Robert Denham Pinnock

The sodium channel β‐subunit SCN3b modulates the kinetics of SCN5a and is expressed heterogeneously in sheep heart

1 Cardiac sodium channels are composed of a pore‐forming α‐subunit, SCN5a, and one or more auxiliary β‐subunits. The aim of this study was to investigate the role of the recently discovered member of the β‐subunit family, SCN3b, in the heart. 2 Northern blot and Western blot studies show that SCN3b is highly expressed in the ventricles and Purkinje fibres but not in atrial tissue. This is in contrast to the uniform expression of SCN1b throughout the heart. 3 Co‐expression of SCN3b with the cardiac‐specific α‐subunit SCN5a in Xenopus oocytes resulted in a threefold increase in the level of functional sodium channel expression, similar to that observed when SCN1b was co‐expressed with SCN5a. These results suggest that both SCN1b and SCN3b improve the efficiency with which the mature channel is targeted to the plasma membrane. 4 When measured in cell‐attached oocyte macropatches, SCN3b caused a significant depolarising shift in the half‐voltage of steady‐state inactivation compared to SCN5a alone or SCN5a + SCN1b. The half‐voltage of steady‐state activation was not significantly different between SCN5a alone and SCN5a + SCN3b or SCN5a + SCN1b. 5 The rates of inactivation for SCN5a co‐expressed with either subunit were not significantly different from that for SCN5a alone. However, recovery from inactivation at −90 mV was significantly faster for SCN5a + SCN1b compared to SCN5a + SCN3b, and both were significantly faster than SCN5a alone. 6 Thus, SCN1b and SCN3b have distinctive effects on the kinetics of activation and inactivation, which, in combination with the different patterns of expression of SCN3b and SCN1b, could have important consequences for the integrated electrical activity of the intact heart.

Gabapentin inhibits excitatory synaptic transmission in the hyperalgesic spinal cord.

In the present study we tested the effects of the antihyperalgesic compound gabapentin on dorsal horn neurones in adult spinal cord. Slices were taken from control and hyperalgesic animals suffering from streptozocin‐induced diabetic neuropathy. At concentrations up to 100 μM, bath application failed to affect the resting membrane properties of dorsal horn neurones taken from both groups of animal. In contrast, bath application of gabapentin dramatically reduced the magnitude of the excitatory postsynaptic current (EPSC) in neurones taken from hyperalgesic animals without altering the magnitude of the EPSC in control animals. Using a paired pulse stimulation protocol, together with analysis of miniature EPSCs, it was possible to demonstrate that gabapentin mediated these effects via a pre‐synaptic site of action.

Glucose‐receptive neurones in the rat ventromedial hypothalamus express KATP channels composed of Kir6.1 and SUR1 subunits

1 Patch‐clamp recordings were made from rat ventromedial hypothalamic neurones in slices of brain tissue in vitro. In cell‐attached recordings, removal of extracellular glucose or metabolic inhibition with sodium azide reduced the firing rate of a subpopulation of cells through the activation of a 65 pS channel that was blocked by the sulphonylureas tolbutamide and glibenclamide. 2 In whole‐cell patch‐clamp recordings, in the absence of ATP in the electrode solution, glucose‐receptive neurones gradually hyperpolarized due to the induction of an outward current at ‐60 mV. This outward current and the resultant hyperpolarization were blocked by the sulphonylureas tolbutamide and glibenclamide. 3 In recordings where the electrode solution contained 4 mM ATP, this outward current was not observed. Under these conditions, 500 μM diazoxide was found to induce an outward current that was blocked by tolbutamide. 4 In cell‐attached recordings diazoxide and the active fragment of leptin (leptin 22–56) reduced the firing rate of glucose‐receptive neurones by the activation of a channel with similar properties to that induced by removal of extracellular glucose. 5 Reverse transcription followed by the polymerase chain reaction using cytoplasm from single glucose‐receptive neurones demonstrated the expression of the ATP‐sensitive potassium (KATP) channel subunits Kir6.1 and SUR1 but not Kir6.2 or SUR2. 6 It is concluded that glucose‐receptive neurones within the rat ventromedial hypothalamus exhibit a KATP channel current with pharmacological and molecular properties similar to those reported in other tissues.

Identification of MEK1 as a novel target for the treatment of neuropathic pain

In the present study we have attempted to identify changes in gene expression which are associated with neuropathic pain using subtractive suppression hybridization analysis of the lumbar spinal cord of animals suffering streptozocin induced diabetic neuropathy. Using this approach, we found a significant up‐regulation of several key components of the extracellular signal‐regulated kinase (ERK) cascade. These findings were confirmed by Western blot analysis, which demonstrated that the levels of active ERK1 and 2 correlated with the onset of streptozocin‐induced hyperalgesia. Intrathecal administration of the selective MAPK/ERK‐kinase (MEK) inhibitor PD 198306 dose‐dependently (1–30 μg) blocked static allodynia in both the streptozocin and the chronic constriction injury (CCI) models of neuropathic pain. The antihyperalgesic effects of PD 198306, in both the streptozocin and CCI models of neuropathic pain, correlated with a reduction in the elevated levels of active ERK1 and 2 in lumbar spinal cord. Intraplantar administration of PD 198306 had no effect in either model of hyperalgesia, indicating that changes in the activation of ERKs and the effect of MEK inhibition are localized to the central nervous system. In summary, we have demonstrated for the first time that the development of neuropathic pain is associated with an increase in the activity of the MAPK/ERK‐kinase cascade within the spinal cord and that enzymes in this pathway represent potential targets for the treatment of this condition.

β3, a novel auxiliary subunit for the voltage-gated sodium channel, is expressed preferentially in sensory neurons and is upregulated in the chronic constriction injury model of neuropathic pain

Adult dorsal root ganglia (DRG) have been shown to express a wide range of voltage‐gated sodium channel α‐subunits. However, of the auxiliary subunits, β1 is expressed preferentially in only large‐ and medium‐diameter neurons of the DRG while β2 is absent in all DRG cells. In view of this, we have compared the distribution of β1 in rat DRG and spinal cord with a novel, recently cloned β1‐like subunit, β3. In situ hybridization studies demonstrated high levels of β3 mRNA in small‐diameter c‐fibres, while β1 mRNA was virtually absent in these cell types but was expressed in 100% of large‐diameter neurons. In the spinal cord, β3 transcript was present specifically in layers I/II (substantia gelatinosa) and layer X, while β1 mRNA was expressed in all laminae throughout the grey matter. Since the pattern of β3 expression in DRG appears to correlate with the TTX‐resistant voltage‐gated sodium channel subunit PN3, we co‐expressed the two subunits in Xenopus oocytes. In this system, β3 caused a 5‐mV hyperpolarizing shift in the threshold of activation of PN3, and a threefold increase in the peak current amplitude when compared with PN3 expressed alone. On the basis of these results, we examined the expression of β‐subunits in the chronic constriction injury model of neuropathic pain. Results revealed a significant increase in β3 mRNA expression in small‐diameter sensory neurons of the ipsilateral DRG. These results show that β3 is the dominant auxiliary sodium channel subunit in small‐diameter neurons of the rat DRG and that it is significantly upregulated in a model of neuropathic pain.

Developmental expression of the novel voltage-gated sodium channel auxiliary subunit β3, in rat CNS

1 We have compared the mRNA distribution of sodium channel alpha subunits known to be expressed during development with the known auxiliary subunits Naβ1.1 and Naβ2.1 and the novel, recently cloned subunit, β3. 2 In situ hybridisation studies demonstrated high levels of Nav1.2, Nav1.3, Nav1.6 and β3 mRNA at embryonic stages whilst Naβ1.1 and Naβ2.1 mRNA was absent throughout this period. 3 Naβ1.1 and Naβ2.1 expression occurred after postnatal day 3 (P3), increasing steadily in most brain regions until adulthood. β3 expression differentially decreased after P3 in certain areas but remained high in the hippocampus and striatum. 4 Emulsion‐dipped slides showed co‐localisation of β3 with Nav1.3 mRNA in areas of the CNS suggesting that these subunits may be capable of functional interaction. 5 Co‐expression in Xenopus oocytes revealed that β3 could modify the properties of Nav1.3; β3 changed the equilibrium of Nav1.3 between the fast and slow gating modes and caused a negative shift in the voltage dependence of activation and inactivation. 6 In conclusion, β3 is shown to be the predominant β subunit expressed during development and is capable of modulating the kinetic properties of the embryonic Nav1.3 subunit. These findings provide new information regarding the nature and properties of voltage‐gated sodium channels during development.

Kappa-bungarotoxin blocks an alpha-bungarotoxin-sensitive nicotinic receptor in the insect central nervous system.

Snake venom kappa-neurotoxins are selective antagonists of nicotinic acetylcholine responses in avian, murine and bovine neurons, and have been used as probes for functionally defined vertebrate neuronal nicotinic receptors. The actions of kappa-bungarotoxin, a kappa-neurotoxin, have now been examined at a central invertebrate nicotinic receptor. kappa-Bungarotoxin is a potent antagonist (IC50 = 100 nM) of nicotinic responses, producing a long-lasting blockade of insect nicotinic acetylcholine receptors. The blockade appears to be competitive, and voltage-clamp experiments on an identified cockroach motorneuron indicate that the actions of kappa-bungarotoxin are not dependent on membrane potential. alpha-Bungarotoxin is also a potent antagonist at the cockroach central nicotinic receptor, and binds (Kd = 4.3 nM) to a nicotinic site in cockroach nervous tissue. kappa-Bungarotoxin recognizes this invertebrate nicotinic site with high affinity (Ki = 27 nM). A comparison of the pharmacological properties of insect nicotinic receptors with those of functionally defined receptors identified by kappa-neurotoxins in avian autonomic ganglia reveals several similarities. However, a striking exception is alpha-bungarotoxin, which is the most potent antagonist examined at cockroach nicotinic receptors, but fails to recognize functional autonomic ganglia nicotinic receptors even at very high concentrations. It is concluded that kappa-neurotoxins can be used as selective probes for neuronal nicotinic receptors in both vertebrates and invertebrates. Although invertebrates diverged from vertebrates over 600 million years ago, the results indicate that the neuronal nicotinic receptors found in species as diverse as cockroach and chick retain considerable structural similarity, and thus neuronal nicotinic receptors appear to be highly conserved membrane proteins.

Bombesin-like peptides depolarize rat hippocampal interneurones through interaction with subtype 2 bombesin receptors.

1 Whole‐cell patch‐clamp recordings were made from visually identified hippocampal interneurones in slices of rat brain tissue in vitro. Bath application of the bombesin‐like neuropeptides gastrin‐releasing peptide (GRP) or neuromedin B (NMB) produced a large membrane depolarization that was blocked by pre‐incubation with the subtype 2 bombesin (BB2) receptor antagonist [D‐Phe6,Des‐Met14]bombesin‐(6‐14)ethyl amide. 2 The inward current elicited by NMB or GRP was unaffected by K+ channel blockade with external Ba2+ or by replacement of potassium gluconate in the electrode solution with caesium acetate. 3 Replacement of external NaCl with Tris‐HCl significantly reduced the magnitude of the GRP‐induced current at ‐60 mV. In contrast, replacement of external NaCl with LiCl had no effect on the magnitude of this current. 4 Photorelease of caged GTPγS inside neurones irreversibly potentiated the GRP‐induced current at ‐60 mV. Similarly, bath application of the phospholipase C (PLC) inhibitor U‐73122 significantly reduced the size of the inward current induced by GRP. 5 Reverse transcription followed by the polymerase chain reaction using cytoplasm from single hippocampal interneurones demonstrated the expression of BB2 receptor mRNA together with glutamate decarboxylase (GAD67). 6 Although bath application of GRP or NMB had little or no effect on the resting membrane properties of CA1 pyramidal cells per se, these neuropeptides produced a dramatic increase in the number and amplitude of miniature inhibitory postsynaptic currents in these cells in a TTX‐sensitive manner.

Tachykinins increase [3H]acetylcholine release in mouse striatum through multiple receptor subtypes

Tachykinins have been suggested to play a significant role in the mammalian striatum, at least in part by the control of acetylcholine release from cholinergic interneurons. In the present study, we have examined the ability of known tachykinin agonists and antagonists to modulate the activity of these interneurons in mouse striatal slices. Using whole-cell patch-clamp recordings, the selective neurokinin-1, neurokinin-2 and neurokinin-3 receptor agonists [sar9,Met(O2)11]substance P, [beta-ala8]neurokinin A(4-10) and senktide each produced a dose-dependent depolarization of visually identified cholinergic interneurons that was retained under conditions designed to interrupt synaptic transmission. The nature of these neurons and the expression of multiple tachykinin receptors was confirmed using single-cell reverse transcriptase-polymerase chain reaction analysis. Using in vitro superfusion techniques, the selective neurokinin-1, neurokinin-2 and neurokinin-3 receptor agonists [sar9,Met(O2)11]substance P, [beta-ala8]neurokinin A(4-10) and senktide, respectively, each produced a dose-dependent increase in acetylcholine release, the selectivity of which was confirmed using the neurokinin-1, neurokinin-2 and neurokinin-3 receptor antagonists SR140333, GR94800 and SR142801 (100 nM). U73122 (10 microM), a phospholipase C inhibitor, blocked [sar9,Met(O2)11]substance P- and senktide-induced acetylcholine release, but had no effect on [beta-ala8]neurokinin A(4-10)-induced release. The protein kinase C inhibitors chelerythrine and Ro-31-8220 (both 1 microM) significantly inhibited responses induced by all three agonists. These findings indicate that tachykinins modulate the activity of mouse striatal cholinergic interneurons. Furthermore, neurokinin-2 receptors are shown to perform a role in mouse that has not been identified previously in other species.

Tissue distribution and functional expression of the human voltage-gated sodium channel β3 subunit

Abstract. This study investigated the distribution of β3 in human tissues and the functional effects of the human β3 subunit on the gating properties of brain and skeletal muscle α subunits. Using RT-PCR of human cDNA panels, β3 message was detected in brain, heart, kidney, lung, pancreas and skeletal muscle. Both αIIA and SkM1 expressed in Xenopus oocytes inactivated with a time course described by two exponential components representing fast and slow gating modes, while co-expression of human β3 with αIIA or SkM1 significantly increased the proportion of channels operating by the fast gating mode. In the presence of β3 a greater proportion of αIIA or SkM1 current was described by the fast time constant for both inactivation and recovery from inactivation. β3 caused a hyperpolarizing shift in the voltage dependence of inactivation of αIIA and reduced the slope factor. The voltage dependence of inactivation of SkM1 was described by a double Boltzmann equation. However, SkM1 co-expressed with β3 was described by a single Boltzmann equation similar to one of the Boltzmann components for SkM1 expressed alone, with a small positive shift in V1/2 value and reduced slope factor. This is the first study demonstrating that β3 is expressed in adult mammalian skeletal muscle and can functionally couple to the skeletal muscle α subunit, SkM1.