Pore-forming Alpha Subunit Research Articles

Critical regulation of the voltage-gated sodium channel, Nav1.4, by PIP2.

Voltage-gated sodium (NaV) channels are vital to the electrical signalling of cells. The voltage-dependent activation of NaV channels is followed almost instantaneously by fast inactivation, setting a timer on the excitability of tissues. NaV channels are protein complexes that consist of a large, pore forming alpha subunit and are often associated with auxiliary beta subunits. The alpha subunit is made up of four tandem repeat domains (DI-IV) that consist of six transmembrane segments (S1-S6) with S4 in each domain serving as its putative voltage sensor. The gating cycle of NaV channels is subject to tight regulation, with dysfunction leading to several disease states. Most ion channels are regulated by the membrane phospholipid, phosphatidylinositol (4,5) bisphosphate [PI(4,5)P2]; however, it is not known whether PI(4,5)P2 modulates the activity of NaV channels. Of the nine (NaV1.1 - NaV1.9) family members that have been identified and classified based on tissue distribution, we studied NaV1.4 which is the isoform primarily expressed in skeletal muscle. We used the optogenetically activated inositide phosphatase, pseudojanin, an enzymatic chimera of inositol polyphosphate 5 phosphatase E (INPP5E), which converts PI(4,5)P2 to PI4P and the S. cerevisiae sac1 phosphatase, which dephosphorylates PI(4)P, to evaluate the role of PI(4,5)P2 in NaV1.4 channel gating and regulation. Through a combination of patch-clamp recording and optogenetics we show that dephosphorylating PI(4,5)P2 shifts the voltage-dependent gating of NaV1.4 to more hyperpolarized membrane potentials. PI(4,5)P2 depletion also augments the late current that persists after fast inactivation, a phenotype that is associated with several mutations that lead to myotonia. Through this work, we provide evidence that PI(4,5)P2 is a necessary co-factor in the gating of NaV1.4 channels.

Functional evidence of a novel mechanism of allosteric modulation of the voltage gated sodium channel

Voltage-gated sodium (Nav) channels are a molecular determinant of the action potential. Despite the canonical role of the pore-forming alpha subunit in enabling this function, protein:protein interactions (PPI) between the alpha subunit and Nav channel auxiliary proteins are necessary to reconstitute the full physiological function of the Nav channel. In the brain, one salient example of such a PPI occurs between the Nav1.6 channel and fibroblast growth factor 14 (FGF14). This PPI controls electrical outputs of medium spiny neurons (MSN), which are the principal cell type of the nucleus accumbens, a clinically relevant brain region involved in regulating motivation.

Validating the Significance of Ser1487 in the Beta-Adrenergic Regulation of the Alpha Subunit of the Cardiac L-Type Calcium Channel Using a CRISPR/Cas9 Mouse Model

Beta-adrenergic receptor stimulation increases calcium influx through the pore forming alpha subunit of the cardiac L-type Ca2+ channel (CaV1.2). Chronic activation of the CaV1.2 can lead to development of cardiac hypertrophy and arrhythmia formation; yet the molecular mechanism remains unresolved.

Activation of BK Channels Prevents Hepatic Stellate Cell Activation and Liver Fibrosis Through the Suppression of TGFβ1/SMAD3 and JAK/STAT3 Profibrotic Signaling Pathways.

Large-conductance and Ca2+-activated K+ (BK) channels are expressed in human hepatic stellate cells (HSCs), where they have roles in normal hepatic microcirculation, as well as in portal hypertension in liver cirrhosis through the regulation of contractility in activated HSCs. Nevertheless, whether BK channel activity exerts protective effects against aberrant HSC activation and hepatic fibrosis is unknown. Here, we report that BK channels are expressed in activated primary rat HSCs as well as in a human HSC line. Moreover, whole-cell K+ currents recorded from activated HSCs were markedly increased by exposure to rottlerin, a BK channel-specific activator, but were inhibited by treatment with the BK channel-specific inhibitor, paxilline, suggesting that BK channels are functional in activated HSCs. Overexpression but not downregulation of the BK channel pore-forming alpha subunit, KCNMA1, led to reduced migration and collagen expression in activated HSCs. Consistently, rottlerin treatment suppressed the fibrogenic cell function both in vitro and in CCl4-induced liver fibrosis in vivo. Microarray and pathway analysis, combined with a luciferase reporter assay and western blotting, further showed that rottlerin treatment led to a significant downregulation of the profibrotic TGFβ1/SMAD3 and JAK/STAT3 signaling pathways, both in vitro and in vivo. Our findings not only link BK channel function to profibrotic signaling pathways, but also provide evidence that BK channel activation represents a promising therapeutic strategy for the treatment of liver fibrosis.

Proestrus Differentially Regulates Expression of Ion Channel and Calcium Homeostasis Genes in GnRH Neurons of Mice.

In proestrus, the changing gonadal hormone milieu alters the physiological properties of GnRH neurons and contributes to the development of the GnRH surge. We hypothesized that proestrus also influences the expression of different ion channel genes in mouse GnRH neurons. Therefore, we performed gene expression profiling of GnRH neurons collected from intact, proestrous and metestrous GnRH-GFP transgenic mice, respectively. Proestrus changed the expression of 37 ion channel and 8 calcium homeostasis-regulating genes. Voltage-gated sodium channels responded with upregulation of three alpha subunits (Scn2a1, Scn3a, and Scn9a). Within the voltage-gated potassium channel class, Kcna1, Kcnd3, Kcnh3, and Kcnq2 were upregulated, while others (Kcna4, Kcnc3, Kcnd2, and Kcng1) underwent downregulation. Proestrus also had impact on inwardly rectifying potassium channel subunits manifested in enhanced expression of Kcnj9 and Kcnj10 genes, whereas Kcnj1, Kcnj11, and Kcnj12 subunit genes were downregulated. The two-pore domain potassium channels also showed differential expression with upregulation of Kcnk1 and reduced expression of three subunit genes (Kcnk7, Kcnk12, and Kcnk16). Changes in expression of chloride channels involved both the voltage-gated (Clcn3 and Clcn6) and the intracellular (Clic1) subtypes. Regarding the pore-forming alpha-1 subunits of voltage-gated calcium channels, two (Cacna1b and Cacna1h) were upregulated, while Cacna1g showed downregulation. The ancillary subunits were also differentially regulated (Cacna2d1, Cacna2d2, Cacnb1, Cacnb3, Cacnb4, Cacng5, Cacng6, and Cacng8). In addition, ryanodine receptor 1 (Ryr1) gene was downregulated, while a transient receptor potential cation channel (Trpm3) gene showed enhanced expression. Genes encoding proteins regulating the intracellular calcium homeostasis were also influenced (Calb1, Hpca, Hpcal1, Hpcal4, Cabp7, Cab 39l, and Cib2). The differential expression of genes coding for ion channel proteins in GnRH neurons at late proestrus indicates that the altering hormone milieu contributes to remodeling of different kinds of ion channels of GnRH neurons, which might be a prerequisite of enhanced cellular activity of GnRH neurons and the subsequent surge release of the neurohormone.

Mining Protein Evolution for Insights into Mechanisms of Voltage-Dependent Sodium Channel Auxiliary Subunits.

Voltage-gated sodium channel (VGSC) beta (β) subunits have been called the "overachieving" auxiliary ion channel subunit. Indeed, these subunits regulate the trafficking of the sodium channel complex at the plasma membrane and simultaneously tune the voltage-dependent properties of the pore-forming alpha-subunit. It is now known that VGSC β-subunits are capable of similar modulation of multiple isoforms of related voltage-gated potassium channels, suggesting that their abilities extend into the broader voltage-gated channels. The gene family for these single transmembrane immunoglobulin beta-fold proteins extends well beyond the traditional VGSC β1-β4 subunit designation, with deep roots into the cell adhesion protein family and myelin-related proteins - where inherited mutations result in a myriad of electrical signaling disorders. Yet, very little is known about how VGSC β-subunits support protein trafficking pathways, the basis for their modulation of voltage-dependent gating, and, ultimately, their role in shaping neuronal excitability. An evolutionary approach can be useful in yielding new clues to such functions as it provides an unbiased assessment of protein residues, folds, and functions. An approach is described here which indicates the greater emergence of the modern β-subunits roughly 400 million years ago in the early neurons of Bilateria and bony fish, and the unexpected presence of distant homologues in bacteriophages. Recent structural breakthroughs containing α and β eukaryotic sodium channels containing subunits suggest a novel role for a highly conserved polar contact that occurs within the transmembrane segments. Overall, a mixture of approaches will ultimately advance our understanding of the mechanism for β-subunit interactions with voltage-sensor containing ion channels and membrane proteins.

THE ROLE OF GLYCOSYLATION IN CARDIAC REPOLARIZATION

The human ether-a-go-go-related gene (hERG) encodes the pore-forming alpha subunit of the rapidly activating delayed rectifier potassium current (IKr), which is important for cardiac repolarization. Dysfunction of hERG due to drug block or mutations can result in reduced IKr, which is associated with the development of Long QT Syndrome (LQTS). LQTS is a cardiac arrhythmia seen on an ECG as an increased QT interval, and can result in syncope or sudden death. hERG has 6 transmembrane domains, ranging from S1 to S6, and a complex N-linked oligosaccharide chain which resides on the extracellular S5 pore linker region. Glycosylation chains have been associated with numerous roles in cell and protein function, such as protection from protease-induced degradation. The role of the glycosylation chain found on hERG in the channel’s function and expression has not yet been fully revealed, and could provide insight into the cellular mechanisms behind hERG-related LQTS. In the current study, we aim to investigate the relationship between hERG susceptibility to protease degradation and the presence of the glycosylation chain. Human Embryonic Kidney Cells expressing hERG channels were cultured with the glycosylation-reducing agents tunicamycin (an antibiotic that prevents the addition of N-linked glycosylation) and neuraminidase (an exoglycosidase that removes sialic acid residues). Tunicamycin and neuraminidase-treated cells were then subjected to protease degradation by Proteinase K (PK) and Calpain-1. hERG protease susceptibility was measured either through Western blot fragment intensity quantification or through whole cell voltage clamp analysis. Scorpion toxin BeKm-1 was added to cell culture media with PK, then washed out prior to protease susceptibility analysis. We demonstrate that unglycosylated hERG channels are more susceptible to protease-induced degradation by PK and Calpain-1. Removal of the end-chain sialic acid residues using the enzyme neuraminidase did not alter the glycosylation-mediated protease protection afforded to wild-type hERG channels. Additionally, we showed that the scorpion toxin BeKm-1 requires the glycosylation chain to protect hERG channels from PK cut. Our findings indicate that the glycosylation chain found on mature hERG channels plays a key role in protecting the channel from protease digestion, and suggests a glycosylation-mediated mechanism for the BeKm-1-mediated hERG channel protection from PK cut.

Live-Cell Imaging of Midchannel Proteolysis of Neuronal Surface L-Type Calcium Channels

We recently identified midchannel proteolysis as a novel homeostatic mechanism of negative feedback regulation of neuronal voltage-gated calcium channels (VGCCs): the main body of the pore-forming alpha1 subunit of L-type VGCCs, Cav1.2, is cleaved, resulting in Cav1.2 fragment-channels that separate but remain on the plasma membrane, and can be detected by confocal microscopy in fixed neurons [Michailidis et al, Neuron, 82 (2014), 1045-57]. The proteolysis is bidirectionally regulated by channel activity, depends on calpain and the ubiquitin-proteasome system, and increases with age. To investigate the kinetics of midchannel proteolysis and the fate of the resulting fragment-channels, we performed live-cell confocal microscopy. In live cultured rat hippocampal neurons, we visualized surface Cav1.2 labeled with extracellular fluorescent probes placed in different internal repeat domains of the alpha1 subunit. The results reveal robust movements of surface Cav1.2 and its domains, suggesting midchannel proteolysis and continuous dynamic regulation of surface channels.

A Fluorogen-Activating Biosensor for Analysis of BK Channel Traffic and Surface Residency

The regulation of ion channels is critical to numerous physiological processes, especially neuronal function. The large conductance voltage and calcium-activated potassium (BK) channel can modulate neuronal excitability, neurotransmitter release, and may be involved in the development of epilepsy; following a seizure, hippocampal BK channels are more highly trafficked to the plasma membrane. In addition to the pore-forming alpha subunits, which are sufficient to confer channel function, Beta subunits can be incorporated to alter gating and trafficking. Studies of the brain-specific Beta4 subunit effects on surface expression have produced conflicting results, likely due to alternative splicing in the alpha subunit. Fluorogen-activating peptides (FAPs) developed in our lab are well-suited to study protein trafficking; consisting of a ScFv-derived fusion tag and fluorogen dyes added to the cellular media, the association of these two cognate parts results in bright, specific fluorescence. By using a FAP-tagged BKα construct and a pair of fluorogen dyes, we have developed a method for two-color labeling of surface-resident and intracellular BK channels in live cells, allowing for rapid analysis of protein localization by flow cytometry and direct imaging. Using this system in stably expressing HEK293 cells, we found that modulation of the C-terminal region by kinase activity exerted strong changes in surface expression. Taken together with previous observations that these same kinases alter channel gating, this provides a synergistic model for BK-mediated hyperexcitability. A common confound in BK channel research is due to the vast number of splicing isoforms, each having different sensitivities to post-translational modifications. To that end, we are currently generating a knock-in mouse model in which our FAP-tagged BKα is included in all splice variants to examine trafficking changes in Vivo as well as analysis of the behavior of single channels.

0265: A novel SCN5A mutation associated with exercise-induced polymorphic ventricular arrhythmias resembling CPVT

Cardiac sodium channel is a complex which includes the poreforming alpha subunit and regulatory proteins that allow sodium influx during the depolarization phase of the ventricular action potential. The alpha subunit, Nav1.5, is encoded by SCN5A . Mutations in this gene are responsible for a wide spectrum of hereditary arrhythmias such as cardiac conduction disease and atrial fibrillation. A mutation in SCN5A , leading to a p.I141V substitution, was identified in a large multigenerational family with an arrhythmia syndrome resembling catecholaminergic polymorphic ventricular tachycardia through whole-exome sequencing. The purpose of this study was to identify the molecular mechanisms linking the p.I141V mutation to this arrhythmia. To evaluate the incidence of this substitution on Nav1.5 function, whole-cell patch-clamp experiments were performed on HEK293 cells transfected with the human alpha and beta subunits. The presence of the p.I141V mutation shifted the voltage-dependence of steady state activation towards more negative potentials. Thus, we studied the effect of this mutation on the sodium window current. Compared to the WT, the p.I141V window current exhibited a larger peak and this peak was shifted towards more negative potentials. To investigate the functional consequences of the cardiac hyperexcitability due to the p.I141V mutation, we incorporated biophysical properties of the mutant into atrial, Purkinje and ventricular cell models. The computational analyses imply that the biophysical changes caused by the p.I141V mutation accelerates the spontaneous rate in the Purkinje cell model, and reduce the excitation threshold leading to an increase of the excitability of the cardiac cells. In conclusion, the present study demonstrates that mutations in SCN5A also may result in exercise-induced polymorphic ventricular premature complexes and tachycardia resembling CPVT.

KCNE1 Separates the Main Voltage Sensor Movement and Channel Opening in KCNQ1/KCNE1 Channels

The IKs channel is a slowly activating potassium channel that generates one of the potassium currents that limits the duration of the cardiac action potential. The IKs channel consists of 4 pore forming alpha subunits (KCNQ1) and 2-4 beta subunits (KCNE1). The four KCNQ1 subunits form functional potassium channels, but the KCNE1 beta subunit is necessary to recapitulate the slow activation kinetics of the IKs channel. The mechanism by which KCNE1 slows the kinetics of KCNQ1 channels is a matter of current controversy. Here, we use a combination of voltage clamp fluorometry (VCF) and gating current measurements to show that IKs channel activation occurs in two steps: (1) mutually independent voltage sensor movements in the four KCNQ1 subunits generate the main gating charge movement and underlie the delay in the activation time course of the KCNQ1/KCNE1 currents, (2) a slower and concerted conformational change of all four voltage sensors and the gate, which opens the KCNQ1/KCNE1 channel. Gating currents develop with a similar time and voltage dependence as the first fluorescence component, as if the first component reports on the main S4 charge movement. In contrast to other Kv channels, the voltage dependences of the main voltage sensor movement and channel opening are separated by over 100 mV. The two activation steps in KCNQ1/KCNE1 channels can be farther separated by a disease-causing mutation in KCNE1. We determine rates and voltage dependence of the gating transitions to construct a model for KCNQ1/KCNE1 channels. Our model is consistent with that KCNQ1/KCNE1 channel has a fast S4 movement at negative voltages that moves the majority of gating charge and a slower second conformational change at positive voltages that moves a smaller amount of gating charge and opens the gate.

Calorimetric and NMR Characterization of a Rem2 G-Domain Interaction with the Sh3-GK Core of the Ca2+ Channel Beta4 Subunit

The RGK (Rad, Rem1, Rem2 and Gem/Kir) subfamily of small GTPases has potent inhibitory effects on high voltage-activated Ca2+ channels. The inhibition has been shown to occur through a mechanism that is dependent on the Ca2+ channel beta subunit. In this study, we characterized an interaction between the G domain of Rem2 and the beta4 guanylate kinase (GK) domain using NMR and isothermal titration calorimetry (ITC) techniques. We determined that the interaction is endothermic, has a stoichiometry of 1, and a Kd 156 uM, which is three orders of magnitude weaker than the beta4 interaction with the pore-forming alpha1 subunit. NMR chemical shift perturbation analysis reveals that Rem2 interacts exclusively with the GK domain of the SH3-GK core by way of residues located in the former GMP binding region of the inactive kinase. The site is composed of residues just proximal to 310 helix sequences 2 and 3, and residues in alpha helix 6. Thus, the RGK binding site is clearly distinct from the binding site for the alpha1 subunit AID peptide. Site-directed mutagenesis of surface residues combined with ITC indicate that multiple GK sequences contribute to Rem2 binding collectively. These results support the idea that Ca2+ channel inhibition by Rem2 is unlikely to be the result of direct competition with alpha1 binding to the beta subunit. Instead, it argues in favor of a mechanism in which beta subunit-anchored Rem2 executes its inhibitory effect through other contacts with alpha1 subunits. This study demonstrates that NMR combined with ITC can provide detailed information on important weak protein-protein interactions involved in Ca2+ channel regulation.

Sodium Channel β1 Subunits: Overachievers of the Ion Channel Family

Voltage gated Na+ channels in mammals contain a pore-forming alpha subunit and one or more beta subunits. There are five mammalian beta subunits in total: beta1, beta1B, beta2, beta3, and beta4, encoded by four genes: SCN1B-SCN4B. With the exception of the SCN1B splice variant, beta1B, the subunits are type I topology transmembrane proteins. In contrast, beta1B lacks a transmembrane domain and is a secreted protein. A growing body of work shows that VGSC beta subunits are multifunctional. While they do not form the ion channel pore, beta subunits alter gating, voltage-dependence, and kinetics of VGSC alpha subunits and thus regulate cellular excitability in vivo. In addition to their roles in channel modulation, beta subunits are members of the immunoglobulin superfamily of cell adhesion molecules and regulate cell adhesion and migration. Beta subunits are also substrates for sequential proteolytic cleavage by secretases. An example of the multifunctional nature of beta subunits is beta1, encoded by SCN1B, that plays a critical role in neuronal migration and pathfinding during brain development, and whose function is dependent on Na+ current and gamma-secretase activity. Functional deletion of SCN1B results in Dravet Syndrome, a severe and intractable pediatric epileptic encephalopathy. Beta subunits are emerging as key players in a wide variety of pathophysiologies, including epilepsy, cardiac arrhythmia, multiple sclerosis, Huntington's disease, neuropsychiatric disorders, neuropathic and inflammatory pain, and cancer. Beta subunits mediate multiple signaling pathways on different timescales, regulating electrical excitability, adhesion, migration, pathfinding, and transcription. Importantly, some beta subunit functions may operate independent of alpha subunits. Thus, beta subunits perform critical roles during development and disease. As such, they may prove useful in disease diagnosis and therapy.

β4 Modulates NaV1.2 Toxin Pharmacology

The voltage-gated sodium (Nav) channel signaling complex contains up to four distinct beta-subunits (β1-β4) that influence the functional properties of the pore-forming alpha-subunit. While investigating whether beta-subunits also influence ligand interactions, we found that β4 can drastically alter the response of the neuronal rNav1.2a isoform to spider and scorpion toxins that target paddle motifs within Nav channel voltage sensors. In conjunction with the β4 crystal structure, we utilized the altered sensitivity to a tarantula toxin as a tool to probe the interaction between rNav1.2a and β4. As a result, we identified 58Cys as an exposed residue that when mutated eliminates the influence of β4 on rNav1.2a toxin pharmacology. In addition, we exploited β4-induced alterations in toxin sensitivity to probe the interaction between rNav1.2a and a β4 mutant that mimics a β1 mutation implicated in epilepsy (C121W). We found that although this mutant still folds and traffics to the membrane, its interaction with the Nav channel is lost. The principles emerging from this work: 1) help explain tissue-dependent variations in Nav channel pharmacology; 2) enable the mechanistic interpretation of beta-subunit-related disorders; and 3) provide new opportunities to design molecules capable of correcting aberrant beta-subunit behavior.

Voltage-Gated Calcium Channels Exist and Function as Fragment-Channels in Neuronal Plasma Membranes

We reveal a novel form of regulation of neuronal L-type VGCCs: cleavage of the main body of the pore-forming alpha1 subunit (Cavalpha1) resulting in Cavalpha1 fragment-channels that separate but remain on the plasma membrane. This “core” proteolysis inversely correlates with channel activity, is developmentally regulated, and causes down-regulation and biophysical alterations of VGCC currents. Recombinant Cavalpha1 fragment-channels do not form active channels on their own, yet when properly paired, they produce currents with distinct biophysical properties. These fragments also influence the activity and/or properties of full-length VGCCs. Core proteolysis represents a new paradigm of feedback regulation of VGCCs that would profoundly affect cell excitability, synaptic transmission, and cellular calcium signaling in physiological and pathological states.

Calcium-Mediated Tailspin of Calmodulin on the IQ Motif of the Neuronal Voltage-Dependent Sodium Channel Nav1.2

NaV1.2 is regulated by calmodulin (CaM), an essential calcium sensor with two homologous domains (N and C). CaM binds tightly to an IQ motif in the intracellular C-terminal tail of the pore-forming alpha subunit of NaV1.2 as well as to the inactivation gate. The IQ motif interacts with the “semi-open” cleft of apo CaM: I inserts into the cleft, while Q contacts the FG-turn of CaM (2KXW). To learn how CaM-IQ responds to increases in intracellular [Ca2+], we determined equilibrium constants for apo and calcium-saturated CaM binding to biosensors containing mutated IQ motif sequences sandwiched between YFP and CFP. Their quantum yields permit resolution of Kd values close to 1 nM from equilibrium titrations. Changes of NaV1.2 residues making close contacts with apo CaM were anticipated to diminish binding of both apo and calcium-saturated CaM. However, the quantitative effects differed by orders of magnitude. The affinity for calcium-saturated CaM dropped by factors of 10-100, while effects on apo CaM binding were more severe. Thus, the CaM-IQ interface differs dramatically depending on calcium-saturation of CaM. NMR studies of a complex of (Ca2+)2-CaM-C-domain bound to the IQ motif showed that calcium binding opens CaM but also causes it to pivot by 180° so that it binds to the IQ motif in the opposite direction. The I of the IQ motif contacts the “open” hydrophobic cleft of (Ca2+)2-CaM, but the Q points towards the linker between the N- and C-domains of CaM. In conjunction with 2KXW, this new structure provides the first pair of high resolution structures for apo and calcium-saturated CaM bound to a single IQ motif. Support: NIH R01 GM57001 and Carver Charitable Trust Grant 01-224.

Sodium channel β2 subunit promotes filopodia-like processes and expansion of the dendritic tree in developing rat hippocampal neurons

The β2 auxiliary subunit of voltage-gated sodium channels (VGSCs) appears at early stages of brain development. It is abundantly expressed in the mammalian central nervous system where it forms complexes with different channel isoforms, including Nav1.2. From the structural point of view, β2 is a transmembrane protein: at its extracellular N-terminus an Ig-like type C2 domain mediates the binding to the pore-forming alpha subunit with disulfide bonds and the interactions with the extracellular matrix. Given this structural versatility, β2 has been suggested to play multiple functions ranging from channel targeting to the plasma membrane and gating modulation to control of cell adhesion. We report that, when expressed in Chinese Hamster Ovary cells CHO-K1, the subunit accumulates at the perimetral region of adhesion and particularly in large lamellipodia-like membrane processes where it induces formation of filopodia-like structures. When overexpressed in developing embryonic rat hippocampal neurons in vitro, β2 specifically promotes formation of filopodia-like processes in dendrites leading to expansion of the arborization tree, while axonal branching remains unaltered. In contrast to this striking and highly specific effect on dendritic morphology, the targeting of functional sodium channels to the plasma membrane, including the preferential localization of Nav1.2 at the axon, and their gating properties are only minimally affected. From these and previously reported observations it is suggested that β2, among its multiple functions, may contribute to promote dendritic outgrowth and to regulate neuronal wiring at specific stages of neuronal development.

Reduced PIP2 Binding to KCNJ2 (M307I) Channels is Linked to Type 1 Andersen-Tawil Syndrome

Background: Inward rectifier potassium channels play a key role in setting and maintaining the resting membrane potential and regulating excitability in various tissues. Kir2.x subfamily members mediate the cardiac inward rectifier potassium current (IK1). KCNJ2 encodes Kir2.1, the pore-forming alpha subunit responsible for cardiac IK1, and the mutations in this gene are associated with type 1 Andersen-Tawil Syndrome (ATS1). A Kir2.1 missense mutation, M307I, has been identified in a Korean family with ATS1. We found that the ATS1-associated M307I mutation is a loss-of-function mutation in KCNJ2 that mediates a dominant-negative effect on wild-type (WT) channels. M307I is located in the intracellular C-terminal domain in a region known to be associated with putative phosphatidylinositol 4,5-bisphosphate (PIP2) binding and channel trafficking. Here we explored the mechanisms underlying the dominant-negative effect of the mutation.Methods and Results: Human Kir2.1 was subcloned into pFlag-CMV vector and pFlag-Kir2.1-M307I was generated by site-direct mutagenesis. The Flag-Kir2.1-WT and Flag-Kir2.1-M307I were expressed in HEK293 cells and affinity purified. PIP2-binding was assessed using a Lipid-bead-protein pull-down assay with cell lysate and Protein-lipid overlay assay with purified proteins. The electrophysiological data showed that the M307I mutant channel significantly reduces whole cell current densities when co-expressed with Kir2.1-WT channels. Immunofluorescence (IF) staining assays reveal that M307I channels exhibit normal membrane trafficking. PIP2 binding assays show that Flag-Kir2.1-M307I channels exhibit dramatically decreased binding to PIP2 compared to WT channels.Conclusions: M307I is an ATS1-associated, loss-of-function missense mutation in KCNJ2 that mediates a dominant-negative effect on WT channels by reducing PIP2 binding to the channel.

Gem Stabilizes Voltage-Gated Calcium Channels in the Inactivated State: Implications for Human Disease

RGK proteins (Rad, Rem1, Rem2 and Gem/Kir) are small GTPases that often colocalize with and strongly inhibit high-voltage activated (HVA) calcium channels (L-, N-, P/Q- and R-type). The mechanism of inhibition is largely unclear. The pore-forming alpha-1 subunit of HVA calcium channels requires the beta subunit for cell surface trafficking and gating. We previously showed direct binding between alpha-1 and Gem and obtained a Gem-insensitive chimeric P/Q alpha-1containing the IIS1-S3 region of a Gem-resistant, low-voltage activated T-type channel (Fan et al., PNAS, 2010). In this study, we investigated the cause of this insensitivity. Since the IIS1-S3 region of P/Q alpha-1 has too few cytosolic amino acids to be a Gem binding site, and since we had identified a different alpha-1 region that binds Gem, we focused on altered biophysical properties of the chimera. We observed that the chimeric channel inactivated much slower than WT P/Q channel did; we therefore examined whether slow inactivation was responsible for Gem insensitivity. Mutating residues known to increase channel inactivation enhanced Gem inhibition, whereas mutating residues known to slow channel inactivation weakened or abolished Gem inhibition. Replacing beta3 with beta2a, an auxiliary subunit known to slow channel inactivation, also weakened Gem inhibition. Furthermore, recovery from inactivation of WT P/Q channels in the presence of Gem was markedly slower than without Gem. Thus, Gem appeared to stabilize the channel in the inactivated state. These results shed light on a new factor - altered regulation by RGK proteins - that may contribute to HVA calcium channel-related channelopathies. Indeed, we found that familial hemiplegic migraine mutations that speed P/Q channel inactivation strengthened Gem inhibition, whereas Timothy syndrome mutations that slow channel inactivation greatly weakened Gem inhibition.

Calmodulin Discrimination Between Voltage-Dependent Sodium Channel IQ Motifs

Voltage-dependent sodium channels in the NaV1 family control action potentials in neurons and muscle. They are regulated by calmodulin (CaM), an essential eukaryotic calcium sensor that contains two highly homologous domains (N and C). NaV channelopathies include epilepsy, Long QT syndrome, ventricular fibrillation, familial autism, and pain insensitivity. Some mutations are in or near an IQ motif (IQxxxBGxxxB, B = K, R) in the intracellular C-terminal tail of the pore-forming alpha subunit where CaM binds tightly. In the 11 residues of each NaV IQ motif, only the Q is completely conserved. In NaV1.2 (2KXW) and NaV1.5 (2L53), both apo and Ca2+-saturated CaM anchor to the IQ motif via C. However, Ca2+ binding to CaM changes its conformation and lowers its affinity for the IQ motif. To determine how CaM translates changes in intracellular [Ca2+] into conformational work, and how it regulates multiple isoforms of NaV, we are using NMR and fluorescence to test how CaM discriminates between IQ motifs of NaV and how disease-causing mutations uniquely affect binding of apo and calcium-saturated N and C. To determine energetic differences in CaM binding to IQ motifs, we embedded each sequence into an auto-fluorescent biosensor (YFP-IQ-CFP) to determine their affinity for CaM, and each CaM domain individually. Biosensor quantum yield permits resolution of dissociation constants close to 1 nM from equilibrium titrations. Based on thermodynamic and structural studies of (Ca2+)4-CaM bound to IQ motifs in the CaV family, a homology model is proposed for (Ca2+)4-CaM bound to NaV1.2. NIH R01 GM57001 and Carver Charitable Trust Grant 01-224.91