1. We have compared the mRNA distribution of sodium channel alpha subunits known to be expressed during development with the known auxiliary subunits Nab1.1 and Nab2.1 and the novel, recently cloned subunit, b3. 2.In situ hybridisation studies demonstrated high levels of Nav1.2, Nav1.3, Nav1.6 and b3 mRNA at embryonic stages whilst Nab1.1 and Nab2.1 mRNA was absent throughout this period.3. Nab1.1 and Nab2.1 expression occurred after postnatal day 3 (P3), increasing steadily in most brain regions until adulthood. b3 expression differentially decreased after P3 in certain areas but remained high in the hippocampus and striatum.4. Emulsion-dipped slides showed co-localisation of b3 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 b3 could modify the properties of Nav1.3; b3 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, b3 is shown to be the predominant b 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.

Adult dorsal root ganglia (DRG) have been shown to express a wide range of voltage-gated sodium channel alpha-subunits. However, of the auxiliary subunits, beta1 is expressed preferentially in only large- and medium-diameter neurons of the DRG while beta2 is absent in all DRG cells. In view of this, we have compared the distribution of beta1 in rat DRG and spinal cord with a novel, recently cloned beta1-like subunit, beta3. In situ hybridization studies demonstrated high levels of beta3 mRNA in small-diameter c-fibres, while beta1 mRNA was virtually absent in these cell types but was expressed in 100% of large-diameter neurons. In the spinal cord, beta3 transcript was present specifically in layers I/II (substantia gelatinosa) and layer X, while beta1 mRNA was expressed in all laminae throughout the grey matter. Since the pattern of beta3 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, beta3 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 beta-subunits in the chronic constriction injury model of neuropathic pain. Results revealed a significant increase in beta3 mRNA expression in small-diameter sensory neurons of the ipsilateral DRG. These results show that beta3 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.

1. Single channel current recordings were used to study the characteristics of a large conductance Ca2+-activated K+ (BKCa) channel present in neurones acutely dissociated from the rat motor cortex. Application of ATP to the intracellular surface of excised inside-out patches produced a large, concentration-dependent increase in BKCa channel activity.2. This ATP-mediated activation was dependent upon the presence of Mg2+ in the intracellular bathing solution and was diminished by the phosphatases 2,3-butanedione monoxime (BDM) or alkaline phosphatase and by the protein kinase inhibitors staurosporine, H-7 and PKI.3. ADP stimulated BKCa channel activity in a Mg2+-dependent manner, an action also inhibited by the concomitant application of PKI or BDM. The effect of ADP was reduced by application of hexokinase and glucose or by application of the adenylate kinase inhibitor Ap5A.4. Of other nucleotides tested, only CTP consistently activated BKca channel activity.5. Using the cell-attached configuration, bath application of forskolin or dibutyryl cAMP stimulated BKCa channel activity. 6. It is concluded that BKCa channel activity in the rat motor cortex is subject to modulation by the activity of a closely associated kinase. The ability of cAMP activators to stimulate BKCa channel activity in the intact cell suggests that this system may be of physiological importance.Large conductance Ca2+-activated K+ (BKCa) channels have been identified in many cell types including neurones, smooth and skeletal muscle, kidney tubules, and both endocrine and exocrine glands (Latorre, Oberhauser, Labarca & Alvarez, 1989). Although the precise role of these channels is not clear, they have been implicated in a number of important physiological processes such as the regulation of secretion from endocrine and exocrine glands, the co-ordination of membrane excitability in neurones and the control of K+ ion movement across epithelia.The regulation of BKCa channel activity is complex, with both a rise in intracellular Ca2+ and membrane depolarization producing an increase in channel activity. Additionally, it has recently been shown that conditions which favour protein phosphorylation can also enhance BKca channel activity in a number of tissues (Sadoshima, Akaike, Kanaide & Nakamura, 1988; Kume, Takei, Tokuno & Tomita, 1989). However, the most widely studied example of BKCa channel regulation by phosphorylation has been obtained using synaptosomal membranes from the rat brain incorporated into lipid bilayers (Farley & Rudy, 1988 P(r) = 100 n! pr(l _ p)n r r!(n -r)! where P(r) is the percentage time that 0, 1, 2... n channels are open, n is the total number of channels in the membrane patch, r is the number of channels open and p is the probability that anygiven channel is open.In order to assess the pH sensitivity of BKca channel activity, the data obtained in the presence of varying conditions of intracellular pH were fitted by non-linear regression to the following equation:where P. is the channel activity at the test pH, pK is the ...

No abstract

Whole‐cell patch clamp recordings were made from rat ventromedial hypothalamic neurones in slices of brain tissue in vitro. Bath application of 50 μm (1S, 3R)‐1‐aminocyclopentane‐1,3‐dicarboxylic acid (1S,3R‐ACPD) depolarized all neurones tested by activation of an inward current of approximately 55 pA at −60 mV. The inward current elicited by 1S,3R‐ACPD was unaffected by K+ channel blockade with external Cs+, Ba2+ or TEA. However, the current was significantly reduced by replacement of the external NaCl with either Tris‐HCl or LiCl. The 1S,3R‐ACPD‐induced current was reduced by the heavy metal ions Ni2+ or La3+ and also by the Na+–Ca2+ exchange current inhibitor 3′,4′‐dichlorobenzamil. The effects of 1S,3R‐ACPD were mimicked by the group I metabotropic agonist 3,5‐dihydroxyphenylglycine (DHPG) but not by the group III selective agonist, l‐2‐amino‐4‐phosphonobutanoate (l‐AP4). Furthermore, the effects of 1S,3R‐ACPD were inhibited by the metabotropic antagonists α‐methyl‐4‐carboxyphenylglycine (MCPG) and 1‐aminoindan‐1,5‐dicarboxylic acid (AIDA) but not by the presynaptic metabotropic receptor antagonists α‐methyl‐4‐phosphonophenylglycine (MPPG) or α‐methyl‐4‐tetrazolylphenylglycine (MTPG). Photorelease of caged GDPβS inside neurones irreversibly blocked the 1S,3R‐ACPD‐induced current whilst photolysis of caged GTPγS inside neurones irreversibly potentiated this current. The PLC inhibitor U‐73122 significantly reduced the size of the inward current induced by 1S,3R‐ACPD. This effect was not mimicked by the inactive analogue U‐73343. Flash photolysis of the caged calcium chelator diazo‐2 inside neurones diminished the response to 1S,3R‐ACPD. It is concluded that group I metabotropic glutamate receptors depolarize neurones in the VMH by activation of a Na+–Ca2+ exchange current through a G‐protein coupled increase in intracellular Ca2+.