An SK3 Channel/nWASP/Abi-1 Complex Is Involved in Early Neurogenesis

BACKGROUND: The stabilization or regulated reorganization of the actin cytoskeleton is essential for cellular structure and function. Recently, we could show that the activation of the SK3-channel that represents the predominant SK-channel in neural stem cells, leads to a rapid local outgrowth of long filopodial processes. This observation indicates that the rearrangement of the actin based cytoskeleton via membrane bound SK3-channels might selectively be controlled in defined micro compartments of the cell. PRINCIPAL FINDINGS: We found two important proteins for cytoskeletal rearrangement, the Abelson interacting protein 1, Abi-1 and the neural Wiskott Aldrich Syndrome Protein, nWASP, to be in complex with SK3- channels in neural stem cells (NSCs). Moreover, this interaction is also found in spines and postsynaptic compartments of developing primary hippocampal neurons and regulates neurite outgrowth during early phases of differentiation. Overexpression of the proteins or pharmacological activation of SK3 channels induces obvious structural changes in NSCs and hippocampal neurons. In both neuronal cell systems SK3 channels and nWASP act synergistic by strongly inducing filopodial outgrowth while Abi-1 behaves antagonistic to its interaction partners. CONCLUSIONS: Our results give good evidence for a functional interplay of a trimeric complex that transforms incoming signals via SK3-channel activation into the local rearrangement of the cytoskeleton in early steps of neuronal differentiation involving nWASP and Abi-1 actin binding proteins

Discovery of novel activators of large-conductance calcium-activated potassium channels for the treatment of cerebellar ataxia

Impaired cerebellar Purkinje neuron firing resulting from reduced expression of large-conductance calcium-activated potassium (BK) channels is a consistent feature in models of inherited neurodegenerative spinocerebellar ataxia (SCA). Restoring BK channel expression improves motor function and delays cerebellar degeneration, indicating that BK channels are an attractive therapeutic target. Current BK channel activators lack specificity and potency and are therefore poor templates for future drug development. We implemented an automated patch clamp platform for high-throughput drug discovery of BK channel activators using the Nanion SyncroPatch 384PE system. We screened over 15,000 compounds for their ability to increase BK channel current amplitude under conditions of lower intracellular calcium that is present in disease. We identified several novel BK channel activators that were then retested on the SyncroPatch 384PE to generate concentration-response curves (CRCs). Compounds with favorable CRCs were subsequently tested for their ability to improve irregular cerebellar Purkinje neuron spiking, characteristic of BK channel dysfunction in SCA1 mice. We identified a novel BK channel activator, 4-chloro-N-(5-chloro-2-cyanophenyl)-3-(trifluoromethyl)benzene-1-sulfonamide (herein renamed BK-20), that exhibited a more potent half-maximal activation of BK current (pAC50 = 4.64) than NS-1619 (pAC50 = 3.7) at a free internal calcium concentration of 270 nM in a heterologous expression system and improved spiking regularity in SCA1 Purkinje neurons. BK-20 had no activity on small-conductance calcium-activated potassium (SK)1-3 channels but interestingly was a potent blocker of the T-type calcium channel, Cav3.1 (IC50 = 1.05 μM). Our work describes both a novel compound for further drug development in disorders with irregular Purkinje spiking and a unique platform for drug discovery in degenerative ataxias. SIGNIFICANCE STATEMENT: Motor impairment associated with altered Purkinje cell spiking due to dysregulation of large-conductance calcium-activated potassium (BK) channel expression and function is a shared feature of disease in many degenerative ataxias. BK channel activators represent an outstanding therapeutic agent for ataxia. We have developed a high-throughput platform to screen for BK channel activators and identified a novel compound that can serve as a template for future drug development for the treatment of these disabling disorders

Epac signaling is required for hippocampus-dependent memory retrieval

Previously we uncovered a critical role for norepinephrine and β1-adrenergic signaling in hippocampus-dependent memory retrieval. Because the β1 receptor couples to Gs, we examine here whether cAMP is also required for contextual memory retrieval. Using pharmacologic and genetic approaches to manipulate cAMP and downstream signaling, we demonstrate that cAMP and two of its targets, protein kinase A (PKA) and exchange protein activated by cAMP (Epac), are both required for retrieval. These findings demonstrate that cAMP signaling through Epac (as well as PKA) plays an essential role in cognition

Inhibition of the cation channel TRPV4 improves bladder function in mice and rats with cyclophosphamide-induced cystitis

Reduced functional bladder capacity and concomitant increased micturition frequency (pollakisuria) are common lower urinary tract symptoms associated with conditions such as cystitis, prostatic hyperplasia, neurological disease, and overactive bladder syndrome. These symptoms can profoundly affect the quality of life of afflicted individuals, but available pharmacological treatments are often unsatisfactory. Recent work has demonstrated that the cation channel TRPV4 is highly expressed in urothelial cells and plays a role in sensing the normal filling state of the bladder. In this article, we show that the development of cystitis-induced bladder dysfunction is strongly impaired in Trpv4−/− mice. Moreover, we describe HC-067047, a previously uncharacterized, potent, and selective TRPV4 antagonist that increases functional bladder capacity and reduces micturition frequency in WT mice and rats with cystitis. HC-067047 did not affect bladder function in Trpv4−/− mice, demonstrating that its in vivo effects are on target. These results indicate that TRPV4 antagonists may provide a promising means of treating bladder dysfunction