Phosphoinositide 3-kinase γ (PI3Kγ) signaling engaged by β-adrenergic receptors is pivotal in the regulation of myocardial contractility and remodeling. However, the role of PI3Kγ in catecholamine-induced arrhythmia is currently unknown.
Methods and Results
Mice lacking PI3Kγ (PI3Kγ−/−) showed runs of premature ventricular contractions on adrenergic stimulation that could be rescued by a selective β2-adrenergic receptor blocker and developed sustained ventricular tachycardia after transverse aortic constriction. Consistently, fluorescence resonance energy transfer probes revealed abnormal cAMP accumulation after β2-adrenergic receptor activation in PI3Kγ−/− cardiomyocytes that depended on the loss of the scaffold but not of the catalytic activity of PI3Kγ. Downstream from β-adrenergic receptors, PI3Kγ was found to participate in multiprotein complexes linking protein kinase A to the activation of phosphodiesterase (PDE) 3A, PDE4A, and PDE4B but not of PDE4D. These PI3Kγ-regulated PDEs lowered cAMP and limited protein kinase A–mediated phosphorylation of L-type calcium channel (Cav1.2) and phospholamban. In PI3Kγ−/− cardiomyocytes, Cav1.2 and phospholamban were hyperphosphorylated, leading to increased Ca2+ spark occurrence and amplitude on adrenergic stimulation. Furthermore, PI3Kγ−/− cardiomyocytes showed spontaneous Ca2+ release events and developed arrhythmic calcium transients.
PI3Kγ coordinates the coincident signaling of the major cardiac PDE3 and PDE4 isoforms, thus orchestrating a feedback loop that prevents calcium-dependent ventricular arrhythmia.
arrhythmias, cardiac; class II phosphatidylinositol 3-kinases; 3′,5′-cyclic-AMP phosphodiesterases; cyclic AMP-dependent protein kinases; receptors, adrenergic beta-2
Voltage-gated calcium (Ca2+) channels are key transducers of membrane potential changes into intracellular Ca2+ transients that initiate many physiological events. There are ten members of the voltage-gated Ca2+ channel family in mammals, and they serve distinct roles in cellular signal transduction. The CaV1 subfamily initiates contraction, secretion, regulation of gene expression, integration of synaptic input in neurons, and synaptic transmission at ribbon synapses in specialized sensory cells. The CaV2 subfamily is primarily responsible for initiation of synaptic transmission at fast synapses. The CaV3 subfamily is important for repetitive firing of action potentials in rhythmically firing cells such as cardiac myocytes and thalamic neurons. This article presents the molecular relationships and physiological functions of these Ca2+ channel proteins and provides information on their molecular, genetic, physiological, and pharmacological properties.
The activity of a voltage-gated calcium channel is enhanced by the binding of an effector ready to respond to a calcium signal. This effector checkpoint mechanism may be a unifying theme in voltage-gated calcium channel regulation.
Increases in intracellular Mg2+ (Mg2+i), as observed in transient cardiac ischemia, decrease L-type Ca2+ current of mammalian ventricular myocytes (VMs). However, cardiac ischemia is associated with an increase in sympathetic tone, which could stimulate L-type Ca2+ current. Therefore, the effect of Mg2+i on L-type Ca2+ current in the context of increased sympathetic tone was unclear. We tested the impact of increased Mg2+i on the β-adrenergic stimulation of L-type Ca2+ current. Exposure of acutely dissociated adult VMs to higher Mg2+i concentrations decreased isoproterenol stimulation of the L-type Ca2+ current from 75 ± 13% with 0.8 mM Mg2+i to 20 ± 8% with 2.4 mM Mg2+i. We activated this signaling cascade at different steps to determine the site or sites of Mg2+i action. Exposure of VMs to increased Mg2+i attenuated the stimulation of L-type Ca2+ current induced by activation of adenylyl cyclase with forskolin, inhibition of cyclic nucleotide phosphodiesterases with isobutylmethylxanthine, and inhibition of phosphoprotein phosphatases I and IIA with calyculin A. These experiments ruled out significant effects of Mg2+i on these upstream steps in the signaling cascade and suggested that Mg2+i acts directly on CaV1.2 channels. One possible site of action is the EF-hand in the proximal C-terminal domain, just downstream in the signaling cascade from the site of regulation of CaV1.2 channels by protein phosphorylation on the C terminus. Consistent with this hypothesis, Mg2+i had no effect on enhancement of CaV1.2 channel activity by the dihydropyridine agonist (S)-BayK8644, which activates CaV1.2 channels by binding to a site formed by the transmembrane domains of the channel. Collectively, our results suggest that, in transient ischemia, increased Mg2+i reduces stimulation of L-type Ca2+ current by the β-adrenergic receptor by directly acting on CaV1.2 channels in a cell-autonomous manner, effectively decreasing the metabolic stress imposed on VMs until blood flow can be reestablished.
Developing an understanding of the mechanism of voltage-gated ion channels in molecular terms requires knowledge of the structure of the active and resting conformations. Although the active-state conformation is known from x-ray structures, an atomic resolution structure of a voltage-dependent ion channel in the resting state is not currently available. This has motivated various efforts at using computational modeling methods and molecular dynamics (MD) simulations to provide the missing information. A comparison of recent computational results reveals an emerging consensus on voltage-dependent gating from computational modeling and MD simulations. This progress is highlighted in the broad context of preexisting work about voltage-gated channels.
Sudden unexpected death in epilepsy (SUDEP) is the most common cause of death in intractable epilepsies, but physiological mechanisms that lead to SUDEP are unknown. Dravet syndrome (DS) is an infantile-onset intractable epilepsy caused by heterozygous loss-of-function mutations in the SCN1A gene, which encodes brain type-I voltage-gated sodium channel NaV1.1. We studied the mechanism of premature death in Scn1a heterozygous KO mice and conditional brain- and cardiac-specific KOs. Video monitoring demonstrated that SUDEP occurred immediately following generalized tonic-clonic seizures. A history of multiple seizures was a strong risk factor for SUDEP. Combined video-electroencephalography-electrocardiography revealed suppressed interictal resting heart-rate variability and episodes of ictal bradycardia associated with the tonic phases of generalized tonic-clonic seizures. Prolonged atropine-sensitive ictal bradycardia preceded SUDEP. Similar studies in conditional KO mice demonstrated that brain, but not cardiac, KO of Scn1a produced cardiac and SUDEP phenotypes similar to those found in DS mice. Atropine or N-methyl scopolamine treatment reduced the incidence of ictal bradycardia and SUDEP in DS mice. These findings suggest that SUDEP is caused by apparent parasympathetic hyperactivity immediately following tonic-clonic seizures in DS mice, which leads to lethal bradycardia and electrical dysfunction of the ventricle. These results have important implications for prevention of SUDEP in DS patients.
Haploinsufficiency of the SCN1A gene encoding voltage-gated sodium channel NaV1.1 causes Dravet Syndrome (DS), a childhood neuropsychiatric disorder including recurrent intractable seizures, cognitive deficit, and autism-spectrum behaviors. The neural mechanisms responsible for cognitive deficit and autism-spectrum behaviors in DS are poorly understood. Here we show that mice with Scn1a haploinsufficiency display hyperactivity, stereotyped behaviors, social interaction deficits, and impaired context-dependent spatial memory. Olfactory sensitivity is retained, but novel food odors and social odors are aversive to Scn1a+/− mice. GABAergic neurotransmission is specifically impaired by this mutation, and selective deletion of NaV1.1 channels in forebrain interneurons is sufficient to cause these behavioral and cognitive impairments. Remarkably, treatment with low-dose clonazepam, a positive allosteric modulator of GABAA receptors, completely rescued the abnormal social behaviors and deficits in fear memory in DS mice, demonstrating that they are caused by impaired GABAergic neurotransmission and not by neuronal damage from recurrent seizures. These results demonstrate a critical role for NaV1.1 channels in neuropsychiatric functions and provide a potential therapeutic strategy for cognitive deficit and autism-spectrum behaviors in DS.
In excitable cells, voltage-gated sodium (NaV) channels activate to initiate action potentials and then undergo fast and slow inactivation processes that terminate their ionic conductance1,2. Inactivation is a hallmark of NaV channel function and is critical for control of membrane excitability3, but the structural basis for this process has remained elusive. Here we report crystallographic snapshots of the wild-type NavAb channel from Arcobacter butzleri captured in two potentially inactivated states at 3.2 Å resolution. Compared to previous structures of NavAb S6-cysteine mutants4, the pore-lining S6 helices and the intracellular activation gate have undergone significant rearrangements in which one pair of S6 segments has collapsed toward the central pore axis and the other S6 pair has moved outward to produce a striking dimer-of-dimers configuration. An increase in global structural asymmetry is observed throughout our wild-type NavAb models, reshaping the ion selectivity filter at the extracellular end of the pore, the central cavity and its residues analogous to the mammalian drug receptor site, and the lateral pore fenestrations. The voltage-sensing domains also shift around the perimeter of the pore module in NavAb, and local structural changes identify a conserved interaction network that connects distant molecular determinants involved in NaV channel gating and inactivation. These potential inactivated-state structures provide new insights into NaV channel gating and novel avenues to drug development and therapy for a range of debilitating NaV channelopathies.
Mutations in voltage-gated sodium channels are associated with epilepsy syndromes with a wide range of severity. Complete loss of function in the Nav1.1 channel encoded by the SCN1A gene is associated with severe myoclonic epilepsy in infancy (SMEI), a devastating infantile-onset epilepsy with ataxia, cognitive dysfunction, and febrile and afebrile seizures resistant to current medications. Genetic mouse models of SMEI have been created that strikingly recapitulate the SMEI phenotype including age and temperature dependence of seizures and ataxia. Loss-of-function in Nav1.1 channels results in severely impaired sodium current and action potential firing in hippocampal γ-aminobutyric acid (GABA)ergic interneurons without detectable changes in excitatory pyramidal neurons. The resulting imbalance between excitation and inhibition likely contributes to hyperexcitability and seizures. Reduced sodium current and action potential firing in cerebellar Purkinje neurons likely contributes to comorbid ataxia. A mechanistic understanding of hyperexcitability, seizures, and comorbidities such as ataxia has led to novel strategies for treatment.
Severe myoclonic epilepsy in infancy; SCN1A; Febrile seizures; Ataxia; Mouse genetic model
The Hodgkin-Huxley studies of the action potential, published 60 years ago, are a central pillar of modern neuroscience research, ranging from molecular investigations of the structural basis of ion channel function to the computational implications at circuit level. In this Symposium Review, we aim to demonstrate the ongoing impact of Hodgkin’s and Huxley’s ideas. The Hodgkin-Huxley model established a framework in which to describe the structural and functional properties of ion channels, including the mechanisms of ion permeation, selectivity, and gating. At a cellular level, the model is used to understand the conditions that control both the rate and timing of action potentials, essential for neural encoding of information. Finally, the Hodgkin-Huxley formalism is central to computational neuroscience to understand both neuronal integration and circuit level information processing, and how these mechanisms might have evolved to minimize energy cost.
We evaluated the ability of the ketogenic diet (KD) to improve thresholds to flurothyl-induced seizures in two mouse lines with Scn1a mutations: one that models Dravet syndrome (DS) and another that models genetic (generalized) epilepsy with
febrile seizures plus (GEFS+).
At postnatal day 21, mouse models of DS and GEFS+ were fasted for 12–14 hours and then placed on either a 6:1 KD or a standard diet (SD) for two weeks. At the end of the two-week period, we measured thresholds to seizures induced by the chemiconvulsant flurothyl. Body weight, β-hydroxybutyrate (BHB) levels, and glucose levels were also recorded every two days over a two-week period in separate cohorts of mutant and wild-type mice that were either on the KD or the SD.
Mice on the KD gained less weight and exhibited significantly higher BHB levels compared to mice on the SD. Importantly, thresholds to flurothyl-induced seizures were restored to more normal levels in both mouse lines after two weeks on the KD.
These results indicate that the KD may be an effective treatment for refractory patients with SCN1A mutations. The availability of mouse models of DS and GEFS+ also provides an opportunity to better understand the mechanism of action of the KD, which may facilitate the development of improved treatments.
Dravet syndrome; genetic epilepsy with febrile seizures plus; mouse models; ketogenic diet; SCN1A
Membrane depolarization and intracellular Ca2+ transients generated by activation of voltage-gated Na+ and Ca2+ channels are local signals, which initiate physiological processes such as action potential conduction, synaptic transmission, and excitation-contraction coupling. Targeting of effector proteins and regulatory proteins to ion channels is an important mechanism to ensure speed, specificity, and precise regulation of signaling events in response to local stimuli. This article reviews experimental results showing that Na+ and Ca2+ channels form local signaling complexes, in which effector proteins, anchoring proteins, and regulatory proteins interact directly with ion channels. The intracellular domains of these channels serve as signaling platforms, mediating their participation in intracellular signaling processes. These protein-protein interactions are important for regulation of cellular plasticity through modulation of Na+ channel function in brain neurons, for short-term synaptic plasticity through modulation of presynaptic CaV2 channels, and for the fight-or-flight response through regulation of postsynaptic CaV1 channels in skeletal and cardiac muscle. These localized signaling complexes are essential for normal function and regulation of electrical excitability, synaptic transmission, and excitation-contraction coupling.
The voltage-gated sodium channel Nav1.6 plays unique roles in the nervous system, but its functional properties and neuromodulation are not as well established as for NaV1.2 channels. We found no significant differences in voltage-dependent activation or fast inactivation between NaV1.6 and NaV1.2 channels expressed in non-excitable cells. In contrast, the voltage dependence of slow inactivation was more positive for Nav1.6 channels, they conducted substantially larger persistent sodium currents than Nav1.2 channels, and they were much less sensitive to inhibtion by phosphorylation by cAMP-dependent protein kinase and protein kinase C. Resurgent sodium current, a hallmark of Nav1.6 channels in neurons, was not observed for NaV1.6 expressed alone or with the auxiliary β4 subunit. The unique properties of NaV1.6 channels, together with the resurgent currents that they conduct in neurons, make these channels well-suited to provide the driving force for sustained repetitive firing, a crucial property of neurons.
Although the extracellular matrix plays an important role in regulating use-dependent synaptic plasticity, the underlying molecular mechanisms are poorly understood. Here we examined the synaptic function of hyaluronic acid (HA), a major component of the extracellular matrix. Enzymatic removal of HA with hyaluronidase reduced nifedipine-sensitive whole-cell Ca2+ currents, decreased Ca2+ transients mediated by L-type voltage-dependent Ca2+ channels (L-VDCCs) in postsynaptic dendritic shafts and spines, and abolished an L-VDCC-dependent component of long-term potentiation (LTP) at the CA3-CA1 synapses in the hippocampus. Adding exogenous HA, either by bath perfusion or via local delivery near recorded synapses, completely rescued this LTP component. In a heterologous expression system, exogenous HA rapidly increased currents mediated by Cav1.2, but not Cav1.3, subunit-containing L-VDCCs, whereas intrahippocampal injection of hyaluronidase impaired contextual fear conditioning. Our observations unveil a previously unrecognized mechanism by which the perisynaptic extracellular matrix influences use-dependent synaptic plasticity through regulation of dendritic Ca2+ channels.
Voltage-gated sodium channels initiate electrical signaling in excitable cells and are the molecular targets for drugs and disease mutations, but the structural basis for their voltage-dependent activation, ion selectivity, and drug block is unknown. Here, we report the crystal structure of a voltage-gated Na+-channel from Arcobacter butzleri (NavAb) captured in a closed-pore conformation with four activated voltage-sensors at 2.7 Å resolution. The arginine gating charges make multiple hydrophilic interactions within the voltage-sensor, including unanticipated hydrogen bonds to the protein backbone. Comparisons to previous open-pore potassium channel structures suggest that the voltage-sensor domains and the S4-S5 linkers dilate the central pore by pivoting together around a hinge at the base of the pore module. The NavAb selectivity filter is short, ~6.5 Å wide, and water-filled, with four acidic side-chains surrounding the narrowest part of the ion conduction pathway. This unique structure presents a high field-strength anionic coordination site, which confers Na+-selectivity through partial dehydration via direct interaction with glutamate side-chains. Fenestrations in the sides of the pore module are unexpectedly penetrated by fatty acyl chains that extend into the central cavity, and these portals are large enough for the entry of small, hydrophobic pore-blocking drugs.
Voltage-gated ion channels generate electrical signals in species from bacteria to man. Their voltage-sensing modules are responsible for initiation of action potentials and graded membrane potential changes in response to synaptic input and other physiological stimuli. Extensive structure-function studies, structure determination, and molecular modeling are now converging on a sliding-helix mechanism for electromechanical coupling in which outward movement of gating charges in the S4 transmembrane segments catalyzed by sequential formation of ion pairs pulls the S4-S5 linker, bends the S6 segment, and opens the pore. Impairment of voltage-sensor function by mutations in Na+ channels contributes to several ion channelopathies, and gating pore current conducted by mutant voltage sensors in NaV1.4 channels is the primary pathophysiological mechanism in Hypokalemic Periodic Paralysis. The emerging structural model for voltage sensor function opens the way to development of a new generation of ionchannel drugs that act on voltage sensors rather than blocking the pore.
β-Adrenergic receptors (β-ARs) enhance cardiac contractility by increasing cAMP levels and activating PKA. PKA increases Ca2+-induced Ca2+ release via phosphorylation of L-type Ca2+ channels (LTCCs) and ryanodine receptor 2. Multiple cyclic nucleotide phosphodiesterases (PDEs) regulate local cAMP concentration in cardiomyocytes, with PDE4 being predominant for the control of β-AR–dependent cAMP signals. Three genes encoding PDE4 are expressed in mouse heart: Pde4a, Pde4b, and Pde4d. Here we show that both PDE4B and PDE4D are tethered to the LTCC in the mouse heart but that β-AR stimulation of the L-type Ca2+ current (ICa,L) is increased only in Pde4b–/– mice. A fraction of PDE4B colocalized with the LTCC along T-tubules in the mouse heart. Under β-AR stimulation, Ca2+ transients, cell contraction, and spontaneous Ca2+ release events were increased in Pde4b–/– and Pde4d–/– myocytes compared with those in WT myocytes. In vivo, after intraperitoneal injection of isoprenaline, catheter-mediated burst pacing triggered ventricular tachycardia in Pde4b–/– mice but not in WT mice. These results identify PDE4B in the CaV1.2 complex as a critical regulator of ICa,L during β-AR stimulation and suggest that distinct PDE4 subtypes are important for normal regulation of Ca2+-induced Ca2+ release in cardiomyocytes.
During the fight-or-flight response, the sympathetic nervous system stimulates L-type calcium ion (Ca2+) currents conducted by CaV1 channels through activation of β-adrenergic receptors, adenylyl cyclase, and phosphorylation by adenosine 3′,5′-monophosphate–dependent protein kinase [also known as protein kinase A (PKA)], increasing contractility of skeletal and cardiac muscles. We reconstituted this regulation of cardiac CaV1.2 channels in non-muscle cells by forming an autoinhibitory signaling complex composed of CaV1.2Δ1800 (a form of the channel truncated at the in vivo site of proteolytic processing), its noncovalently associated distal carboxyl-terminal domain, the auxiliary α2δ1 and β2b subunits, and A-kinase anchoring protein 15 (AKAP15). A factor of 3.6 range of CaV1.2 channel activity was observed from a minimum in the presence of protein kinase inhibitors to a maximum upon activation of adenylyl cyclase. Basal CaV1.2 channel activity in unstimulated cells was regulated by phosphorylation of serine-1700 and threonine-1704, two residues located at the interface between the distal and the proximal carboxyl-terminal regulatory domains, whereas further stimulation of channel activity through the PKA signaling pathway only required phosphorylation of serine-1700. Our results define a conceptual framework for CaV1.2 channel regulation and identify sites of phosphorylation that regulate channel activity.
Sympathetic stimulation of the heart increases the force of contraction and rate of ventricular relaxation by triggering PKA-dependent phosphorylation of proteins that regulate intracellular calcium. We hypothesized that scaffolding of cAMP signaling complexes by AKAP5 is required for efficient sympathetic stimulation of calcium transients.
We examined the function of AKAP5 in the β-adrenergic signaling cascade.
Methods and Results
We used calcium imaging and electrophysiology to examine the sympathetic response of cardiomyocytes isolated from wild type and AKAP5 mutant animals. The β-adrenergic regulation of calcium transients and the phosphorylation of substrates involved in calcium handling were disrupted in AKAP5 knockout cardiomyocytes. The scaffolding protein, AKAP5 (also called AKAP150/79), targets adenylyl cyclase, PKA, and calcineurin to a caveolin 3-associated complex in ventricular myocytes that also binds a unique sub-population of Cav1.2 L-type calcium channels. Only the caveolin 3-associated Cav1.2 channels are phosphorylated by PKA in response to sympathetic stimulation in wild type heart. However, in the AKAP5 knockout heart the organization of this signaling complex is disrupted, adenylyl cyclase 5/6 no longer associates with caveolin 3 in the T-tubules, and non-caveolin 3 associated calcium channels become phosphorylated after β-adrenergic stimulation although this does not lead to an enhanced calcium transient. The signaling domain created by AKAP5 is also essential for the PKA-dependent phosphorylation of ryanodine receptors and phospholambam.
These findings identify an AKAP5-organized signaling module that is associated with caveolin 3 and is essential for sympathetic stimulation of the calcium transient in adult heart cells.
AKAP; Ca2+ channels; adenylyl cyclase; cAMP; PKA
Hypokalemic periodic paralysis and normokalemic periodic paralysis are caused by mutations of the gating charge–carrying arginine residues in skeletal muscle NaV1.4 channels, which induce gating pore current through the mutant voltage sensor domains. Inward sodium currents through the gating pore of mutant R666G are only ∼1% of central pore current, but substitution of guanidine for sodium in the extracellular solution increases their size by 13- ± 2-fold. Ethylguanidine is permeant through the R666G gating pore at physiological membrane potentials but blocks the gating pore at hyperpolarized potentials. Guanidine is also highly permeant through the proton-selective gating pore formed by the mutant R666H. Gating pore current conducted by the R666G mutant is blocked by divalent cations such as Ba2+ and Zn2+ in a voltage-dependent manner. The affinity for voltage-dependent block of gating pore current by Ba2+ and Zn2+ is increased at more negative holding potentials. The apparent dissociation constant (Kd) values for Zn2+ block for test pulses to −160 mV are 650 ± 150 µM, 360 ± 70 µM, and 95.6 ± 11 µM at holding potentials of 0 mV, −80 mV, and −120 mV, respectively. Gating pore current is blocked by trivalent cations, but in a nearly voltage-independent manner, with an apparent Kd for Gd3+ of 238 ± 14 µM at −80 mV. To test whether these periodic paralyses might be treated by blocking gating pore current, we screened several aromatic and aliphatic guanidine derivatives and found that 1-(2,4-xylyl)guanidinium can block gating pore current in the millimolar concentration range without affecting normal NaV1.4 channel function. Together, our results demonstrate unique permeability of guanidine through NaV1.4 gating pores, define voltage-dependent and voltage-independent block by divalent and trivalent cations, respectively, and provide initial support for the concept that guanidine-based gating pore blockers could be therapeutically useful.
Mutations in the voltage-gated sodium channel SCN1A are responsible for a number of seizure disorders including Generalized Epilepsy with Febrile Seizures Plus (GEFS+) and Severe Myoclonic Epilepsy of Infancy (SMEI). To determine the effects of SCN1A mutations on channel function in vivo, we generated a bacterial artificial chromosome (BAC) transgenic mouse model that expresses the human SCN1A GEFS+ mutation, R1648H. Mice with the R1648H mutation exhibit a more severe response to the proconvulsant kainic acid compared with mice expressing a control Scn1a transgene. Electrophysiological analysis of dissociated neurons from mice with the R1648H mutation reveal delayed recovery from inactivation and increased use-dependent inactivation only in inhibitory bipolar neurons, as well as a hyperpolarizing shift in the voltage dependence of inactivation only in excitatory pyramidal neurons. These results demonstrate that the effects of SCN1A mutations are cell type-dependent and that the R1648H mutation specifically leads to a reduction in interneuron excitability.
Sodium channel; SCN1A; GEFS+; SMEI; epilepsy; mutation
L-type Ca2+ currents conducted by Cav1.2 channels initiate excitation–contraction coupling in cardiac myocytes. Intracellular Mg2+ (Mgi) inhibits the ionic current of Cav1.2 channels. Because Mgi is altered in ischemia and heart failure, its regulation of Cav1.2 channels is important in understanding cardiac pathophysiology. Here, we studied the effects of Mgi on voltage-dependent inactivation (VDI) of Cav1.2 channels using Na+ as permeant ion to eliminate the effects of permeant divalent cations that engage the Ca2+-dependent inactivation process. We confirmed that increased Mgi reduces peak ionic currents and increases VDI of Cav1.2 channels in ventricular myocytes and in transfected cells when measured with Na+ as permeant ion. The increased rate and extent of VDI caused by increased Mgi were substantially reduced by mutations of a cation-binding residue in the proximal C-terminal EF-hand, consistent with the conclusion that both reduction of peak currents and enhancement of VDI result from the binding of Mgi to the EF-hand (KD ≈ 0.9 mM) near the resting level of Mgi in ventricular myocytes. VDI was more rapid for L-type Ca2+ currents in ventricular myocytes than for Cav1.2 channels in transfected cells. Coexpression of Cavβ2b subunits and formation of an autoinhibitory complex of truncated Cav1.2 channels with noncovalently bound distal C-terminal domain (DCT) both increased VDI in transfected cells, indicating that the subunit structure of the Cav1.2 channel greatly influences its VDI. The effects of noncovalently bound DCT on peak current amplitude and VDI required Mgi binding to the proximal C-terminal EF-hand and were prevented by mutations of a key divalent cation-binding amino acid residue. Our results demonstrate cooperative regulation of peak current amplitude and VDI of Cav1.2 channels by Mgi, the proximal C-terminal EF-hand, and the DCT, and suggest that conformational changes that regulate VDI are propagated from the DCT through the proximal C-terminal EF-hand to the channel-gating mechanism.
The IUPHAR database (IUPHAR-DB) integrates peer-reviewed pharmacological, chemical, genetic, functional and anatomical information on the 354 nonsensory G protein-coupled receptors (GPCRs), 71 ligand-gated ion channel subunits and 141 voltage-gated-like ion channel subunits encoded by the human, rat and mouse genomes. These genes represent the targets of approximately one-third of currently approved drugs and are a major focus of drug discovery and development programs in the pharmaceutical industry. IUPHAR-DB provides a comprehensive description of the genes and their functions, with information on protein structure and interactions, ligands, expression patterns, signaling mechanisms, functional assays and biologically important receptor variants (e.g. single nucleotide polymorphisms and splice variants). In addition, the phenotypes resulting from altered gene expression (e.g. in genetically altered animals or in human genetic disorders) are described. The content of the database is peer reviewed by members of the International Union of Basic and Clinical Pharmacology Committee on Receptor Nomenclature and Drug Classification (NC-IUPHAR); the data are provided through manual curation of the primary literature by a network of over 60 subcommittees of NC-IUPHAR. Links to other bioinformatics resources, such as NCBI, Uniprot, HGNC and the rat and mouse genome databases are provided. IUPHAR-DB is freely available at http://www.iuphar-db.org.
Voltage sensing by voltage-gated sodium channels determines the electrical excitability of cells, but the molecular mechanism is unknown. β-Scorpion toxins bind specifically to neurotoxin receptor site 4 and induce a negative shift in the voltage dependence of activation through a voltage sensor-trapping mechanism. Kinetic analysis showed that β-scorpion toxin binds to the resting state, and subsequently the bound toxin traps the voltage sensor in the activated state in a voltage-dependent but concentration-independent manner. The rate of voltage sensor trapping can be fit by a two-step model, in which the first step is voltage-dependent and correlates with the outward gating movement of the IIS4 segment, whereas the second step is voltage-independent and results in shifted voltage dependence of activation of the channel. Mutations of Glu779 in extracellular loop IIS1–S2 and both Glu837 and Leu840 in extracellular loop IIS3–S4 reduce the binding affinity of β-scorpion toxin. Mutations of positively charged and hydrophobic amino acid residues in the IIS4 segment do not affect β-scorpion toxin binding but alter voltage dependence of activation and enhance β-scorpion toxin action. Structural modeling with the Rosetta algorithm yielded a three-dimensional model of the toxin-receptor complex with the IIS4 voltage sensor at the extracellular surface. Our results provide mechanistic and structural insight into the voltage sensor-trapping mode of scorpion toxin action, define the position of the voltage sensor in the resting state of the sodium channel, and favor voltage-sensing models in which the S4 segment spans the membrane in both resting and activated states.
Cav2.1 channels, which mediate P/Q-type Ca2+ currents, undergo Ca2+/calmodulin (CaM)-dependent inactivation and facilitation that can significantly alter synaptic efficacy. Here we report that the neuronal Ca2+-binding protein 1 (CaBP1) modulates Cav2.1 channels in a manner that is markedly different from modulation by CaM. CaBP1 enhances inactivation, causes a depolarizing shift in the voltage dependence of activation, and does not support Ca2+-dependent facilitation of Cav2.1 channels. These inhibitory effects of CaBP1 do not require Ca2+, but depend on the CaM-binding domain in the α1 subunit of Cav2.1 channels (α12.1). CaBP1 binds to the CaM-binding domain, co-immunoprecipitates with α12.1 from transfected cells and brain extracts, and colocalizes with α12.1 in discrete microdomains of neurons in the hippocampus and cerebellum. Our results identify an interaction between Ca2+ channels and CaBP1 that may regulate Ca2+-dependent forms of synaptic plasticity by inhibiting Ca2+ influx into neurons.