In vivo selective inhibition of TRPC6 by antagonist BI 749327 ameliorates fibrosis and dysfunction in cardiac and renal disease.

Transient receptor potential canonical type 6 (TRPC6) is a nonselective receptor-operated cation channel that regulates reactive fibrosis and growth signaling. Increased TRPC6 activity from enhanced gene expression or gain-of-function mutations contribute to cardiac and/or renal disease. Despite evidence supporting a pathophysiological role, no orally bioavailable selective TRPC6 inhibitor has yet been developed and tested in vivo in disease models. Here, we report an orally bioavailable TRPC6 antagonist (BI 749327; IC50 13 nM against mouse TRPC6, t1/2 8.5-13.5 hours) with 85- and 42-fold selectivity over the most closely related channels, TRPC3 and TRPC7. TRPC6 calcium conductance results in the stimulation of nuclear factor of activated T cells (NFAT) that triggers pathological cardiac and renal fibrosis and disease. BI 749327 suppresses NFAT activation in HEK293T cells expressing wild-type or gain-of-function TRPC6 mutants (P112Q, M132T, R175Q, R895C, and R895L) and blocks associated signaling and expression of prohypertrophic genes in isolated myocytes. In vivo, BI 749327 (30 mg/kg/day, yielding unbound trough plasma concentration ∼180 nM) improves left heart function, reduces volume/mass ratio, and blunts expression of profibrotic genes and interstitial fibrosis in mice subjected to sustained pressure overload. Additionally, BI 749327 dose dependently reduces renal fibrosis and associated gene expression in mice with unilateral ureteral obstruction. These results provide in vivo evidence of therapeutic efficacy for a selective pharmacological TRPC6 inhibitor with oral bioavailability and suitable pharmacokinetics to ameliorate cardiac and renal stress-induced disease with fibrosis.

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.

Localization of PIP2 activation gate in inward rectifier K+ channels.

Ion channels respond to changes in transmembrane voltage or ligand concentration by opening or closing an activation gate. In voltage-gated K+ channels, this gate has been localized to an intracellular bundle crossing. Here we examined whether this bundle crossing, or the more internal cytoplasmic pore, acts as a gate for PIP2 activation of inward rectifier K+ (Kir) channels expressed in Xenopus laevis oocytes. We studied the open/closed state-dependence of the accessibility of intracellular cationic modifiers to a position (residue Ile176 in the TM2 helix of Kir2.1) more external to the bundle crossing. Cd2+ blocked I176C mutant channels much more weakly in the closed state than in the open state, but Ag+ and sulfhydryl-specific methanethiosulfonate reagents modified the channels with similar rates in both states. These results suggest that the TM2 helices undergo conformation changes upon PIP2 binding/unbinding, but neither they nor the cytoplasmic pore close fully to form a physical gate for K+ conduction.

A single amino acid mutation attenuates rundown of voltage-gated calcium channels.

The activity of voltage-gated calcium channels (VGCCs) decreases with time in whole-cell and inside-out patch-clamp recordings. In this study we found that substituting a single amino acid (I1520) at the intracellular end of IIIS6 in the alpha(1) subunit of P/Q-type Ca(2+) channels with histidine or aspartate greatly attenuated channel rundown in inside-out patch-clamp recordings. The homologous mutations also slowed rundown of N- and L-type Ca(2+) channels, albeit to a lesser degree. In P/Q-type channels, the attenuation of rundown is accompanied by an increased apparent affinity for phosphatidylinositol-4,5-bisphosphate, which has been shown to be critical for maintaining Ca(2+) channel activity [L. Wu, C.S. Bauer, X.-G. Zhen, C. Xie, J. Yang, Dual regulation of voltage-gated calcium channels by PtdIns(4,5)P2. Nature 419 (2002) 947-952]. Furthermore, the histidine mutation significantly stabilized the open state, making the channels easier to open, slower to close, harder to inactivate and faster to recover from inactivation. Our finding that mutation of a single amino acid can greatly attenuate rundown provides an easy and efficient way to slow the rundown of VGCCs, facilitating functional studies that require direct access to the cytoplasmic side of the channel.

Localization of the activation gate of a voltage-gated Ca2+ channel.

Ion channels open and close in response to changes in transmembrane voltage or ligand concentration. Recent studies show that K+ channels possess two gates, one at the intracellular end of the pore and the other at the selectivity filter. In this study we determined the location of the activation gate in a voltage-gated Ca2+ channel (VGCC) by examining the open/closed state dependence of the rate of modification by intracellular methanethiosulfonate ethyltrimethylammonium (MTSET) of pore-lining cysteines engineered in the S6 segments of the alpha1 subunit of P/Q type Ca2+ channels. We found that positions above the putative membrane/cytoplasm interface, including two positions below the corresponding S6 bundle crossing in K+ channels, showed pronounced state-dependent accessibility to internal MTSET, reacting approximately 1,000-fold faster with MTSET in the open state than in the closed state. In contrast, a position at or below the putative membrane/cytoplasm interface was modified equally rapidly in both the open and closed states. Our results suggest that the S6 helices of the alpha1 subunit of VGCCs undergo conformation changes during gating and the activation gate is located at the intracellular end of the pore.

Functional architecture of the inner pore of a voltage-gated Ca2+ channel.

The inner pore of voltage-gated Ca2+ channels (VGCCs) is functionally important, but little is known about the architecture of this region. In K+ channels, this part of the pore is formed by the S6/M2 transmembrane segments from four symmetrically arranged subunits. The Ca2+ channel pore, however, is formed by four asymmetric domains of the same (alpha1) subunit. Here we investigated the architecture of the inner pore of P/Q-type Ca2+ channels using the substituted-cysteine accessibility method. Many positions in the S6 segments of all four repeats of the alpha1 subunit (Ca(v)2.1) were modified by internal methanethiosulfonate ethyltrimethylammonium (MTSET). However, the pattern of modification does not fit any known sequence alignment with K+ channels. In IIS6, five consecutive positions showed clear modification, suggesting a likely aqueous crevice and a loose packing between S6 and S5 segments, a notion further supported by the observation that some S5 positions were also accessible to internal MTSET. These results indicate that the inner pore of VGCCs is indeed formed by the S6 segments but is different from that of K+ channels. Interestingly some residues in IIIS6 and IVS6 whose mutations in L-type Ca2+ channels affect the binding of dihydropyridines and phenylalkylamines and are thought to face the pore appeared not to react with internal MTSET. Probing with qBBr, a rigid thiol-reactive agent with a dimension of 12 angstroms x 10 angstroms x 6 angstroms suggests that the inner pore can open to >10 angstroms. This work provides an impetus for future studies on ion permeation, gating, and drug binding of VGCCs.

AVP(4-8) Enhances PKC and MAPK Activities in SK-N-SH Cells.

AVP(4-8), one of endogenous metabolite of argipressin(AVP) in brain, can enhance learning and memory. To understand further the molecular mechanism of its function, human neuroblastoma SK-N-SH cell line was chosen as a model to study its signal transduction pathway. Radioligand binding assay showed the existence of binding sites for AVP(4-8) on SK-N-SH cells. The activity of PKC and MAPK in SK cells was significantly enhanced by AVP(4-8), and the enhancement of PKC and MAPK was suppressed by ZDC(C)PR, an antagonist of AVP(4-8).

Arginine Vasopressin(4-8) Mobilizes Intracellular Calcium in C6 Glioma Cells.

To understand the mechanism of neurotrophic action of neuropeptide ZNC(C)PR, which could affect growth of C6 cells, fluorescent dye Fluo-3 and confocal laser scanning microscope were used to assay the intracellular calcium in C6 glioma cells. It was found that ZNC(C)PR and it's analogue NLPR could mobilize intracellular calcium in a dose-dependent manner. The ZNC(C)PR antagnist, ZDC(C)PR, could inhibit the process, and the extracellular calcium did not influence it.

Dual regulation of voltage-gated calcium channels by PtdIns(4,5)P2.

Voltage-gated calcium channels (VGCCs) conduct calcium into cells after membrane depolarization and are vital for diverse biological events. They are regulated by various signalling pathways, which has profound functional consequences. The activity of VGCCs decreases with time in whole-cell and inside-out patch-clamp recordings. This rundown reflects persistent intrinsic modulation of VGCCs in intact cells. Although several mechanisms have been reported to contribute to rundown of L-type channels, the mechanism of rundown of other types of VGCC is poorly understood. Here we show that phosphatidylinositol-4,5-bisphosphate (PtdIns(4,5)P2), an essential regulator of ion channels and transporters, is crucial for maintaining the activity of P/Q- and N-type channels. Activation of membrane receptors that stimulate hydrolysis of PtdIns(4,5)P2 causes channel inhibition in oocytes and neurons. PtdIns(4,5)P2 also inhibits P/Q-type channels by altering the voltage dependence of channel activation and making the channels more difficult to open. This inhibition is alleviated by phosphorylation by protein kinase A. The dual actions of PtdIns(4,5)P2 and the crosstalk between PtdIns(4,5)P2 and protein kinase A set up a dynamic mechanism through which the activity of VGCCs can be finely tuned by various neurotransmitters, hormones and trophic factors.