The voltage gated sodium channel comprises a pore-forming a subunit and regulatory b subunits. We report here the identification and characterization of a novel splicing variant of the human b 1 subunit, termed b 1B . The 807 bp open reading frame of the human b 1B subunit encodes a 268 residue protein with a calculated molecular mass of 30.4 kDa. The novel human b 1B subunit shares an identical N-terminal half (residues 1-149) with the human b 1 subunit, but contains a novel C-terminal half (residues 150-268) of less than 17% sequence identity with the human b 1 subunit. The C-terminal region of the human b 1B is also significantly different from that of the rat b 1A subunit, sharing less than 33% sequence identity. Tissue distribution studies reveal that the human b 1B subunit is expressed predominantly in human brain, spinal cord, dorsal root ganglion and skeletal muscle. Functional studies in oocytes demonstrate that the human b 1B subunit increases the ionic current when coexpressed with the tetrodotoxin sensitive channel, Na V 1.2, without significantly changing voltage dependent kinetics and steady-state properties, thus distinguishing it from the human b 1 and rat b 1A subunits.Keywords: sodium channel; b 1B subunit; splicing variant.By mediating the rapid entry of sodium ions into excitable cells in response to voltage changes across the plasma membrane, voltage gated sodium channels (VGSCs) play a fundamental role in the control of neuronal excitability in the central and peripheral nervous systems. The VGSC is a heteromeric protein complex that comprises at least a large (200-300 kDa) pore-forming a subunit and several smaller (30-40 kDa) regulatory b subunits [1][2][3][4]. It is well known that sodium channel a subunits determine the basic properties of the channel, while b subunits modulate the channel properties. Functional studies in a heterologous system have demonstrated that, depending on the type of coexpressed a subunit, b subunits are able to modulate almost all aspects of the channel properties, including voltage dependent gating, activation and inactivation, as well as greatly increasing the number of functional channels present on the plasma membrane [5,6]. Currently, at least nine different a subunits, three b subunits, and a splicing variant of the b 1 subunit, rat b 1A [7], have been cloned and characterized.The rat b 1A subunit is a splicing variant of the b 1 subunit via intron retention. The N-terminal half of the b 1A subunit is identical to that of the rat b 1 subunit, whereas its C-terminal half, encoded by a retained intron with an in-frame stop codon, is completely different from that of the rat b 1 subunit (to which it shows less than 17% identity). Coexpression of the rat b 1A subunit with the pore forming alpha subunit, Na V 1.2, in Chinese hamster lung 1610 cells, increased the sodium current density and produced subtle changes in voltage dependent activation and inactivation [7]. To further explore the function and physiological relevance of the sodium channel b 1 splicing...
Two mutations in the M2 region of the human á4 neuronal nicotinic subunit -á4(S248F) and á4(776ins3) -have been linked to autosomal dominant nocturnal frontal lobe epilepsy (ADNFLE) (Steinlein et al. 1995(Steinlein et al. , 1997. The á4(S248F) mutation is a serine to phenylalanine substitution at position 248 in the human á4 nicotinic subunit. The á4(776ins3) mutation is a 3-base pair insertion that adds a leucine at position 259 in the amino acid sequence of the human á4 subunit. Photo-affinity labelling and structurefunction experiments show that the M2 region of the nicotinic subunits forms the conducting pore of the receptor (reviewed in Karlin & Akabas, 1995). Thus, both ADNFLE mutations lie in the conducting pore of the nicotinic receptor. ADNFLE patients suffer from brief and occasionally violent nocturnal seizures . However, the physiological mechanism responsible for these seizures has not been established. Previous studies show that the predominant brain nicotinic receptor subtype is á4â2 (Whiting & Lindstrom, 1987;Flores et al. 1991;Whiting et al. 1991). Receptors formed by co-expressing á4(S248F) or á4(776ins3) subunits with wild-type (WT) â2 subunits in Xenopus oocytes (Weiland et al. 1996; Steinlein et al. 1997;Kuryatov et al. 1997) differ from the WT receptor in several ways but no common effects of the two mutations on the acetylcholine (ACh) response have been reported previously. To determine whether the ADNFLE mutations have any common effects on the ACh response, we constructed two rat homologues (S252F and +L264) of the human ADNFLE mutations á4(S248F) and á4(777ins3), co-expressed them with rat â2 subunits in Xenopus oocytes, and studied the properties of the expressed receptors. We also constructed the rat double mutation V247I:S252F, which combined the S252F mutation with a second V247I mutation that converted the only rat/human residue substitution in the á4 M2 region to the corresponding human residue. All three Journal of Physiology (1998) 1. We constructed rat homologues (S252F and +L264) of two human á4 nicotinic mutations -á4(S248F) and á4(777ins3) -that have been linked to autosomal dominant nocturnal frontal lobe epilepsy (ADNFLE) and co-expressed them with wild-type rat â2 subunits in Xenopus oocytes. 2. The S252F and +L264 mutations had three common effects on the ACh response. First, they caused use-dependent potentiation of the response during a train of brief 100 nÒ ACh pulses. Second, they delayed the rise times of the 5-15 nÒ (+L264) and 30 nÒ (S252F) ACh responses. Third, they reduced extracellular Ca¥-induced increases in the 30 ìÒ ACh response. 3. Beside these shared effects, the S252F mutation also reduced the channel burst duration measured from voltage-jump relaxations, enhanced steady-state desensitization and reduced the single-channel conductance. In contrast, the +L264 mutation prolonged the channel burst duration, did not affect desensitization and slightly increased single-channel conductance. Neither mutation affected the number of surface receptors measured b...
1 We studied the pharmacological properties of native rat brain and heterologously expressed rat a4b2 nicotinic receptors immunoprecipitated onto a ®xed substrate with the anti-a4 antibody mAb 299. 2 Immunodepletion with the anti-b2 antibody mAb 270 showed that 89% of the mAb-299-precipitated rat brain receptors contained b2.
In comparison with a control group with ACS, patients who presented with TSCM have higher levels of anxiety but not depression.
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