TRPC1 transcript variants, inefficient nonsense-mediated decay and low up-frameshift-1 in vascular smooth muscle cells.

BACKGROUND: Transient Receptor Potential Canonical 1 (TRPC1) is a widely-expressed mammalian cationic channel with functional effects that include stimulation of cardiovascular remodelling. The initial aim of this study was to investigate variation in TRPC1-encoding gene transcripts. RESULTS: Extensive TRPC1 transcript alternative splicing was observed, with exons 2, 3 and 5-9 frequently omitted, leading to variants containing premature termination codons. Consistent with the predicted sensitivity of such variants to nonsense-mediated decay (NMD) the variants were increased by cycloheximide. However it was notable that control of the variants by NMD was prominent in human embryonic kidney 293 cells but not human vascular smooth muscle cells. The cellular difference was attributed in part to a critical protein in NMD, up-frameshift-1 (UPF1), which was found to have low abundance in the vascular cells. Rescue of UPF1 by expression of exogenous UPF1 was found to suppress vascular smooth muscle cell proliferation. CONCLUSIONS: The data suggest: (i) extensive NMD-sensitive transcripts of TRPC1; (ii) inefficient clearance of aberrant transcripts and enhanced proliferation of vascular smooth muscle cells in part because of low UPF1 expression.

A sphingosine-1-phosphate-activated calcium channel controlling vascular smooth muscle cell motility.

In a screen of potential lipid regulators of transient receptor potential (TRP) channels, we identified sphingosine-1-phosphate (S1P) as an activator of TRPC5. We explored the relevance to vascular biology because S1P is a key cardiovascular signaling molecule. TRPC5 is expressed in smooth muscle cells of human vein along with TRPC1, which forms a complex with TRPC5. Importantly, S1P also activates the TRPC5-TRPC1 heteromultimeric channel. Because TRPC channels are linked to neuronal growth cone extension, we considered a related concept for smooth muscle. We find S1P stimulates smooth muscle cell motility, and that this is inhibited by E3-targeted anti-TRPC5 antibody. Ion permeation involving TRPC5 is crucial because S1P-evoked motility is also suppressed by the channel blocker 2-aminoethoxydiphenyl borate or a TRPC5 ion-pore mutant. S1P acts on TRPC5 via two mechanisms, one extracellular and one intracellular, consistent with its bipolar signaling functions. The extracellular effect appears to have a primary role in S1P-evoked cell motility. The data suggest S1P sensing by TRPC5 calcium channel is a mechanism contributing to vascular smooth muscle adaptation.

Differential Localization of Two Alternatively Spliced GABAA Receptor gamma2-Subunit mRNAs in the Chick Brain.

Two variants of the GABAA receptor gamma2 subunit are known to exist, which differ by the presence (gamma2L) or absence (gamma2S) of eight amino acids in the presumed intracellular loop between the third and fourth membrane-spanning domains. These variants have been shown to be generated by alternative splicing of the gamma2-subunit primary gene transcript in mouse (Kofuji et al., J. Neurochem., 56, 713 - 715, 1991), and in bovine and human (Whiting et al., Proc. Natl. Acad. Sci. USA, 87, 9966 - 9970, 1990) brain. We describe here the cloning, from chick (Gallus domesticus) brain, of cDNAs that encode the gamma2L and gamma2S subunits, and report on the regional and cellular localization of the corresponding mRNAs as revealed by in situ hybridization histochemistry with transcript-specific oligonucleotide probes. While the two transcripts are found to be colocalized throughout the chick neuroaxis, certain nuclei (for example, the nucleus isthmi, pars magnocellularis, the nucleus isthmi, pars parvocellularis, the nucleus solitarius and the paleostriatum primitivum) are found to contain predominantly either the gamma2S- or the gamma2L-subunit mRNA. We conclude that receptors that contain either the gamma2S or the gamma2L subunit occur, and that these probably have functionally different roles in the modulation of neurotransmission in the central nervous system. In addition, our data indicate that certain cells may produce both transcripts. Consequently, these will have either a single receptor subtype that contains both a gamma2S and a gamma2L subunit, or two receptor subtypes, one of which contains a gamma2S subunit and the other a gamma2L subunit.

The benzodiazepine site of the GABAA receptor: an old target with new potential?

The gamma-aminobutyric acid-A (GABA(A)) receptors is the target for the most widely prescribed sleep medicines. It is a ligand-gated ion channel, activated by the amino acid neurotransmitter GABA, which normally results in hyperpolarization of neurons leading to reduced action potential firing, and thereby a reduction in neuronal activity. It has a rich pharmacology with a number of separate modulator binding sites. The best studied of these is the benzodiazepine site. Modulation of GABA(A) receptor activity by benzodiazepines produces sedative, hypnotic, anxiolytic and anticonvulsant activities. Short half-life benzodiazepines such as triazolam have been particularly useful in treating insomnia, but concerns have been raised regarding tolerance potential and dependence liability of classical benzodiazepines, which has led to reduced prescribing of these agents. In recent years, the treatment of sleep disorders has moved towards the use of non-benzodiazepine sedative hypnotics. These agents act at the same site on the GABA(A) receptor, but feature less of the problems associated with classical benzodiazepines. Recent progress in our understanding of the diversity and pharmacology of GABA(A) receptor subtypes has provided a rational explanation for the efficacy of these compounds. Findings from preclinical studies reveal promising avenues for the design of better therapeutics in the near future.

Neurotrophin-induced preprotachykinin-A gene promoter modulation in organotypic rat spinal cord culture.

To study regulation of the preprotachykinin-A gene promoter, we utilised a biolistic gene transfer protocol to deliver a DNA construct that incorporates a portion of the preprotachykinin-A gene promoter and an enhanced green fluorescent protein reporter gene into neonatal rat spinal cord organotypic slices. The ability of the neurokinin-1 receptor agonist [Sar9,Met(O2)11]-substance P, nerve growth factor and brain derived neurotrophic factor to modulate positively preprotachykinin-A gene promoter construct activity, as indicated by de novo enhanced green fluorescent protein expression, was determined. Treatment of organotypic slices with [Sar9, Met(O2)11]-substance P (10 microm, P < 0.05), nerve growth factor (200 ng/mL, P < 0.001) or brain derived neurotrophic factor (200 ng/mL, P < 0.02) significantly increased the proportion of cytomegaloviral promoter-DsRed transfected cells (used to visualise total transfected cells) that co-expressed enhanced green fluorescent protein. The distribution of enhanced green fluorescent protein/DsRed-positive neurones across spinal laminae was broadly in line with the known distribution of spinal Trk and neurokinin-1 receptors. These data suggest a modulated activity of the preprotachykinin-A gene promoter in spinal neurones in vitro by substance P and/or neurotrophins. The functional consequences of such transcriptional changes within central peptidergic circuitry and their relevance to chronic pain are considered.

Sensing of lysophospholipids by TRPC5 calcium channel.

TRPC calcium channels are emerging as a ubiquitous feature of vertebrate cells, but understanding of them is hampered by limited knowledge of the mechanisms of activation and identity of endogenous regulators. We have revealed that one of the TRPC channels, TRPC5, is strongly activated by common endogenous lysophospholipids including lysophosphatidylcholine (LPC) but, by contrast, not arachidonic acid. Although TRPC5 was stimulated by agonists at G-protein-coupled receptors, TRPC5 activation by LPC occurred downstream and independently of G-protein signaling. The effect was not due to the generation of reactive oxygen species or because of a detergent effect of LPC. LPC activated TRPC5 when applied to excised membrane patches and thus has a relatively direct action on the channel structure, either because of a phospholipid binding site on the channel or because of sensitivity of the channel to perturbation of the bilayer by certain lipids. Activation showed dependence on side-chain length and the chemical head-group. The data revealed a previously unrecognized lysophospholipid-sensing capability of TRPC5 that confers the property of a lipid ionotropic receptor.

Cloning of the human TASK-2 (KCNK5) promoter and its regulation by chronic hypoxia.

The tandem P domain potassium channel family includes five members of the acid-sensing subfamily, TASK. TASK channels are active at resting potential and are inhibited by extracellular protons, suggesting they function as acid sensors and control excitability/ion homeostasis. Indeed, TASK-2 (KCNK5) has been shown to control excitability, volume regulation, bicarbonate handling, and apoptosis in a variety of tissues. With such diverse functions being ascribed to TASK-2, it is important to understand long-term as well as short-term regulation of this important channel. Thus, we have cloned the TASK-2 promoter, demonstrated that its transcriptional activity is dependent upon pO(2), shown that deletion of overlapping consensus binding sites for NF-kappaB/Elk-1 ablates this O(2) sensitivity, and proved that Elk-1 binds preferentially to this site. Furthermore, the consequences of chronic hypoxia on natively expressed TASK-2 are decreased steady-state mRNA and cell depolarization showing that TASK-2 contributes to the excitability of this important lung cell type.

A model of organotypic rat spinal slice culture and biolistic transfection to elucidate factors that drive the preprotachykinin-A promoter.

The tachykinin substance P (SP) is a neuropeptide that is expressed in some nociceptive primary sensory afferents and in discrete populations of spinal cord neurons. Expression of spinal SP and the preprotachykinin-A (PPT-A) gene that encodes SP exhibits plasticity in response to conditions such as peripheral inflammation but the mechanisms that regulate expression are poorly understood. We have developed a spinal cord organotypic culture system that is suitable for the analysis of PPT-A gene promoter activity following biolistic transfection of recombinant DNA constructs. Spinal cord organotypic slices showed good viability over a 7-day culture period. Immunostaining for phenotypic markers such as NeuN and beta-III tubulin demonstrated preservation of neurons and their structure, although there was evidence of axotomy-induced down-regulation of NeuN in certain neuronal populations. Neurokinin-1 receptor (NK-1R) immunostaining in laminae I and III was similar to that seen in acute slices. Biolistic transfection was used to introduce DNA constructs into neurons of these organotypic cultures. Following transfection with a construct in which expression of enhanced green fluorescent protein (EGFP) is controlled by the PPT-A promoter, we showed that induction of neuronal activity by administration of a forskolin analogue/high K(+) (10 microM/10 mM) for 24 h resulted in a fourfold increase in the number of EGFP-positive cells. Similarly, a twofold increase was obtained after treatment with the NK-1R-specific agonist [Sar(9),Met (O(2))(11)]-substance P (10 microM). These data demonstrate the usefulness of this model to study physiological and pharmacological factors relevant to nociceptive processing that can modulate PPT-A promoter activity.

Post-transcriptional control of human maxiK potassium channel activity and acute oxygen sensitivity by chronic hypoxia.

Various cardiorespiratory diseases (e.g. congestive heart failure, emphysema) result in systemic hypoxia and patients consequently demonstrate adaptive cellular responses which predispose them to conditions such as pulmonary hypertension and stroke. Central to many affected excitable tissues is activity of large conductance, Ca2+-activated K+ (maxiK) channels. We have studied maxiK channel activity in HEK293 cells stably co-expressing the most widely distributed of the human alpha- and beta-subunits that constitute these channel following maneuvers which mimic severe hypoxia. At all [Ca2+]i, chronic hypoxia (approximately 18 mm Hg, 72 h) increased K+ current density, most markedly at physiological [Ca2+]i K+ currents in cells cultured in normoxia showed a [Ca2+]i-dependent sensitivity to acute hypoxic inhibition ( approximately 25 mm Hg, 3 min). However, chronic hypoxia dramatically changed the Ca2+ sensitivity of this acute hypoxic inhibitory profile such that low [Ca2+]i could sustain an acute hypoxic inhibitory response. Chronic hypoxia caused no change in alpha-subunit immunoreactivity with Western blotting but evoked a 3-fold increase in beta-subunit expression. These observations were fully supported by immunocytochemistry, which also suggested that chronic hypoxia augmented alpha/beta-subunit co-localization at the plasma membrane. Using a novel nuclear run-on assay and RNase protection we found that chronic hypoxia did not alter mRNA production rates or steady-state levels, which suggests that this important environmental cue modulates maxiK channel function via post-transcriptional mechanisms.