Base pair numbers are expressed relative to transcription start sites. that are believed to represent a major M-channel isoform in the CNS and peripheral nervous system (Delmas and Brown, 2005). The importance of the appropriate functioning of Kv7.2 and Kv7.3 in the nervous system is highlighted by the fact that mutations in and are associated with benign familial neonatal convulsions, an autosomal dominant neonatal epilepsy (Charlier et al., 1998; Singh et al., 1998; Yang et al., 1998; Biervert and Steinlein, 1999). The M-current is also one of the key players in nociceptive transmission; inhibition of M-current leads to membrane depolarization and hyperexcitability of nociceptive neurons (Passmore et al., 2003; Crozier et al., 2007; Linley et al., 2008; Liu et al., 2010), an important determinant of many pain conditions. Pharmacological openers of Kv7.2/Kv7.3 channels therefore now represent important analgesic targets (Surti and Jan, 2005; Gribkoff, 2008; Pyridoxamine 2HCl Wickenden and McNaughton-Smith, 2009) because current therapies are not efficacious for the majority of inflammatory and chronic pain conditions. Despite their importance, very little is known Pyridoxamine 2HCl about the mechanisms responsible for regulating and expression. Here we use a functional assay to identify important regulatory regions within the and genes. We show that both and contain GC box motifs and provide evidence that their transcription is enhanced by the Sp1 transcription factor. We also show that expression of both and is repressed by REST (repressor element 1-silencing Pyridoxamine 2HCl transcription factor) and that expression of REST in neurons is sufficient to repress and Mouse monoclonal to EphA5 expression, inhibiting functional expression of the M-current and resulting in hyperexcitable neurons. We show that REST levels are increased in dorsal root ganglia (DRG) neurons in response to inflammatory mediators and that Kv7.2 levels and M-current density are reduced, suggesting a potential role in regulating inflammatory pain responses. Neuronal expression of REST is increased in response to sustained neuronal hyperactivity, i.e., in epileptic insults (Palm et al., 1998), cerebral ischemia (Calderone et al., 2003), and a model of neuropathic pain (Uchida et al.); we therefore suggest that, by repressing and expression, REST may contribute to chronic overexcitability of neuronal circuits seen in epilepsy and chronic pain. Materials and Methods Cell culture. SHSY-5Y and HEK293 cells were grown in DMEM/F-12 supplemented with 10% fetal calf serum (PAA Laboratories), 6 g/liter penicillin, 10 g/liter streptomycin, and 2 mm l-glutamine at 37C and 5% CO2. DRG neurons were cultured as described previously (Crozier et al., 2007; Linley et al., 2008; Liu et al., 2010); briefly, ganglia were extracted from 7- to 10-d-old rats from all spinal levels. Ganglia were enzymatically dissociated in HBSS supplemented with collagenase type 1A (1.5 mg/ml) and dispase (15 mg/ml; Invitrogen) at 37C for 15C20 min. Cells were then mechanically triturated, washed twice by centrifugation (800 rpm for 5 min), resuspended in 800 l of growth medium, and plated onto glass coverslips coated with poly-d-lysine and laminin. DRG neurons were cultured for 2C5 d in a humidified incubator (37C, 5% CO2) in Pyridoxamine 2HCl DMEM supplemented with GlutaMAX I (Invitrogen), 10% fetal bovine serum, penicillin (50 U/ml), and streptomycin Pyridoxamine 2HCl (50 g/ml). For the inflammatory conditions, cells were incubated with the following inflammatory mediators for 48 h: 1 m bradykinin, 1 m histamine, 1 m ATP, 1 m substance P, and 10 m PAR-2 activating peptide. For patch-clamp experiments, cells were washed twice and media were.
