Sensory neurons of the dorsal root ganglia (DRG) express multiple voltage-gated sodium (Na) channels that substantially differ in gating kinetics and pharmacology. Small-diameter (<25 µm) neurons isolated from the rat DRG express a combination of fast tetrodotoxin-sensitive (TTX-S) and slow TTX-resistant (TTX-R) Na currents while large-diameter neurons (>30 µm) predominately express fast TTX-S Na current. Na channel expression was further investigated using single-cell RT-PCR to measure the transcripts present in individually harvested DRG neurons. Consistent with cellular electrophysiology, the small neurons expressed transcripts encoding for both TTX-S (Nav1.1, Nav1.2, Nav1.6, Nav1.7) and TTX-R (Nav1.8, Nav1.9) Na channels. Nav1.7, Nav1.8 and Nav1.9 were the predominant Na channels expressed in the small neurons. The large neurons highly expressed TTX-S isoforms (Nav1.1, Nav1.6, Nav1.7) while TTX-R channels were present at comparatively low levels. A unique subpopulation of the large neurons was identified that expressed TTX-R Na current and high levels of Nav1.8 transcript. DRG neurons also displayed substantial differences in the expression of neurofilaments (NF200, peripherin) and Necl-1, a neuronal adhesion molecule involved in myelination. The preferential expression of NF200 and Necl-1 suggests that large-diameter neurons give rise to thick myelinated axons. Small-diameter neurons expressed peripherin, but reduced levels of NF200 and Necl-1, a pattern more consistent with thin unmyelinated axons. Single-cell analysis of Na channel transcripts indicates that TTX-S and TTX-R Na channels are differentially expressed in large myelinated (Nav1.1, Nav1.6, Nav1.7) and small unmyelinated (Nav1.7, Nav1.8, Nav1.9) sensory neurons.

The relationship between membrane lipid bilayer hydration and acyl chain order was investigated using time-resolved fluorescence spectroscopy. The degree of hydration in the head group region was assessed from fluorescence lifetime data along with fluorescence intensity measurements in D2O, relative to H2O buffer, using N-(5-dimethylaminonaphthalene-1-sulfonyl)dipalmitoylphosphatidylethan ola mine (dansyl-PE). The degree of hydration in the acyl chain region was estimated from its effect on the fluorescence lifetime of 1-palmitoyl-2-[[2-[4-(6-phenyl-trans-1,3,5-hexatrienyl)phenyl]ethyl] carbonyl]-3-sn-phosphatidylcholine (DPH-PC), and acyl chain order was determined from time-resolved anisotropy measurements of the DPH-PC. Comparisons of sn-2 unsaturation with sn-1,2 diunsaturation in phosphatidylcholine (PC) bilayers with the same number of double bonds/PC revealed a marked difference in interchain hydration and acyl chain order but little difference in terms of head group hydration. For diunsaturated dioleoyl-PC (DOPC) bilayers with two double bonds/PC, the DPH-PC fluorescence lifetime data indicated a greater level of interchain hydration than 1-palmitoyl-2-docosahexaenoyl-PC (PDPC) with six double bonds/sn-2 chain. By contrast, the head group hydration for DOPC was markedly less than for PDPC. A similar lack of correlation of effects on the two regions of the bilayer was found with cholesterol, it having opposite effects on interchain and head group hydration. When DPH-PC fluorescence lifetime data for bilayers composed of a range of different lipids was plotted as a function of acyl chain order, a strong correlation of interchain hydration with acyl chain order was revealed that was independent of lipid composition.(ABSTRACT TRUNCATED AT 250 WORDS)

The key metabolite of vitamin D3, 1 alpha,25-dihydroxyvitamin D3 (1,25-D3), induces rapid cellular responses that constitute a so-called "non-genomic" response. This effect is distinguished from its "classic" genomic role in calcium homeostasis involving the nuclear 1,25-D3 receptor. Evidence is presented that protein kinase C (PKC) is directly activated by 1,25-D3 at physiological concentrations (EC50 = 16 +/- 1 nM). The effect was demonstrable with single PKC-alpha, -gamma, and -epsilon isoform preparations, assayed in a system containing only purified enzyme, substrate, co-factors, and lipid vesicles, from which it is inferred that a direct interaction with the enzyme is involved. The finding that calcium-independent isoform PKC-epsilon was also activated by 1,25-D3 shows that the calcium binding C2 domain is not required. The level of 1,25-D3-induced activation, paired with either diacylglycerol or 4 beta-12-O-tetradecanoylphorbol-13-acetate, was greater than that achievable by any individual activator alone, each at a saturating concentration, a result that implies two distinct activator sites on the PKC molecule. Phosphatidylethanolamine present in the lipid vesicles potentiated 4 beta-12-O-tetradecanoylphorbol-13-acetate- and diacylglycerol-induced PKC activities, whereas 1,25-D3-induced activity decreased, consistent with 1,25-D3-activated PKC possessing a distinct conformation. The results suggest that PKC is a "membrane-bound receptor" for 1,25-D3 and that it could be important in the control of non-genomic cellular responses to the hormone.

Despite almost a century of research, the mechanism of anaesthesia remains obscure and there is still no agreement on the location of the site(s) of action. Because the potencies of general anaesthetics increase in proportion to their solubility in olive oil, this led to a consensus that the site is within the cell membrane. This led to theories that lipid bilayer perturbation was the primary event, which was then transmitted to a membrane protein. But at the concentrations used clinically, such perturbations are small. A plausible site would be in or on ion channels at the synapse, where a number of modulatory effects have been described. A possible location for such a site would be at the protein-lipid interface. We report here that anaesthetics inhibit protein kinase C, a key component in signal transduction. The potency is a linear function of the octanol-water partition coefficient (the Meyer-Overton rule of anaesthesia). The effect was obtained in a lipid-free assay, implicating a hydrophobic site in the protein, supporting the contention that a (membrane) protein may be a target for anaesthetic interactions. In a lipid-dependent assay, a potential role of lipids in the protein-site model was demonstrated. The inhibition was absent in the isolated catalytic domain, suggesting that the site of inhibition is on the regulatory subunit, which is unique to protein kinase C.

Nature 364, 82-84). In this study, we show that interaction of n-alkanols and general anesthetics with PKC␣ results in dramatically different effects on membraneassociated compared with lipid-independent enzyme activity. Furthermore, the effects on membrane-associated PKC␣ differ markedly depending on whether activity is induced by diacylglycerol or phorbol ester and also on n-alkanol chain length. PKC␣ contains two distinct phorbol ester binding regions of low and high affinity for the activator, respectively (Slater, S. J., Ho, C., Kelly, M. B., Larkin, J. D., Taddeo, F. J., Yeager, M. D., and Stubbs, C. D. (1996) J. Biol. Chem. 271, 4627-4631). Short chain n-alkanols competed for low affinity phorbol ester binding to the enzyme, resulting in reduced enzyme activity, whereas high affinity phorbol ester binding was unaffected. Long chain n-alkanols not only competed for low affinity phorbol ester binding but also enhanced high affinity phorbol ester binding. Furthermore, long chain n-alkanols enhanced phorbol ester induced PKC␣ activity. This effect of long chain n-alkanols was similar to that of diacylglycerol, although the nalkanols alone were weak activators of the enzyme. The cellular effects of n-alkanols and general anesthetics on PKC-mediated processes will therefore depend in a complex manner on the locality of the enzyme (e.g. cytoskeletal or membrane-associated) and activator type, apart from any isoform-specific differences. Furthermore, effects mediated by interaction with the region on the enzyme possessing low affinity for phorbol esters represent a novel mechanism for the regulation of PKC activity.