Hong-Gang Wang

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.

Targeted Epigenetic Remodeling of Endogenous Loci by CRISPR/Cas9-Based Transcriptional Activators Directly Converts Fibroblasts to Neuronal Cells

Overexpression of exogenous fate-specifying transcription factors can directly reprogram differentiated somatic cells to target cell types. Here, wexa0show that similar reprogramming can also be achieved through the direct activation of endogenous genes using engineered CRISPR/Cas9-based transcriptional activators. We use this approach to induce activation of the endogenous Brn2, Ascl1, and Myt1l genes (BAM factors) to convert mouse embryonic fibroblasts to induced neuronal cells. This direct activation of endogenous genes rapidly remodeled the epigenetic state of the target loci and induced sustained endogenous gene expression during reprogramming. Thus, transcriptional activation and epigenetic remodeling of endogenous master transcription factors are sufficient for conversion between cell types. The rapid and sustained activation of endogenous genes in their native chromatin context by this approach may facilitate reprogramming with transient methods that avoid genomic integration and provides a new strategy for overcoming epigenetic barriers to cell fate specification.

Ca2+/Calmodulin Regulates Trafficking of CaV1.2 Ca2+ Channels in Cultured Hippocampal Neurons

As the Ca2+-sensor for Ca2+-dependent inactivation, calmodulin (CaM) has been proposed, but never definitively demonstrated, to be a constitutive CaV1.2 Ca2+ channel subunit. Here we show that CaM is associated with the CaV1.2 pore-forming α1C subunit in brain in a Ca2+-independent manner. Within its CaM binding pocket, α1C has been proposed to contain a membrane targeting domain. Because ion channel subunits assemble early during channel biosynthesis, we postulated that this association with CaM could afford the opportunity for Ca2+-dependent regulation of membrane targeting. We showed that the isolated domain functioned as a Ca2+/CaM regulated trafficking determinant for CD8 (a model transmembrane protein) using fluorescent-activated cell sorting analysis and, using green fluorescent protein-tagged α1C subunits expressed in cultured hippocampal neurons, that Ca2+/CaM interaction with this domain accelerated trafficking of CaV1.2 channels to distal regions of the dendritic arbor. Furthermore, this Ca2+/CaM-accelerated trafficking was activity dependent. Thus, CaM imparts Ca2+-dependent regulation not only to mature CaV1.2 channels at the cell surface but also to steps during channel biosynthesis.

Structural analyses of Ca2+/CaM interaction with NaV channel C-termini reveal mechanisms of calcium-dependent regulation

Ca2+ regulates voltage-gated Na+ (NaV) channels and perturbed Ca2+ regulation of NaV function is associated with epilepsy syndromes, autism, and cardiac arrhythmias. Understanding the disease mechanisms, however, has been hindered by a lack of structural information and competing models for how Ca2+ affects NaV channel function. Here, we report the crystal structures of two ternary complexes of a human NaV cytosolic C-terminal domain (CTD), a fibroblast growth factor homologous factor, and Ca2+/calmodulin (Ca2+/CaM). These structures rule out direct binding of Ca2+ to the NaV CTD, and uncover new contacts between CaM and the NaV CTD. Probing these new contacts with biochemical and functional experiments allows us to propose a mechanism by which Ca2+ could regulate NaV channels. Further, our model provides hints towards understanding the molecular basis of the neurologic disorders and cardiac arrhythmias caused by NaV channel mutations.

Ca2+/CaM Controls Ca2+-Dependent Inactivation of NMDA Receptors by Dimerizing the NR1 C Termini

Ca2+ influx through NMDA receptors (NMDARs) leads to channel inactivation, which limits Ca2+ entry and protects against excitotoxicity. Extensive functional data suggests that this Ca2+-dependent inactivation (CDI) requires both calmodulin (CaM) binding to the C0 cassette of the NR1 subunits C terminus (CT) and regulation by α-actinin-2, but a molecular understanding of CDI has been elusive. Here we used a number of methods to analyze the molecular nature of the interaction among CaM, α-actinin-2, and the NR1 CT. We found that a single CaM binds to two NR1 CTs in a Ca2+-dependent manner and promotes their reversible “dimerization.” Expressed NMDARs containing NR1 concatamers in which the NR1 C termini are “uncoupled” display markedly reduced CDI. In contrast to current models, α-actinin-2 does not bind to the NR1 CT. We propose a new model for CDI in which the noncanonical Ca2+/CaM-dependent dimerization of the two NR1 subunits inactivates the channel by propagating a conformational change from the short NR1 CT to the nearby channel pore.

Rem2-Targeted shRNAs Reduce Frequency of Miniature Excitatory Postsynaptic Currents without Altering Voltage-Gated Ca2+ Currents

Ca2+ influx through voltage-gated Ca2+ channels (VGCCs) plays important roles in neuronal cell development and function. Rem2 is a member of the RGK (Rad, Rem, Rem2, Gem/Kir) subfamily of small GTPases that confers potent inhibition upon VGCCs. The physiologic roles of RGK proteins, particularly in the brain, are poorly understood. Rem2 was implicated in synaptogenesis through an RNAi screen and proposed to regulate Ca2+ homeostasis in neurons. To test this hypothesis and uncover physiological roles for Rem2 in the brain, we investigated the molecular mechanisms by which Rem2 knockdown affected synaptogenesis and Ca2+ homeostasis in cultured rat hippocampal neurons. Expression of a cocktail of shRNAs targeting rat Rem2 (rRem2) reduced the frequency of miniature excitatory postsynaptic currents (mEPSCs) measured 10 d after transfection (14 d in vitro), but did not affect mEPSC amplitude. VGCC current amplitude after rRem2-targeted knockdown was not different from that in control cells, however, at either 4 or 10 d post transfection. Co-expression of a human Rem2 that was insensitive to the shRNAs targeting rRem2 was unable to prevent the reduction in mEPSC frequency after rRem2-targeted knockdown. Over-expression of rRem2 resulted in 50% reduction in VGCC current, but neither the mEPSC frequency nor amplitude was affected. Taken together, the observed effects upon synaptogenesis after shRNA treatment are more likely due to mechanisms other than modulation of VGCCs and Ca2+ homeostasis, and may be independent of Rem2. In addition, our results reveal a surprising lack of contribution of VGCCs to synaptogenesis during early development in cultured hippocampal neurons.

The Auxiliary Subunit KChIP2 is an Essential Regulator of Homeostatic Excitability

Background: The necessity for, or redundancy of, distinctive KChIP proteins is not known. Results: Deletion of KChIP2 leads to increased susceptibility to epilepsy and to a reduction in IA and increased excitability in pyramidal hippocampal neurons. Conclusion: KChIP2 is essential for homeostasis in hippocampal neurons. Significance: Mutations in K+ channel auxiliary subunits may be loci for epilepsy. The somatodendritic IA (A-type) K+ current underlies neuronal excitability, and loss of IA has been associated with the development of epilepsy. Whether any one of the four auxiliary potassium channel interacting proteins (KChIPs), KChIP1–KChIP4, in specific neuronal populations is critical for IA is not known. Here we show that KChIP2, which is abundantly expressed in hippocampal pyramidal cells, is essential for IA regulation in hippocampal neurons and that deletion of Kchip2 affects susceptibility to limbic seizures. The specific effects of Kchip2 deletion on IA recorded from isolated hippocampal pyramidal neurons were a reduction in amplitude and shift in the V½ for steady-state inactivation to hyperpolarized potentials when compared with WT neurons. Consistent with the relative loss of IA, hippocampal neurons from Kchip2−/− mice showed increased excitability. WT cultured neurons fired only occasional single action potentials, but the average spontaneous firing rate (spikes/s) was almost 10-fold greater in Kchip2−/− neurons. In slice preparations, spontaneous firing was detected in CA1 pyramidal neurons from Kchip2−/− mice but not from WT. Additionally, when seizures were induced by kindling, the number of stimulations required to evoke an initial class 4 or 5 seizure was decreased, and the average duration of electrographic seizures was longer in Kchip2−/− mice compared with WT controls. Together, these data demonstrate that the KChIP2 is essential for physiologic IA modulation and homeostatic stability and that there is a lack of functional redundancy among the different KChIPs in hippocampal neurons.

A novel NaV1.5 voltage sensor mutation associated with severe atrial and ventricular arrhythmias.

BACKGROUNDnInherited autosomal dominant mutations in cardiac sodium channels (NaV1.5) cause various arrhythmias, such as long QT syndrome and Brugada syndrome. Although dozens of mutations throughout the protein have been reported, there are few reported mutations within a voltage sensor S4 transmembrane segment and few that are homozygous. Here we report analysis of a novel lidocaine-sensitive recessive mutation, p.R1309H, in the NaV1.5 DIII/S4 voltage sensor in a patient with a complex arrhythmia syndrome.nnnMETHODS AND RESULTSnWe expressed the wild type or mutant NaV1.5 heterologously for analysis with the patch-clamp and voltage clamp fluorometry (VCF) techniques. p.R1309H depolarized the voltage-dependence of activation, hyperpolarized the voltage-dependence of inactivation, and slowed recovery from inactivation, thereby reducing the channel availability at physiologic membrane potentials. Additionally, p.R1309H increased the late Na(+) current. The location of the mutation in DIIIS4 prompted testing for a gating pore current. We observed an inward current at hyperpolarizing voltages that likely exacerbates the loss-of-function defects at resting membrane potentials. Lidocaine reduced the gating pore current.nnnCONCLUSIONSnThe p.R1309H homozygous NaV1.5 mutation conferred both gain-of-function and loss-of-function effects on NaV1.5 channel activity. Reduction of a mutation-induced gating pore current by lidocaine suggested a therapeutic mechanism.

The two-pore-domain potassium channel TREK-1 mediates cardiac fibrosis and diastolic dysfunction

Cardiac two-pore domain potassium channels (K2P) exist in organisms from Drosophila to humans; however, their role in cardiac function is not known. We identified a K2P gene, CG8713 (sandman), in a Drosophila genetic screen and show that sandman is critical to cardiac function. Mice lacking an ortholog of sandman, TWIK-related potassium channel (TREK-1, also known Kcnk2), exhibit exaggerated pressure overload–induced concentric hypertrophy and alterations in fetal gene expression, yet retain preserved systolic and diastolic cardiac function. While cardiomyocyte-specific deletion of TREK-1 in response to in vivo pressure overload resulted in cardiac dysfunction, TREK-1 deletion in fibroblasts prevented deterioration in cardiac function. The absence of pressure overload–induced dysfunction in TREK-1–KO mice was associated with diminished cardiac fibrosis and reduced activation of JNK in cardiomyocytes and fibroblasts. These findings indicate a central role for cardiac fibroblast TREK-1 in the pathogenesis of pressure overload–induced cardiac dysfunction and serve as a conceptual basis for its inhibition as a potential therapy.

59. Multiplex Gene Activation by CRISPR/Cas9-Based Transcription Factors for the Direct Conversion of Fibroblasts to a Neuronal Phenotype

The reprogramming of cell lineage between mature somatic cell types has a significant potential to advance disease modeling, drug discovery, and gene and cell therapies for regenerative medicine. Several groups have established methods to reprogram cell phenotype through the ectopic delivery of cDNAs encoding master regulatory transcription factors. These factors can target epigenetically silenced genes and activate transcriptional networks specific to other cell lineages.The recent advances made in engineering the CRISPR/Cas9 system as a programmable transcriptional regulator provide an opportunity to study alternate methods to achieve cell reprogramming. The dCas9-based transcription factors (dCas9-TFs) can attain multiplex activation and repression of endogenous genes. Its simplicity of use enables a standardized method of regulating endogenous transcriptional networks in any cell type of interest. The ability of dCas9-TFs to target virtually any region of the genome within the native chromatin context provides an opportunity to study how non-coding regulatory elements and epigenetic signatures influence reprogramming outcomes.Here we demonstrate the capacity of dCas9-TFs to drive the direct conversion of murine embryonic fibroblasts (MEFs) to cells of a neuronal phenotype. We utilized a dCas9-TF with both N-terminal and C-terminal VP64 transactivation domains and demonstrated a 10-fold improvement in activation of target genes compared to dCas9-TFs with only a single VP64. Using this dCas9-TF variant, we demonstrated the multiplex activation of BRN2, ASCL1, and MYT1L (BAM factors) in MEFs. These three factors have been shown to generate functional neurons when expressed ectopically in MEFs.When compared to overexpression of the transgenes, dCas9-TFs induced significantly less mRNA and protein expression of the BAM factors, as determined by qRT-PCR and immunofluorescence staining. However, by day 14 in culture following the delivery of dCas9-TFs targeting the endogenous BAM factors, we identified cells positive for the pan-neuronal markers TUJ1 and MAP2 that exhibited complex neuronal morphologies. Furthermore, we found that dCas9-TFs were able to generate 23% more TUJ1/MAP2-co-positive cells than that achieved through the ectopic delivery of the BAM factors. Electrophysiological recordings of these cells identified single, action potential-like responses to step-depolarization by current clamp in the whole cell configuration. The cells also displayed calcium transients in response to KCl-induced depolarization, measured by monitoring fluorescence intensity of a GCaMP reporter.Ongoing work is focused on improving the functional maturity of the cells and addressing the kinetics of gene activation and epigenetic reprogramming at the target loci. We hypothesize that the targeted activation at the endogenous loci may more deterministically reprogram the chromatin to marks of active transcription that stabilize gene expression.