CaM-dependent Inactivation Research Articles

RyR2 Serine-2030 PKA Site Governs Ca2+ Release Termination and Ca2+ Alternans.

PKA (protein kinase A)-mediated phosphorylation of cardiac RyR2 (ryanodine receptor 2) has been extensively studied for decades, but the physiological significance of PKA phosphorylation of RyR2 remains poorly understood. Recent determination of high-resolution 3-dimensional structure of RyR2 in complex with CaM (calmodulin) reveals that the major PKA phosphorylation site in RyR2, serine-2030 (S2030), is located within a structural pathway of CaM-dependent inactivation of RyR2. This novel structural insight points to a possible role of PKA phosphorylation of RyR2 in CaM-dependent inactivation of RyR2, which underlies the termination of Ca2+ release and induction of cardiac Ca2+ alternans. We performed single-cell endoplasmic reticulum Ca2+ imaging to assess the impact of S2030 mutations on Ca2+ release termination in human embryonic kidney 293 cells. Here we determined the role of the PKA site RyR2-S2030 in a physiological setting, we generated a novel mouse model harboring the S2030L mutation and carried out confocal Ca2+ imaging. We found that mutations, S2030D, S2030G, S2030L, S2030V, and S2030W reduced the endoplasmic reticulum luminal Ca2+ level at which Ca2+ release terminates (the termination threshold), whereas S2030P and S2030R increased the termination threshold. S2030A and S2030T had no significant impact on release termination. Furthermore, CaM-wild-type increased, whereas Ca2+ binding deficient CaM mutant (CaM-M [a loss-of-function CaM mutation with all 4 EF-hand motifs mutated]), PKA, and Ca2+/CaMKII (CaM-dependent protein kinase II) reduced the termination threshold. The S2030L mutation abolished the actions of CaM-wild-type, CaM-M, and PKA, but not CaMKII, in Ca2+ release termination. Moreover, we showed that isoproterenol and CaM-M suppressed pacing-induced Ca2+ alternans and accelerated Ca2+ transient recovery in intact working hearts, whereas CaM-wild-type exerted an opposite effect. The impact of isoproterenol was partially and fully reversed by the PKA inhibitor N-[2-(p-bromocinnamylamino)ethyl]-5-isoquinoline-sulfonamide and the CaMKII inhibitor N-[2-[N-(4-chlorocinnamyl)-N-methylaminomethyl]phenyl]-N-(2-hydroxyethyl)-4-methoxybenzenesulfonamide individually and together, respectively. S2030L abolished the impact of CaM-wild-type, CaM-M, and N-[2-(p-bromocinnamylamino)ethyl]-5-isoquinoline-sulfonamide-sensitive component, but not the N-[2-[N-(4-chlorocinnamyl)-N-methylaminomethyl]phenyl]-N-(2-hydroxyethyl)-4-methoxybenzenesulfonamide-sensitive component, of isoproterenol.

Calmodulin variant E140G associated with long QT syndrome impairs CaMKIIδ autophosphorylation and L-type calcium channel inactivation

Long QT syndrome (LQTS) is a human inherited heart condition that can cause life-threatening arrhythmia including sudden cardiac death. Mutations in the ubiquitous Ca2+-sensingprotein calmodulin (CaM) are associated with LQTS, but the molecular mechanism by which these mutations lead to irregular heartbeats is not fully understood. Here, we use a multidisciplinary approach including protein biophysics, structural biology, confocal imaging, and patch-clamp electrophysiology to determine the effect of the disease-associated CaM mutation E140G on CaM structure and function. We present novel data showing that mutant-regulated CaMKIIδ kinase activity is impaired with a significant reduction in enzyme autophosphorylation rate. We report the first high-resolution crystal structure of a LQTS-associated CaM variant in complex with the CaMKIIδ peptide, which shows significant structural differences, compared to the WT complex. Furthermore, we demonstrate that the E140G mutation significantly disrupted Cav1.2 Ca2+/CaM-dependent inactivation, while cardiac ryanodine receptor (RyR2) activity remained unaffected. In addition, we show that the LQTS-associated mutation alters CaM's Ca2+-binding characteristics, secondary structure content, and interaction with key partners involved in excitation-contraction coupling (CaMKIIδ, Cav1.2, RyR2). In conclusion, LQTS-associated CaM mutation E140G severely impacts the structure-function relationship of CaM and its regulation of CaMKIIδ and Cav1.2. This provides a crucial insight into the molecular factors contributing to CaM-mediated arrhythmias with a central role for CaMKIIδ.

Divergent Ca2+/calmodulin feedback regulation of CaV1 and CaV2 voltage-gated calcium channels evolved in the common ancestor of Placozoa and Bilateria

CaV1 and CaV2 voltage-gated calcium channels evolved from an ancestral CaV1/2 channel via gene duplication somewhere near the stem animal lineage. The divergence of these channel types led to distinguishing functional properties that are conserved among vertebrates and bilaterian invertebrates and contribute to their unique cellular roles. One key difference pertains to their regulation by calmodulin (CaM), wherein bilaterian CaV1 channels are uniquely subject to pronounced, buffer-resistant Ca2+/CaM-dependent inactivation, permitting negative feedback regulation of calcium influx in response to local cytoplasmic Ca2+ rises. Early diverging, nonbilaterian invertebrates also possess CaV1 and CaV2 channels, but it is unclear whether they share these conserved functional features. The most divergent animals to possess both CaV1 and CaV2 channels are placozoans such as Trichoplax adhaerens, which separated from other animals over 600 million years ago shortly after their emergence. Hence, placozoans can provide important insights into the early evolution of CaV1 and CaV2 channels. Here, we build upon previous characterization of Trichoplax CaV channels by determining the cellular expression and ion-conducting properties of the CaV1 channel orthologue, TCaV1. We show that TCaV1 is expressed in neuroendocrine-like gland cells and contractile dorsal epithelial cells. In vitro, this channel conducts dihydropyridine-insensitive, high-voltage–activated Ca2+ currents with kinetics resembling those of rat CaV1.2 but with left-shifted voltage sensitivity for activation and inactivation. Interestingly, TCaV1, but not TCaV2, exhibits buffer-resistant Ca2+/CaM-dependent inactivation, indicating that this functional divergence evolved prior to the emergence of bilaterian animals and may have contributed to their unique adaptation for cytoplasmic Ca2+ signaling within various cellular contexts.

The Structural Basis of Calcium-Dependent Inactivation of the Transient Receptor Potential Vanilloid 5 Channel.

The transient receptor potential vanilloid channel subfamily member 5 (TRPV5) is a highly selective calcium ion channel predominately expressed in the kidney epithelium that plays an essential role in calcium reabsorption from renal infiltrate. In order to maintain Ca2+ homeostasis, TRPV5 possesses a tightly regulated negative feedback mechanism, where the ubiquitous Ca2+ binding protein calmodulin (CaM) directly binds to the intracellular TRPV5 C-terminus, thus regulating TRPV5. Here we report on the characterization of the TRPV5 C-terminal CaM binding site and its interaction with CaM at an atomistic level. We have solved the de novo solution structure of the TRPV5 C-terminus in complex with a CaM mutant, creating conditions that mimic the cellular basal Ca2+ state. We demonstrate that under these conditions the TRPV5 C-terminus is exclusively bound to the CaM C-lobe only, while it confers conformational freedom to the CaM N-lobe. We also show that at elevated calcium levels, additional interactions between the TRPV5 C-terminus and CaM N-lobe occur, resulting in formation of a tight 1:1 complex, effectively making the N-lobe the calcium sensor. Together, these data are consistent with and support the novel model for Ca2+/CaM-dependent inactivation of TRPV channels as proposed by Bate and co-workers [ Bate , N. , et al. ( 2018 ) Biochemistry , ( 57), DOI: 10.1021/acs.biochem.7b01286 ].

A Novel Mechanism for Calmodulin-Dependent Inactivation of Transient Receptor Potential Vanilloid 6.

The paralogues TRPV5 and TRPV6 belong to the vanilloid subfamily of the transient receptor potential (TRP) superfamily of ion channels, and both play an important role in overall Ca2+ homeostasis. The functioning of the channels centers on a tightly controlled Ca2+-dependent feedback mechanism in which the direct binding of the universal Ca2+-binding protein calmodulin (CaM) to the channel's C-terminal tail is required for channel inactivation. We have investigated this interaction at the atomic level and propose that under basal cellular Ca2+ concentrations CaM is constitutively bound to the channel's C-tail via CaM C-lobe only contacts. When the cytosolic Ca2+ concentration increases charging the apo CaM N-lobe with Ca2+, the CaM:TRPV6 complex rearranges and the TRPV6 C-tail further engages the CaM N-lobe via a crucial interaction involving L707. In a cellular context, mutation of L707 significantly increased the rate of channel inactivation. Finally, we present a model for TRPV6 CaM-dependent inactivation, which involves a novel so-called "two-tail" mechanism whereby CaM bridges two TRPV6 monomers resulting in closure of the channel pore.

A Precision Medicine Approach to the Rescue of Function on Malignant Calmodulinopathic Long-QT Syndrome.

Calmodulinopathies comprise a new category of potentially life-threatening genetic arrhythmia syndromes capable of producing severe long-QT syndrome (LQTS) with mutations involving CALM1, CALM2, or CALM3. The underlying basis of this form of LQTS is a disruption of Ca2+/calmodulin (CaM)-dependent inactivation of L-type Ca2+ channels. To gain insight into the mechanistic underpinnings of calmodulinopathies and devise new therapeutic strategies for the treatment of this form of LQTS. We generated and characterized the functional properties of induced pluripotent stem cell-derived cardiomyocytes from a patient with D130G-CALM2-mediated LQTS, thus creating a platform with which to devise and test novel therapeutic strategies. The patient-derived induced pluripotent stem cell-derived cardiomyocytes display (1) significantly prolonged action potentials, (2) disrupted Ca2+ cycling properties, and (3) diminished Ca2+/CaM-dependent inactivation of L-type Ca2+ channels. Next, taking advantage of the fact that calmodulinopathy patients harbor a mutation in only 1 of 6 redundant CaM-encoding alleles, we devised a strategy using CRISPR interference to selectively suppress the mutant gene while sparing the wild-type counterparts. Indeed, suppression of CALM2 expression produced a functional rescue in induced pluripotent stem cell-derived cardiomyocytes with D130G-CALM2, as shown by the normalization of action potential duration and Ca2+/CaM-dependent inactivation after treatment. Moreover, CRISPR interference can be designed to achieve selective knockdown of any of the 3 CALM genes, making it a generalizable therapeutic strategy for any calmodulinopathy. Overall, this therapeutic strategy holds great promise for calmodulinopathy patients as it represents a generalizable intervention capable of specifically altering CaM expression and potentially attenuating LQTS-triggered cardiac events, thus initiating a path toward precision medicine.

Functional Rescue of Calmodulinopathy IPSC-Derived Cardiomyocytes -- a Foray into Personalized Medicine

Calmodulinopathies comprise a growing population of patients with congenital life-threatening cardiac arrhythmias, including severe long-QT syndrome (LQTS). The underlying mutations disrupt Ca2+ binding to calmodulin (CaM), implicating a myriad of Ca2+/CaM targets as potential pathogenic elements. To gain further mechanistic insight, we utilized induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs), a model of congenital cardiac diseases with a patient-specific genetic background. First, we demonstrated that calmodulinopathy iPSC-CMs indeed display (1) significantly prolonged action potentials (APs), (2) increased Ca2+ transient (CaT) amplitudes, (3) delayed CaT upstrokes, and (4) drastically slower CaT decay rates -- all potential components of an arrhythmic substrate and an ideal platform for developing a treatment strategy. Next, we considered the role of the L-type Ca2+ channel (LTCC) in arrhythmogenesis within these cells. Indeed, the iPSC-CMs displayed severely diminished LTCC Ca2+/CaM-dependent inactivation (CDI), a known mechanism for AP prolongation. Finally, taking advantage of the fact that calmodulinopathy patients harbor a mutation in only a single of three redundant CaM genes, we have devised a strategy to selectively suppress the mutant gene while sparing the wild-type counterparts. The extent of this suppression was confirmed via quantitative RNA expression profiling. Importantly, this manipulation produced a functional rescue (restoration of the AP duration) in calmodulinopathy iPSC-CMs in a dose-dependent manner. Moreover, selective knockdown of any of the three genes underlying calmodulinopathies could be achieved. This technique not only promises significant relief of LQT symptoms, but any disease manifestations in these patients as well. Thus, we have expanded the therapeutic toolkit for treatment of calmodulinopathies and initiated a path towards personalized medicine.

Impaired calcium-calmodulin-dependent inactivation of Cav1.2 contributes to loss of sarcoplasmic reticulum calcium release refractoriness in mice lacking calsequestrin 2

AimsIn cardiac muscle, Ca2+ release from sarcoplasmic reticulum (SR) is reduced with successively shorter coupling intervals of premature stimuli, a phenomenon known as SR Ca2+ release refractoriness. We recently reported that the SR luminal Ca2+ binding protein calsequestrin 2 (Casq2) contributes to release refractoriness in intact mouse hearts, but the underlying mechanisms remain unclear. Here, we further investigate the mechanisms responsible for physiological release refractoriness. Methods and resultsGene-targeted ablation of Casq2 (Casq2 KO) abolished SR Ca2+ release refractoriness in isolated mouse ventricular myocytes. Surprisingly, impaired Ca2+-dependent inactivation of L-type Ca2+ current (ICa), which is responsible for triggering SR Ca2+ release, significantly contributed to loss of Ca2+ release refractoriness in Casq2 KO myocytes. Recovery from Ca2+-dependent inactivation of ICa was significantly accelerated in Casq2 KO compared to wild-type (WT) myocytes. In contrast, voltage-dependent inactivation measured by using Ba2+ as charge carrier was not significantly different between WT and Casq2 KO myocytes. Ca2+-dependent inactivation of ICa was normalized by intracellular dialysis of excess apo-CaM (20μM), which also partially restored physiological Ca2+ release refractoriness in Casq2 KO myocytes. ConclusionsOur findings reveal that the intra-SR protein Casq2 is largely responsible for the phenomenon of SR Ca2+ release refractoriness in murine ventricular myocytes. We also report a novel mechanism of impaired Ca2+-CaM-dependent inactivation of Cav1.2, which contributes to the loss of SR Ca2+ release refractoriness in the Casq2 KO mouse model and, therefore, may further increase risk for ventricular arrhythmia in vivo.

Calmodulin mutations associated with long QT syndrome prevent inactivation of cardiac L-type Ca2 + currents and promote proarrhythmic behavior in ventricular myocytes

Recent work has identified missense mutations in calmodulin (CaM) that are associated with severe early-onset long-QT syndrome (LQTS), leading to the proposition that altered CaM function may contribute to the molecular etiology of this subset of LQTS. To date, however, no experimental evidence has established these mutations as directly causative of LQTS substrates, nor have the molecular targets of CaM mutants been identified. Here, therefore, we test whether expression of CaM mutants in adult guinea-pig ventricular myocytes (aGPVM) induces action-potential prolongation, and whether affiliated alterations in the Ca2+ regulation of L-type Ca2+ channels (LTCC) might contribute to such prolongation. In particular, we first overexpressed CaM mutants in aGPVMs, and observed both increased action potential duration (APD) and heightened Ca2+ transients. Next, we demonstrated that all LQTS CaM mutants have the potential to strongly suppress Ca2+/CaM-dependent inactivation (CDI) of LTCCs, whether channels were heterologously expressed in HEK293 cells, or present in native form within myocytes. This attenuation of CDI is predicted to promote action-potential prolongation and boost Ca2+ influx. Finally, we demonstrated how a small fraction of LQTS CaM mutants (as in heterozygous patients) would nonetheless suffice to substantially diminish CDI, and derange electrical and Ca2+ profiles. In all, these results highlight LTCCs as a molecular locus for understanding and treating CaM-related LQTS in this group of patients.

A Unified Framework for Calcium Channel Modulation by Calcium Binding Proteins

Distinguishing between allostery and competition among modulating ligands is challenging for large target molecules. One key example of such ambiguity concerns calcium-binding proteins (CaBPs) that tune signaling molecules regulated by calmodulin (CaM). In L-type Ca2+ channels, CaBPs can potently eliminate CaM-dependent inactivation (CDI). However, the mechanism for this modulation remains controversial. In past, using a live-cell holomolecule approach, we resolved a cyclical allosteric binding scheme for CaM and CaBP4 to CaV1.3 channels. In this scheme, both CaBP4 and CaM can simultaneously bind, resulting in strong inhibition of CDI despite the presence of a covalently attached CaM (a), supporting an allosteric regulatory mechanism. By contrast, Findeisen et al (J Mol. Biol. 425(17):3217-34) showed that fusion of CaM to CaV1.2 channels prevents CaBP1 modulation, thus arguing for a competitive regulatory mechanism. These results are confounded by limited delivery of CaBPs through pipet dialysis. Here, we show that the CDI of CaV1.2 channels with a fused CaM is robustly inhibited by recombinantly expressed CaBP1 and CaBP4 (b). Thus, it appears that the cyclical allosteric scheme first resolved in CaV1.3 now stands as a common framework for CaBP modulation of L-type calcium channels.

How CaBPs Prevail over CaM in Modulating Cav1 Channels

Calcium-binding proteins (CaBPs), a subfamily of calmodulin (CaM)-like proteins, are widely expressed throughout the brain. They are believed to compete with CaM for binding to target molecules, thereby exerting vital modifications of Ca2+/CaM regulation. Yet, deep sequencing of brain reveals CaBP transcript levels 100-10000 times lower than for CaM. How could so little CaBP produce effects in the brain? Here, we examine this fundamental paradox in L-type CaV1.3 Ca2+ channels, whose Ca2+/CaM-dependent inactivation (CDI) can be suppressed by CaBP4. Upon fusion of CaM to CaV1.3 (a), the ultra-high local CaM concentration should occlude CaBP4 access to a common site. Remarkably, separately expressed CaBP4 still eliminates CDI (b), arguing for different CaBP4 and CaM sites. FRET assays map these distinct sites, and establish corresponding association constants for binding. This noncompetitive scheme predicts that micromolar CaBP4 should reduce CDI, even with abundant CaM. To test this, we used patch fluorometry to measure CDI while gauging the concentration of fluorescently labeled CaBP4 (c). CaBP4 indeed halves CDI by ∼0.5 uM (d, measured), with abundant CaM throughout. Hence, CaBP molecules may prevail over CaM by utilizing distinct sites on target molecules across the brain.View Large Image | View Hi-Res Image | Download PowerPoint Slide

The Distal Carboxy Tail (DCT) of CaV1.4 Modulates More than Ca2+/CaM-Dependent Inactivation (CDI)

CaV1.4 L-type Ca2+ channels populate retinal ribbon synapses, and mediate tonic neurotransmitter release. Mutations in the CACNA1F gene encoding CaV1.4 are implicated in congenital stationary night blindness (CSNB2). The K1591X CSNB2 mutation yields a premature stop that deletes the DCT of CaV1.4. Interestingly, the DCT contains a module that competes with Ca2+-free calmodulin (apoCaM) for binding at the IQ-domain of CaV1.4, thereby tuning channel affinity for apoCaM (Nature 463:968). Since only channels ‘charged’ with apoCaM exhibit Ca2+/CaM-mediated inactivation (CDI), wildtype CaV1.4 channels exhibit little or no CDI, while deleting the DCT yields a resurgence of CDI. The latter effect in K1591X channels has suggested that pathogenesis involves abnormally diminished Ca2+ influx with resulting impairment of photoreceptor signaling. Here, however, we observe a dramatic and unrecognized effect of DCT deletion. Single-channel recordings indicate that wild-type CaV1.4 feature diminutive open probability Po (A. Top, exemplar single-channel trace during voltage ramp. Bottom, Po-V relation averaged over multiple patches). By contrast, K1591X channels exhibit strikingly enhanced Po (B). Hence, this form of CSNB2 likely involves an unexpected Ca2+ overload phenomenon, raising the possibility of therapeutics involving CaV1.4 channel blockade.View Large Image | View Hi-Res Image | Download PowerPoint Slide

Alternative Splicing at C Terminus of CaV1.4 Calcium Channel Modulates Calcium-dependent Inactivation, Activation Potential, and Current Density

The Ca(V)1.4 voltage-gated calcium channel is predominantly expressed in the retina, and mutations to this channel have been associated with human congenital stationary night blindness type-2. The L-type Ca(V)1.4 channel displays distinct properties such as absence of calcium-dependent inactivation (CDI) and slow voltage-dependent inactivation (VDI) due to the presence of an autoinhibitory domain (inhibitor of CDI) in the distal C terminus. We hypothesized that native Ca(V)1.4 is subjected to extensive alternative splicing, much like the other voltage-gated calcium channels, and employed the transcript scanning method to identify alternatively spliced exons within the Ca(V)1.4 transcripts isolated from the human retina. In total, we identified 19 alternative splice variations, of which 16 variations have not been previously reported. Characterization of the C terminus alternatively spliced exons using whole-cell patch clamp electrophysiology revealed a splice variant that exhibits robust CDI. This splice variant arose from the splicing of a novel alternate exon (43*) that can be found in 13.6% of the full-length transcripts screened. Inclusion of exon 43* inserts a stop codon that truncates half the C terminus. The Ca(V)1.4 43* channel exhibited robust CDI, a larger current density, a hyperpolarized shift in activation potential by ∼10 mV, and a slower VDI. Through deletional experiments, we showed that the inhibitor of CDI was responsible for modulating channel activation and VDI, in addition to CDI. Calcium currents in the photoreceptors were observed to exhibit CDI and are more negatively activated as compared with currents elicited from heterologously expressed full-length Ca(V)1.4. Naturally occurring alternative splice variants may in part contribute to the properties of the native Ca(V)1.4 channels.

The N Terminus of a Schistosome β Subunit Regulates Inactivation and Current Density of a Cav2 Channel

The β subunit of high voltage-activated Ca(2+) (Ca(v)) channels targets the pore-forming α(1) subunit to the plasma membrane and tunes the biophysical phenotype of the Ca(v) channel complex. We used a combination of molecular biology and whole-cell patch clamp to investigate the functional role of a long N-terminal polyacidic motif (NPAM) in a Ca(v)β subunit of the human parasite Schistosoma mansoni (β(Sm)), a motif that does not occur in other known Ca(v)β subunits. When expressed in human embryonic kidney cells stably expressing Ca(v)2.3, β(Sm) accelerates Ca(2+)/calmodulin-independent inactivation of Ca(v)2.3. Deleting the first 44 amino acids of β(Sm), a region that includes NPAM, significantly slows the predominant time constant of inactivation (τ(fast)) under conditions that prevent Ca(2+)/CaM-dependent inactivation (β(Sm): τ(fast) = 66 ms; β(SmΔ2-44): τ(fast) = 111 ms, p < 0.01). Interestingly, deleting the amino acids that are N-terminal to NPAM (2-24 or 2-17) results in faster inactivation than with an intact N terminus (τ(fast) = 42 ms with β(SmΔ2-17); τ(fast) = 40 ms with β(SmΔ2-24), p < 0.01). This suggests that NPAM is the structural determinant for accelerating Ca(2+)/calmodulin-independent inactivation. We also created three chimeric subunits that contain the first 44 amino acids of β(Sm) attached to mammalian β(1b), β(2a), and β(3) subunits. For any given mammalian β subunit, inactivation was faster if it contained the N terminus of β(Sm) than if it did not. Co-expression of the mammalian α(2)δ-1 subunit resulted in doubling of the inactivation rate, but the effects of NPAM persisted. Thus, it appears that the schistosome Ca(v) channel complex has acquired a new function that likely contributes to reducing the amount of Ca(2+) that enters the cells in vivo. This feature is of potential interest as a target for new antihelminthics.

Developmental changes in calcium/calmodulin‐dependent inactivation of calcium currents at the rat calyx of Held

Ca2+-binding to calmodulin (CaM) causes facilitation and/or inactivation of recombinant Ca2+ channels. At the rat calyx of Held, before hearing onset, presynaptic Ca2+ currents (IpCa) undergo Ca2+/CaM-dependent inactivation during repetitive activation at around 1 Hz, implying that this may be a major cause of short-term synaptic depression. However, it remains open whether the Ca2+/CaM-dependent inactivation of IpCa persists in more mature animals. To address this question, we tested the effect of CaM inhibitors on the activity-dependent modulation of IpCa in calyces, before (postnatal day (P) 7-9) and after (P13-15) hearing onset. Our results indicate that the CaM-dependent IpCa inactivation during low-frequency stimulation, and the ensuing synaptic depression, occur only at calyces in the prehearing period. However, CaM immunoreactivity in P8 and P14 calyces was equally strong. Even at P13-15, high frequency stimulation (200-500 Hz) could induce IpCa inactivation, which was attenuated by EGTA (10 mM) or a CaM inhibitor peptide loaded into the terminal. Furthermore, the CaM inhibitor peptide attenuated a transient facilitation of IpCa preceding inactivation observed at 500 Hz stimulation, whereas it had no effect on sustained IpCa facilitations during trains of 50-200 Hz stimulation. These results suggest that the Ca2+/CaM-dependent IpCa modulation requires a high intraterminal Ca2+ concentration, which can be attained at immature calyces during low frequency stimulation, but only during unusually high frequency stimulation at calyceal terminals in the posthearing period.

Ca2+/CaM-dependent inactivation of the skeletal muscle L-type Ca2+ channel (Cav1.1)

Ca2+-dependent modulation via calmodulin (CaM) has been documented for most high-voltage-activated Ca2+ channels, but whether the skeletal muscle L-type channel (Cav1.1) exhibits this property has been unknown. In this paper, whole-cell current and fluorescent resonance energy transfer (FRET) recordings were obtained from cultured mouse myotubes to test for potential involvement of CaM in function of Cav1.1. When prolonged depolarization (800 ms) was used to evoke Cav1.1 currents in normal myotubes, the fraction of current remaining at the end of the pulse displayed classic signs of Ca2+-dependent inactivation (CDI), including U-shaped voltage dependence, maximal inactivation (approximately 30%) at potentials eliciting maximal inward current, and virtual elimination of inactivation when Ba2+ replaced external Ca2+ or when 10 mM BAPTA was included in the pipette solution. Furthermore, CDI was virtually eliminated (from 30 to 8%) in normal myotubes overexpressing mutant CaM (CaM1234) that does not bind Ca2+, whereas CDI was unaltered in myotubes overexpressing wild-type CaM (CaMwt). In addition, a significant FRET signal (E=4.06%) was detected between fluorescently tagged Cav1.1 and CaMwt coexpressed in dysgenic myotubes, demonstrating for the first time that these two proteins associate in vivo. These findings show that CaM associates with and modulates Cav1.1.

In CA1 Pyramidal Neurons of the Hippocampus Protein Kinase C Regulates Calcium-Dependent Inactivation of NMDA Receptors

The NMDA subtype of the glutamate-gated channel exhibits a high permeability to Ca(2+). The influx of Ca(2+) through NMDA channels is limited by a rapid and Ca(2+)/calmodulin (CaM)-dependent inactivation that results from a competitive displacement of cytoskeleton-binding proteins from the NR1 subunit of the receptor by Ca(2+)/CaM (Zhang et al., 1998; Krupp et al., 1999). The C terminal of this subunit can be phosphorylated by protein kinase C (PKC) (Tingley et al., 1993). The present study sought to investigate whether PKC regulates Ca(2+)-dependent inactivation of the NMDA channel in hippocampal neurons. Activation of endogenous PKC by 4beta-phorbol 12-myristate 13-acetate enhanced peak (I(p)) and depressed steady-state (I(ss)) NMDA-evoked currents, resulting in a reduction in the ratio of these currents (I(ss)/I(p)). We demonstrated previously that PKC activity enhances I(P) via a sequential activation of the focal adhesion kinase cell adhesion kinase beta/proline-rich tyrosine kinase 2 (CAKbeta/Pyk2) and the nonreceptor tyrosine kinase Src (Huang et al., 1999; Lu et al., 1999). Here, we report that the PKC-induced depression of I(ss) is unrelated to the PKC/CAKbeta/Src-signaling pathway but depends on the concentration of extracellular Ca(2+). Intracellular applications of CaM reduced I(ss)/I(p) and occluded the Ca(2+)-dependent effect of phorbol esters on I(ss.) Moreover, increasing the concentration of intracellular Ca(2+) buffer or intracellular application of the inhibitory CaM-binding peptide (KY9) greatly reduced the phorbol ester-induced depression of I(ss). Taken together, these results suggest that PKC enhances Ca(2+)/CaM-dependent inactivation of the NMDA channel, most likely because of a phosphorylation-dependent regulation of interactions between receptor subunits, CaM, and other postsynaptic density proteins.