Geoffrey S. Pitt

Calmodulin supports both inactivation and facilitation of L-type calcium channels

L-type Ca2+ channels support Ca2+ entry into cells, which triggers cardiac contraction, controls hormone secretion from endocrine cells and initiates transcriptional events that support learning and memory. These channels are examples of molecular signal-transduction units that regulate themselves through their own activity. Among the many types of voltage-gated Ca2+ channel, L-type Ca2+ channels particularly display inactivation and facilitation, both of which are closely linked to the earlier entry of Ca2+ ions. Both forms of autoregulation have a significant impact on the amount of Ca2+ that enters the cell during repetitive activity, with major consequences downstream. Despite extensivebiophysical analysis, the molecular basis of autoregulation remains unclear, although a putative Ca2+-binding EF-hand motif, and a nearby consensus calmodulin-binding isoleucine-glutamine (‘IQ’) motif, in the carboxy terminus of the α1C channel subunit have been implicated,. Here we show that calmodulin is a critical Ca2+ sensor for both inactivation and facilitation, and that the nature of the modulatory effect depends on residues within the IQ motif important for calmodulin binding. Replacement of the native isoleucine by alanine removed Ca2+-dependent inactivation and unmasked a strong facilitation; conversion of the same residue to glutamate eliminated both forms of autoregulation. These results indicate that the same calmodulin molecule may act as a Ca2+ sensor for both positive and negative modulation.

Structurally distinct and stage-specific adenylyl cyclase genes play different roles in dictyostelium development

We have isolated two adenylyl cyclase genes, designated ACA and ACG, from Dictyostelium. The proposed structure for ACA resembles that proposed for mammalian adenylyl cyclases: two large hydrophilic domains and two sets of six transmembrane spans. ACG has a novel structure, reminiscent of the membrane-bound guanylyl cyclases. An aca- mutant, created by gene disruption, has little detectable adenylyl cyclase activity and fails to aggregate, demonstrating that cAMP is required for cell-cell communication. cAMP is not required for motility, chemotaxis, growth, and cell division, which are unaffected. Constitutive expression in aca- cells of either ACA or ACG, which is normally expressed only during germination, restores aggregation and the ability to complete the developmental program. ACA expression restores receptor and guanine nucleotide-regulated adenylyl cyclase activity, while activity in cells expressing ACG is insensitive to these regulators. Although they lack ACA, which has a transporter-like structure, the cells expressing ACG secrete cAMP constitutively.

CaMKII tethers to L-type Ca2+ channels, establishing a local and dedicated integrator of Ca2+ signals for facilitation

Ca2+-dependent facilitation (CDF) of voltage-gated calcium current is a powerful mechanism for up-regulation of Ca2+ influx during repeated membrane depolarization. CDF of L-type Ca2+ channels (Cav1.2) contributes to the positive force–frequency effect in the heart and is believed to involve the activation of Ca2+/calmodulin-dependent kinase II (CaMKII). How CaMKII is activated and what its substrates are have not yet been determined. We show that the pore-forming subunit α1C (Cavα1.2) is a CaMKII substrate and that CaMKII interaction with the COOH terminus of α1C is essential for CDF of L-type channels. Ca2+ influx triggers distinct features of CaMKII targeting and activity. After Ca2+-induced targeting to α1C, CaMKII becomes tightly tethered to the channel, even after calcium returns to normal levels. In contrast, activity of the tethered CaMKII remains fully Ca2+/CaM dependent, explaining its ability to operate as a calcium spike frequency detector. These findings clarify the molecular basis of CDF and demonstrate a novel enzymatic mechanism by which ion channel gating can be modulated by activity.

Identification of the components controlling inactivation of voltage-gated Ca2+ channels

Ca(2+)-dependent inactivation (CDI) of L-type voltage-gated Ca(2+) channels limits Ca(2+) entry into neurons, thereby regulating numerous cellular events. Here we present the isolation and purification of the Ca(2+)-sensor complex, consisting of calmodulin (CaM) and part of the channels pore-forming alpha(1C) subunit, and demonstrate the Ca(2+)-dependent conformational shift that underlies inactivation. Dominant-negative CaM mutants that prevent CDI block the sensors Ca(2+)-dependent conformational change. We show how Ile1654 in the CaM binding IQ motif of alpha(1C) forms the link between the Ca(2+) sensor and the downstream inactivation machinery, using the alpha(1C) EF hand motif as a signal transducer to activate the putative pore-occluder, the alpha(1C) I-II intracellular linker.

KCNQ1 Assembly and Function Is Blocked by Long-QT Syndrome Mutations That Disrupt Interaction With Calmodulin

Calmodulin (CaM) has been recognized as an obligate subunit for many ion channels in which its function has not been clearly established. Because channel subunits associate early during channel biosynthesis, CaM may provide a mechanism for Ca2+-dependent regulation of channel formation. Here we show that CaM is a constitutive component of KCNQ1 K+ channels, the most commonly mutated long-QT syndrome (LQTS) locus. CaM not only acts as a regulator of channel gating, relieving inactivation in a Ca2+-dependent manner, but it also contributes to control of channel assembly. Formation of functional tetramers requires CaM interaction with the KCNQ1 C-terminus. This CaM-regulated process is essential: LQTS mutants that disrupt CaM interaction prevent functional assembly of channels in a dominant-negative manner. These findings offer a new mechanism for LQTS defects and provide a basis for understanding novel ways that intracellular Ca2+ and CaM regulate ion channels.

Crystal Structure of the Ternary Complex of a NaV C-Terminal Domain, a Fibroblast Growth Factor Homologous Factor, and Calmodulin

Voltage-gated Na⁺ (Na(V)) channels initiate neuronal action potentials. Na(V) channels are composed of a transmembrane domain responsible for voltage-dependent Na⁺ conduction and a cytosolic C-terminal domain (CTD) that regulates channel function through interactions with many auxiliary proteins, including fibroblast growth factor homologous factors (FHFs) and calmodulin (CaM). Most ion channel structural studies have focused on mechanisms of permeation and voltage-dependent gating but less is known about how intracellular domains modulate channel function. Here we report the crystal structure of the ternary complex of a human Na(V) CTD, an FHF, and Ca²⁺-free CaM at 2.2 Å. Combined with functional experiments based on structural insights, we present a platform for understanding the roles of these auxiliary proteins in Na(V) channel regulation and the molecular basis of mutations that lead to neuronal and cardiac diseases. Furthermore, we identify a critical interaction that contributes to the specificity of individual Na(V) CTD isoforms for distinctive FHFs.

Calcium Channel Function Regulated by the SH3-GK Module in β Subunits

Abstract β subunits of voltage-gated calcium channels (VGCCs) regulate channel trafficking and function, thereby shaping the intensity and duration of intracellular changes in calcium. β subunits share limited sequence homology with the Src homology 3-guanylate kinase (SH3-GK) module of membrane-associated guanylate kinases (MAGUKs). Here, we show biochemical similarities between β subunits and MAGUKs, revealing important aspects of β subunit structure and function. Similar to MAGUKs, an SH3-GK interaction within β subunits can occur both intramolecularly and intermolecularly. Mutations that disrupt the SH3-GK interaction in β subunits alter channel inactivation and can inhibit binding between the α 1 and β subunits. Coexpression of β subunits with complementary mutations in their SH3 and GK domains rescues these deficits through intermolecular β subunit assembly. In MAGUKs, the SH3-GK module controls protein scaffolding. In β subunits, this module regulates the inactivation of VGCCs and provides an additional mechanism for tuning calcium responsiveness.

Accessory Subunit KChIP2 Modulates the Cardiac L-Type Calcium Current

Complex modulation of voltage-gated Ca2+ currents through the interplay among Ca2+ channels and various Ca2+-binding proteins is increasingly being recognized. The K+ channel interacting protein 2 (KChIP2), originally identified as an auxiliary subunit for KV4.2 and a component of the transient outward K+ channel (Ito), is a Ca2+-binding protein whose regulatory functions do not appear restricted to KV4.2. Consequently, we hypothesized that KChIP2 is a direct regulator of the cardiac L-type Ca2+ current (ICa,L). We found that ICa,L density from KChIP2−/− myocytes is reduced by 28% compared to ICa,L recorded from wild-type myocytes (P<0.05). This reduction in current density results from loss of a direct effect on the Ca2+ channel current, as shown in a transfected cell line devoid of confounding cardiac ion currents. ICa,L regulation by KChIP2 was independent of Ca2+ binding to KChIP2. Biochemical analysis suggested a direct interaction between KChIP2 and the CaV1.2 &agr;1C subunit N terminus. We found that KChIP2 binds to the N-terminal inhibitory module of &agr;1C and augments ICa,L current density without increasing CaV1.2 protein expression or trafficking to the plasma membrane. We propose a model in which KChIP2 impedes the N-terminal inhibitory module of CaV1.2, resulting in increased ICa,L. In the context of recent reports that KChIP2 modulates multiple KV and NaV currents, these results suggest that KChIP2 is a multimodal regulator of cardiac ionic currents.

Fibroblast Growth Factor Homologous Factor 13 Regulates Na+ Channels and Conduction Velocity in Murine Hearts

Rationale: Fibroblast growth factor homologous factors (FHFs), a subfamily of fibroblast growth factors (FGFs) that are incapable of functioning as growth factors, are intracellular modulators of Na+ channels and have been linked to neurodegenerative diseases. Although certain FHFs have been found in embryonic heart, they have not been reported in adult heart, and they have not been shown to regulate endogenous cardiac Na+ channels or to participate in cardiac pathophysiology. Objective: We tested whether FHFs regulate Na+ channels in murine heart. Methods and Results: We demonstrated that isoforms of FGF13 are the predominant FHFs in adult mouse ventricular myocytes. FGF13 binds directly to, and colocalizes with, the NaV1.5 Na+ channel in the sarcolemma of adult mouse ventricular myocytes. Knockdown of FGF13 in adult mouse ventricular myocytes revealed a loss of function of NaV1.5-reduced Na+ current density, decreased Na+ channel availability, and slowed NaV1.5-reduced Na+ current recovery from inactivation. Cell surface biotinylation experiments showed ≈45% reduction in NaV1.5 protein at the sarcolemma after FGF13 knockdown, whereas no changes in whole-cell NaV1.5 protein or in mRNA level were observed. Optical imaging in neonatal rat ventricular myocyte monolayers demonstrated slowed conduction velocity and a reduced maximum capture rate after FGF13 knockdown. Conclusion: These findings show that FHFs are potent regulators of Na+ channels in adult ventricular myocytes and suggest that loss-of-function mutations in FHFs may underlie a similar set of cardiac arrhythmias and cardiomyopathies that result from NaV1.5 loss-of-function mutations.

Essential Cavβ modulatory properties are AID-independent

Voltage-gated Ca2+ channel β (Cavβ) subunits have a highly conserved core consisting of interacting Src homology 3 and guanylate kinase domains, and are postulated to exert their effects through AID, the major interaction site in the pore-forming α1 subunit. This stereotypical interaction does not explain how individual Cavβ subunits modulate α1 subunits differentially. Here we show that AID is neither necessary nor sufficient for critical Cavβ regulatory properties. Complete modulation depends on additional contacts that are exclusive of AID and not revealed in recent crystal structures. These data offer a new context for understanding Cavβ modulation, suggesting that the AID interaction orients the Cavβ core so as to permit additional isoform-specific Cavα1-Cavβ interactions that underlie the particular regulation seen with each Cavα1-Cavβ pair, rather than as the main site of regulation.