for Ca²⁺/CaM binding within IQ(1-2-3-4). Thus, we identified and characterized novel direct CaM binding motifs essential for IQGAP1. This finding indicates that IQGAP1 plays a dynamic role via direct interactions with CaM in a Ca²⁺-dependent or -independent manner.     

Separate Elements within a Single IQ-like Motif in Adenylyl Cyclase Type 8 Impart Ca2+/Calmodulin Binding and Autoinhibition

The ubiquitous Ca²⁺-sensing protein calmodulin (CaM) fulfills its numerous signaling functions through a wide range of modular binding and activation mechanisms. By activating adenylyl cyclases (ACs) 1 and 8, Ca²⁺ acting via calmodulin impacts on the signaling of the other major cellular second messenger cAMP. In possessing two CaM-binding domains, a 1-5-8-14 motif at the N terminus and an IQ-like motif (IQlm) at the C terminus, AC8 offers particularly sophisticated regulatory possibilities. The IQlm has remained unexplored beyond the suggestion that it bound CaM, and the larger C2b region of which it is part was involved in the relief of autoinhibition of AC8. Here we attempt to distinguish the function of individual residues of the IQlm. From a complementary approach of in vitro and cell population AC activity assays, as well as CaM binding, we propose that the IQlm alone, and not the majority of the C2b, imparts CaM binding and autoinhibitory functions. Moreover, this duality of function is spatially separated and depends on amino acid side-chain character. Accordingly, residues critical for CaM binding are positively charged and clustered toward the C terminus, and those essential for the maintenance of autoinhibition are hydrophobic and more N-terminal. Secondary structure prediction of the IQlm supports this separation, with an ideally placed break in the α-helical nature of the sequence. We additionally find that the N and C termini of AC8 interact, which is an association specifically abrogated by fully Ca²⁺-bound, but not Ca²⁺-free, CaM. These data support a sophisticated activation mechanism of AC8 by CaM, in which the duality of the IQlm function is critical.The divalent calcium ion, Ca²⁺, plays a key role in modulating cellular processes as diverse as fertilization and apoptosis (1, 2). Ca²⁺ concentrations inside the cell are held ∼100 nm, despite a perpetually higher level of 1–2 mm in the extracellular medium. This steep gradient across the plasma membrane allows for a large influx when Ca²⁺ channels open, with subsequent signaling events that often rely on transduction via Ca²⁺-binding proteins (3). The archetypal Ca²⁺ sensor is calmodulin (CaM),2 a small, acidic protein so strictly conserved that all vertebrate CaM genes encode an identical 148-residue sequence (4). Multifunctional in its downstream effects, CaM can bind to at least 300 target proteins with novel partners continuing to be discovered (5). The list of effectors includes two isoforms of the adenylyl cyclase (AC) superfamily, AC1 and AC8, which comprise the Ca²⁺-stimulable subset of the nine particulate ACs (6). In intact cells, AC8 exhibits a predilection for store-operated, or capacitative, Ca²⁺ entry (CCE) (7, 8). This mode of Ca²⁺ entry is triggered by the emptying of endo/sarcoplasmic reticular stores by physiological or pharmacological stimuli (9, 10). Although the mechanism of the regulation of AC8 in nonexcitable cells by CCE (or voltage-gated Ca²⁺ entry in neurons) can be viewed to rely on facets of cellular compartmentalization (11–13), the detailed molecular mechanism whereby Ca²⁺ stimulates the enzyme is unclear. Consequently, the present investigation addresses the molecular mechanism whereby Ca²⁺, acting via calmodulin, stimulates AC8.The broad features of CaM that hold the key to its multiple regulatory strategies are understood. CaM is organized into two homologous globular domains (or lobes) united by a short linker segment (14). Both the N-terminal (N-lobe) and the C-terminal lobe (C-lobe) include two EF-hands (specialized Ca²⁺-coordinating helix-loop-helix motifs (15)), endowing CaM with four Ca²⁺-binding sites. Ca²⁺ binding to either lobe of CaM induces a structural reconfiguration determined by the two helices of each EF-hand separating from near-antiparallel to perpendicular arrays (14, 16). This exposes hydrophobic trenches in the C- and N-lobes sequentially (because the former has the highest affinity for Ca²⁺), which are notably lined with a disproportionate number of flexible methionine side chains, ready to accommodate a remarkable array of unrelated sequences. Initially, the mode of Ca²⁺-loaded CaM association with targets was considered to be uniform; the N- and C-lobes collapsed around the target peptides of e.g. smooth muscle myosin light chain kinase (17), skeletal muscle myosin light chain kinase (18), and CaMKIIα (19) with the N-lobe favoring the C-terminal target sequence and vice versa. However, CaM has since proven to be more versatile and unpredictable in how it associates with and regulates effectors, with reports of N-lobe-N-terminal/C-lobe-C-terminal interaction (20), target dimerization promoted by CaM (21), CaM binding to fatty acyl modifications (22), and other deviations from the early model.Nevertheless, three main forms of CaM regulation have been proposed as follows: relief of autoinhibition; active site remodeling; and dimerization of target domains (23). Whether the precise mechanism of CaM binding and subsequent regulation of AC8 falls into these categories of CaM regulation is not resolved. A previous study (24) established that AC8 possesses two calmodulin-binding domains (CaMBDs). CaM recognition sequences generally show little homology, although classifications based on relative positions of key hydrophobic residues have been usefully applied (4). The N-terminal CaMBD of AC8 conforms to a “1-5-8-14 motif” having large hydrophobic side chains from Trp, Val, or Ile residues at these spatially conserved sites. The C-terminal CaMBD contains an IQ-like motif (IQlm), in accordance with a signature (IVL)QXXXR(K) arrangement, to which CaM binds directly (25). By truncating one terminus or both termini, Gu and Cooper (24) asserted that the N terminus contributed little to direct CaM regulation of AC8, whereas the C terminus was critical for the maintenance of an auto-inhibited state, which was relieved upon binding of CaM. Thus, functional elements of both autoinhibition and pre-association, imparted by noncontiguous CaMBDs, have been suggested, thereby excluding AC8 from a simple model of CaM binding to an autoinhibitory domain leading to activation, as is sometimes observed (23).Recently, the proposal that the two CaMBDs play separate roles in AC8 activation was reinforced (26). This study suggested that CaM tethering by the N-terminal 1-5-8-14 motif provides the catalytically relevant C-terminal CaMBD with privileged access to CaM, thereby circumventing the need for AC8 to compete for CaM, whose free concentration in the cell is far exceeded by that of its targets (27, 28). 1–14 motifs are more generally employed in relieving autoinhibitory influences (23), so the use by AC8 of a 1-5-8-14 motif as a CaM-tethering site is unusual. The proposal that CaM pre-associates here was based on limited mutagenesis of residues within the 1-5-8-14 motif of AC8 and the use of CaM mutants whose Ca²⁺ binding capability was abolished completely or limited to discrete lobes. In contrast to the 1-5-8-14 motif at the N terminus, very little is known of the IQlm at the C terminus, in terms of the roles of the individual residues in CaM binding or autoinhibition, or even on the interplay between CaMBDs at the N and C termini of AC8.Against this background, the present study sought to assess the contribution of IQlm residues to the function of AC8, focusing on consequences of key mutations on CaM-binding efficiency, regulation by Ca²⁺/CaM, and maintenance of the autoinhibited state. Through this series of experiments, the level of coordination between the IQ-like and 1-5-8-14 motifs became evident. The provision of a pre-associated CaM molecule was found to allow for potentially deleterious mutations of the IQlm to be tolerated. Within this latter motif, Leu¹¹⁹⁶, Val¹¹⁹⁷, and Leu¹²⁰⁰ are residues essential to autoinhibition, whereas the main responsibility of binding CaM directly at the C terminus lies with Arg¹²⁰² and Arg¹²⁰⁴. In this regard, the AC8 IQlm spatially separates the two functions of CaM binding and maintenance of autoinhibition, a separation that is supported by a predicted break in the helicity of the IQlm region. Thus, a new variation is revealed in the manner by which a target exploits the CaM device into a sophisticated activation mechanism.     

Interactions between N and C termini of α1C subunit regulate inactivation of CaV1.2 L-type Ca2+ channel

The modulation and regulation of voltage-gated Ca²⁺ channels is affected by the pore-forming segments, the cytosolic parts of the channel, and interacting intracellular proteins. In this study we demonstrate a direct physical interaction between the N terminus (NT) and C terminus (CT) of the main subunit of the L-type Ca²⁺ channel Ca_V1.2, α_1C, and explore the importance of this interaction for the regulation of the channel. We used biochemistry to measure the strength of the interaction and to map the location of the interaction sites, and electrophysiology to investigate the functional impact of the interaction. We show that the full-length NT (amino acids 1-154) and the proximal (close to the plasma membrane) part of the CT, pCT (amino acids 1508-1669) interact with sub-micromolar to low-micromolar affinity. Calmodulin (CaM) is not essential for the binding. The results further suggest that the NT-CT interaction regulates the channel's inactivation, and that Ca²⁺, presumably through binding to calmodulin (CaM), reduces the strength of NT-CT interaction. We propose a molecular mechanism in which NT and CT of the channel serve as levers whose movements regulate inactivation by promoting changes in the transmembrane core of the channel via S1 (NT) or S6 (pCT) segments of domains I and IV, accordingly, and not as a kind of pore blocker. We hypothesize that Ca²⁺-CaM-induced changes in NT-CT interaction may, in part, underlie the acceleration of Ca_V1.2 inactivation induced by Ca²⁺ entry into the cell.     

Calmodulin mediates Ca2+ sensitivity of sodium channels

Ca2+ has been proposed to regulate Na+ channels through the action of calmodulin (CaM) bound to an IQ motif or through direct binding to a paired EF hand motif in the Nav1 C terminus. Mutations within these sites cause cardiac arrhythmias or autism, but details about how Ca2+ confers sensitivity are poorly understood. Studies on the homologous Cav1.2 channel revealed non-canonical CaM interactions, providing a framework for exploring Na+ channels. In contrast to previous reports, we found that Ca2+ does not bind directly to Na+ channel C termini. Rather, Ca2+ sensitivity appears to be mediated by CaM bound to the C termini in a manner that differs significantly from CaM regulation of Cav1.2. In Nav1.2 or Nav1.5, CaM bound to a localized region containing the IQ motif and did not support the large Ca(2+)-dependent conformational change seen in the Cav1.2.CaM complex. Furthermore, CaM binding to Nav1 C termini lowered Ca2+ binding affinity and cooperativity among the CaM-binding sites compared with CaM alone. Nonetheless, we found suggestive evidence for Ca2+/CaM-dependent effects upon Nav1 channels. The R1902C autism mutation conferred a Ca(2+)-dependent conformational change in Nav1.2 C terminus.CaM complex that was absent in the wild-type complex. In Nav1.5, CaM modulates the Cterminal interaction with the III-IV linker, which has been suggested as necessary to stabilize the inactivation gate, to minimize sustained channel activity during depolarization, and to prevent cardiac arrhythmias that lead to sudden death. Together, these data offer new biochemical evidence for Ca2+/CaM modulation of Na+ channel function.