Cojen Ho

Single-cell analysis of sodium channel expression in dorsal root ganglion neurons

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, and Nav1.7) and TTX-R (Nav1.8 and 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, and 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, and Nav1.7) and small unmyelinated (Nav1.7, Nav1.8, and Nav1.9) sensory neurons.

Interaction of alcohols and anesthetics with protein kinase Calpha.

The key signal transduction enzyme protein kinase C (PKC) contains a hydrophobic binding site for alcohols and anesthetics (Slater, S. J., Cox, K. J. A., Lombardi, J. V., Ho, C., Kelly, M. B., Rubin, E., and Stubbs, C. D. (1993) Nature 364, 82-84). In this study, we show that interaction of n-alkanols and general anesthetics with PKCα results in dramatically different effects on membrane-associated compared with lipid-independent enzyme activity. Furthermore, the effects on membrane-associated PKCα differ markedly depending on whether activity is induced by diacylglycerol or phorbol ester and also on n-alkanol chain length. PKCα 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 PKCα activity. This effect of long chain n-alkanols was similar to that of diacylglycerol, although the n-alkanols 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.

Polyunsaturation in cell membranes and lipid bilayers and its effects on membrane proteins.

The effect of variation of the degree ofcis-unsaturation on cell membrane protein functioning was investigated using a model lipid bilayer system and protein kinase C (PKC). This protein is a key element of signal transduction. Furthermore it is representative of a class of extrinsic membrane proteins that show lipid dependent interactions with cell membranes. To test for dependence of activity on the phospholipid unsaturation, experiments were devised using a vesicle assay system consisting of phosphatidylcholine (PC) and phosphatidylserine (PS) in which the unsaturation was systematically varied. Highly purified PKCα and ε were obtained using the baculovirus-insect cell expression system. It was shown that increased PC unsaturation elevated the activity of PKCα. By contrast, increasing the unsaturation of PSdecreased the activity of PKCα, and to a lesser extent PKCε. This result immediately rules out any single lipid bilayer physical parameter, such as lipid order, underlying the effect. It is proposed that while PC unsaturation effects are explainable on the basis of a contribution to membrane surface curvature stress, the effects of PS unsaturation may be due to specific protein-lipid interactions. Overall, the results indicate that altered phospholipid unsaturation in cell membranes that occurs in certain disease states such as chronic alcoholism, or by dietary manipulations, are likely to have profound effects on signal transduction pathways involving PKC and similar proteins.

Fluorescence techniques for probing water penetration into lipid bilayers

Fluorescence spectroscopy can be used as a highly sensitive and localized probe for hydration in lipid bilayers. Water associates with the head-group region, where it participates in an interlipid network of hydrogen bonds. Deeper in the bilayer, water is contained within acyl-chain packing defects. Fluorescence methodology is available to probe both the interstitial and head-group hydration in lipid bilayers, and results are in good agreement with other techniques. Using fluorescence spectroscopic approaches, cholesterol is shown to dehydrate the acyl-chain region, while hydrating the head-group region. Membrane proteins appear to increase acyl-chain hydration at the protein-lipid interface. Overall fluorescence spectroscopic techniques may be most effective in studying the water content of lipid bilayers and especially of biological membranes.

Modulation of Kv3.4 channel N-type inactivation by protein kinase C shapes the action potential in dorsal root ganglion neurons

Non‐technical summary  The orchestrated activity of an ensemble of voltage‐gated ion channels determines the initiation, shape and duration of the action potential in excitable cells. In primary pain‐sensing neurons, this ensemble includes a high voltage‐activated potassium channel. However, its molecular identity, function and modulation were unknown. Here, we show that the rapidly inactivating Kv3.4 channel underlying the high voltage‐activated potassium current is a major determinant of action potential repolarization. Furthermore, we found that physiological activation of protein kinase C dramatically slows Kv3.4 channel inactivation, which enhances the channels ability to influence action potential repolarization. Based on these results and earlier work, we conclude that phosphorylation of the Kv3.4 channel inactivation gate is a mechanism by which pain‐sensing neurons shape action potential repolarization. This modulation will influence Ca2+‐dependent processes that play vital roles in nociception and might become deregulated in chronic pain.

Inhibition of membrane lipid-independent protein kinase Calpha activity by phorbol esters, diacylglycerols, and bryostatin-1.

The activity of membrane-associated protein kinase C (PKC) has previously been shown to be regulated by two discrete high and low affinity binding regions for diacylglycerols and phorbol esters (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). PKC is also known to interact with both cytoskeletal and nuclear proteins; however, less is known concerning the mode of activation of this non-membrane form of PKC. By using the fluorescent phorbol ester, sapintoxin D (SAPD), PKCα, alone, was found to possess both low and high affinity phorbol ester-binding sites, showing that interaction with these sites does not require association with the membrane. Importantly, a fusion protein containing the isolated C1A/C1B (C1) domain of PKCα also bound SAPD with low and high affinity, indicating that the sites may be confined to this domain rather than residing elsewhere on the enzyme molecule. Both high and low affinity interactions with native PKCα were enhanced by protamine sulfate, which activates the enzyme without requiring Ca2+ or membrane lipids. However, this “non-membrane” PKC activity was inhibited by the phorbol ester 4β-12-O-tetradecanoylphorbol-13-acetate (TPA) and also by the fluorescent analog, SAPD, opposite to its effect on membrane-associated PKCα. Bryostatin-1 and the soluble diacylglycerol, 1-oleoyl-2-acetylglycerol, both potent activators of membrane-associated PKC, also competed for both low and high affinity SAPD binding and inhibited protamine sulfate-induced activity. Furthermore, the inactive phorbol ester analog 4α-TPA (4α-12-O-tetradecanoylphorbol-13-acetate) also inhibited non-membrane-associated PKC. In keeping with these observations, although TPA could displace high affinity SAPD binding from both forms of the enzyme, 4α-TPA was only effective at displacing high affinity SAPD binding from non-membrane-associated PKC. 4α-TPA also displaced SAPD from the isolated C1 domain. These results show that although high and low affinity phorbol ester-binding sites are found on non-membrane-associated PKC, the phorbol ester binding properties change significantly upon association with membranes.

Differential Expression of Sodium Channel β Subunits in Dorsal Root Ganglion Sensory Neurons

Background: Auxiliary β subunits regulate the voltage-gated sodium channels of dorsal root ganglion (DRG) neurons. Results: β subunits are differentially expressed in subpopulations of DRG neurons and regulate Nav1.7 channels in an isoform-specific manner. Conclusion: Differential β subunit expression and isoform-specific regulation have important implications for the sodium currents of DRG neurons. Significance: β subunits are important determinants of sodium channel function and sensory neuron excitability. The small-diameter (<25 μm) and large-diameter (>30 μm) sensory neurons of the dorsal root ganglion (DRG) express distinct combinations of tetrodotoxin sensitive and tetrodotoxin-resistant Na+ channels that underlie the unique electrical properties of these neurons. In vivo, these Na+ channels are formed as complexes of pore-forming α and auxiliary β subunits. The goal of this study was to investigate the expression of β subunits in DRG sensory neurons. Quantitative single-cell RT-PCR revealed that β subunit mRNA is differentially expressed in small (β2 and β3) and large (β1 and β2) DRG neurons. This raises the possibility that β subunit availability and Na+ channel composition and functional regulation may differ in these subpopulations of sensory neurons. To further explore these possibilities, we quantitatively compared the mRNA expression of the β subunit with that of Nav1.7, a TTX-sensitive Na+ channel widely expressed in both small and large DRG neurons. Nav1.7 and β subunit mRNAs were significantly correlated in small (β2 and β3) and large (β1 and β2) DRG neurons, indicating that these subunits are coexpressed in the same populations. Co-immunoprecipitation and immunocytochemistry indicated that Nav1.7 formed stable complexes with the β1–β3 subunits in vivo and that Nav1.7 and β3 co-localized within the plasma membranes of small DRG neurons. Heterologous expression studies showed that β3 induced a hyperpolarizing shift in Nav1.7 activation, whereas β1 produced a depolarizing shift in inactivation and faster recovery. The data indicate that β3 and β1 subunits are preferentially expressed in small and large DRG neurons, respectively, and that these auxiliary subunits differentially regulate the gating properties of Nav1.7 channels.

The effects of phospholipid unsaturation and alcohol perturbation at the protein/lipid interface probed using fluorophore lifetime heterogeneity

The influence of phospholipid unsaturation and perturbation by alcohols, on the membrane protein/lipid interface, was probed using the fluorescence decay properties of 1,6-diphenyl-1,3,5-hexatriene (DPH) and DPH attached to the sn-2 chain of phosphatidylcholine (DPH-PC), in lipid bilayers and microsomal membranes. With microsomal membranes it was found that it was appropriate to describe the fluorescence decay of DPH-PC as a range of decay rates, accomplished by fitting the data to a bimodal fluorescence lifetime distribution. The major lifetime center had a broad distributional width, indicative of excited state fluorophore heterogeneity. The effect was attributable to protein, and by inference, the protein/lipid interface, since in vesicles made from total microsomal lipids (i.e., without protein) the fluorescence decay was homogeneous. Upon addition of ethanol or hexanol the width of the lifetime distribution of the major lifetime center increased, indicating increased environmental heterogeneity. It was confirmed that the effect was manifest at the protein/lipid interface, and not due to lipid-reorganizational factors, since it could also be obtained using a simple lipid bilayer vesicle system with apocytochrome c as a model membrane protein, and DPH instead of DPH-PC. Environmental heterogeneity was also found to increase with increased phosphatidylcholine (sn-2) unsaturation. The environmental heterogeneity at the protein/lipid interface could arise from a combination of varying polarities of amino acid side chains and of water that may intercalate in packing defects on the hydrophobic surface of the protein. Therefore the results could be explained on the basis of an increased degree of hydration at the protein/lipid interface. Such an effect offers a route whereby acyl chain perturbation and increased unsaturation might influence protein conformation and hence function.

Analysis of cell membrane micro-heterogeneity using the fluorescence lifetime of DPH-type fluorophores

Heterogeneity in the lipid organization in lipid bilayers and cell membranes was probed by using the fluorescence decay of 1,6-diphenyl-1,3,5-hexatriene (DPH) and DPH attached to the sn-2 position of phosphatidylcholine (DPH-PC). In the presence of protein, it is proposed that the bulk lipids and boundary lipids can potentially provide distinct enough fluorophore environments for two different lifetime centers to be recovered from the analysis of the fluorescence decay. To test this model experiments were performed with cytochrome b5 in 1-palmitoyl-2-oleoylphosphatidylcholine bilayers. The number of boundary lipids of cytochrome b5 is known from the literature or can be calculated from known dimensions, so that for a known protein:lipid ratio the fraction of lipids in the bulk and boundary lipid regions is known. These values were found to closely correspond to the fractions associated with the lifetime centers recovered from an analysis of the fluorescence decay assuming two major fluorophore populations. This indicated that the DPH distributed in a similar manner to the lipids and that its boundary lipid residency time was greater than the excited state lifetime, showing the validity of the approach. An important requirement was that the protein should influence the fluorophore decay sufficiently enough to enable separate lifetime centers for the bulk and boundary lipid fluorophores to be recovered by the analysis. Attempts were made to analyze the fluorescence decay of DPH in liver plasma membranes and microsomes as arising from two distinct fluorophore populations, however, the basic condition was not satisfied. By contrast, using DPH-PC it was possible to extract two separate lifetime centers. The limitations and potential of this approach are critically assessed and it is concluded that in certain circumstances information pertaining to the protein-lipid interfacial region of membranes can be extracted from fluorescence decay heterogeneity properties.

The use of fluorescent phorbol esters in studies of protein kinase C-membrane interactions.

The family of protein kinase C (PKC) isozymes belongs to a growing class of proteins that become active by associating with membranes containing anionic phospholipids, such as phosphatidylserine. Depending on the particular PKC isoform, this process is mediated by Ca(2+)-binding to a C2 domain and interaction of activators such as 1,2-diacyl-sn-glycerol or phorbol esters with tandem C1 domains. This cooperation between the C1 and C2 domains in inducing the association of PKC with lipid membranes provides the energy for a conformational change that consists of the release of a pseudosubstrate sequence from the active site, culminating in activation. Thus, the properties of the interactions of the C1 and C2 domains with membranes, both as isolated domains, and as modules in the full length PKC isoforms, have been the subject of intense scrutiny. Here, we review the findings of studies in which fluorescent phorbol esters have been utilized to probe the properties of the C1 domains of PKC with respect to the interaction with activators, the subsequent interaction with membranes, and the role of the activating conformational change that leads to activation.