Biomedical subjects
Publications and source records attributed to E W McCleskey.
Every organism with a nervous system can detect changes in temperature. Recent studies on sensory neurons from rats and genetic evidence from nematodes have provided intriguing hints about the molecular basis of thermosensation.
The initial pain from tissue damage may result from the release of cytoplasmic components that act upon nociceptors, the sensors for pain. ATP was proposed to fill this role because it elicits pain when applied intradermally and may be the active compound in cytoplasmic fractions that cause pain. Moreover, ATP opens ligand-gated ion channels (P2X receptors) in sensory neurons and only sensory neurons express messenger RNA for the P2X3 receptor. To test whether ATP contributes to nociception, we developed a tissue culture system that allows comparison of nociceptive (tooth-pulp afferent) and non-nociceptive (muscle-stretch receptor) rat sensory neurons. Low concentrations of ATP evoked action potentials and large inward currents in both types of neuron. Nociceptors had currents that were similar to those of heterologously expressed channels containing P2X3 subunits, and had P2X3 immunoreactivity in their sensory endings and cell bodies. Stretch receptors had currents that differed from those of P2X3 channels, and had no P2X3 immunoreactivity. These results support the theory that P2X3 receptors mediate a form of nociception, but also suggest non-nociceptive roles for ATP in sensory neurons.
We have shown the presence and activity of ATP-gated ion channels (P2X receptors) in nociceptive nerve endings, supporting the theory that these channels mediate some forms of nociception [Cook S.P., Vulchanova L., Hargreaves K. M., Elde R. and McCleskey E. W. (1997) Distinct ATP receptors on pain-sensing and stretch-sensing neurons. Nature 387, 505-508]. The kinetics and pharmacology of ATP-gated currents in nociceptors suggest that the channels are comprised of either homomeric or heteromeric combinations of P2X3 receptors. Consistent with the diverse nature of P2X structure, electrophysiological responses of rat tooth-pulp nociceptors fall into two distinct classes based on desensitization and recovery kinetics. Here, we quantified the dramatic differences in desensitization kinetics of transient and persistent currents. The major component of transient P2X current desensitized with a tau decay = 32 +/- 2 msec, while persistent current desensitized > 100-fold more slowly, tau decay = 4000 +/- 320 msec. Both currents recovered from desensitization in minutes: tau recovery = 4 min for transient current, and tau recovery = 0.7 +/- 0.2 min for persistent current. Persistent current recovery was often accompanied by a current "overrecovery" that averaged ca threefold magnitude prior to desensitization. Comparison of ATP current in elevated Ca2+ext also revealed differences in transient and presistent currents. In 2 mM Ca2+ext medium, decrease of Na+ext resulted in an almost complete reduction of persistent, but not transient, current. Subsequent elevation of Ca2+ext greatly increased the transient, but not persistent, current. Mechanistic explanations for either the increase in transient current magnitude by elevated Ca2+ext, or persistent current overrecovery may reflect endogenous pathways for P2X receptor modulation.
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Opioids have been shown to cause a potent inhibition of neurons in the locus ceruleus (LC) in vivo in brain slices and isolated neurons; however, the kinetics of opioid action have not been described. In this study, we used acutely isolated LC neurons to examine opioid and alpha2-adrenoceptor action on potassium and calcium currents. [Met]Enkephalin (ME), [D-Ser2,Leu5,Thr6]-enkephalin, etorphine, and [D-Ala2,N-Me-Phe4,Gly-ol5]enkephalin increased potassium conductance, whereas morphine and naloxone were antagonists. The time constant of potassium channel activation was approximately 0.7 sec and was the same for each agonist. The amplitude of the current and the time constant of decay were dependent on the agonist, suggesting that agonist efficacy and affinity, respectively, determined these parameters. The amplitude of potassium current induced by the alpha2-adrenoceptor agonist UK14304 was not significantly different from that induced by ME, but the time constant of current activation was half that of ME, and the decline was more rapid. When potassium conductances were blocked with the combination of internal cesium and external barium, opioid and alpha2 agonists had no effect at potentials more negative than -50 mV and decreased barium currents at potentials between -40 and +20 mV. Both morphine and clonidine caused a small inhibition of barium current. In dorsal root ganglion cells, morphine alone had small and inconsistent effects on the calcium current, but it always competitively antagonized the inhibition caused by [D-Ala2,N-Me-Phe4,Gly-ol5]enkephalin. The results in isolated LC neurons suggest 1) the amplitude and time course of the opioid-induced potassium current depend on agonist efficacy and affinity and 2) the coupling of both mu-opioid and alpha2-adrenoceptors to calcium channels seems to be more efficient than that to potassium channels.
Growth of the human malaria parasite, Plasmodium falciparum, within the red blood cell (RBC) requires external Ca++ and is associated with a markedly elevated intracellular Ca++ concentration, [Ca++]i. We used 45Ca++ flux studies and patch clamp recordings to examine the mechanisms responsible for this increased [Ca++]i. The 45Ca++ flux studies indicated that net Ca++ entry into parasitized RBCs (PRBCs) is 18 times faster than into unparasitized ATPase that keeps the [Ca++]i of unparasitized RBCs exceedingly low. Acceleration of the preexisting Ca++ entry, ATPase that keeps the [Ca++] of unparasitized RBCs exceedingly low. Acceleration of the preexisting Ca++ entry, mediated by a divalent cation carrier, also cannot explain Ca++ accumulation in PRBCs: there are fundamental differences in substrate preference and in the effects of external Ca++ on 45Ca++ efflux between unparasitized RBCs and PRBCs. Patch clamp of intact PRBC surface membranes revealed rare unitary channel openings not observed on unparasitized RBCs. With 80 mM of CaCl2 in the patch pipette, this channel carried inward current, suggesting Ca++ entry at a rate comparable with the observed 45Ca++ flux. These data indicate that the malaria parasite induces a novel pathway in the host RBC membrane for Ca++ entry and suggest that this pathway is a Ca++-permeable channel.
Opioid analgesia, the selective suppression of pain without effects on other sensations, also distinguishes between different types of pain: severe, persistent pain is potently inhibited by opioids, but they fail to cohceal the sensation of a pinprick. The cellular basis for this specificity was analyzed by means of patch-clamp experiments performed on fluorescently labeled nociceptive neurons (nociceptors) that innervate rat tooth pulp. Activation of the mu opioid receptor inhibited calcium channels on almost all small nociceptors but had minimal effect on large nociceptors. Somatostatin had the opposite specificity, preferentially inhibiting calcium channels on the large cells. Because persistent pain is mediated by slow-conducting, small nociceptors, opioids are thus likely to inhibit neurotransmitter release only at those primary synapses specialized for persistent pain.
A crude extract from ginseng root inhibits high-threshold, voltage-dependent Ca2+ channels through an unknown receptor linked to a pertussis toxin-sensitive G protein. We now have found the particular compound that seems responsible for the effect: it is a saponin, called ginsenoside Rf (Rf), that is present in only trace amounts within ginseng. At saturating concentrations, Rf rapidly and reversibly inhibits N-type, and other high-threshold, Ca2+ channels in rat sensory neurons to the same degree as a maximal dose of opioids. The effect is dose-dependent (half-maximal inhibition: 40 microM) and it is virtually eliminated by pretreatment of the neurons with pertussis toxin, an inhibitor of G(o) and Gi GTP-binding proteins. Other ginseng saponins--ginsenosides Rb1, Rc, Re, and Rg1--caused relatively little inhibition of Ca2+ channels, and lipophilic components of ginseng root had no effect. Antagonists of a variety of neurotransmitter receptors that inhibit Ca2+ channels fail to alter the effect of Rf, raising the possibility that Rf acts through another G protein-linked receptor. Rf also inhibits Ca2+ channels in the hybrid F-11 cell line, which might, therefore, be useful for molecular characterization of the putative receptor for Rf. Because it is not a peptide and it shares important cellular and molecular targets with opioids, Rf might be useful in itself or as a template for designing additional modulators of neuronal Ca2+ channels.
1. Using patch-clamp methods, we show that brief prepulses to very positive voltages increase (facilitate) the amplitude of current through Ca2+ channels during a subsequent test pulse in some, but not all, dorsal root ganglion (DRG) sensory neurons. The amplitude of this facilitated current generally increases when the Ca2+ channels are inhibited by activation of the mu-opioid receptor. 2. The facilitated current is blocked by omega-conotoxin GVIA, activates in the range of high-threshold Ca2+ channels, and inactivates at relatively negative holding voltages. Thus facilitated current passes through N-type Ca2+ channels, the same channels that are inhibited by opioids and control neurotransmitter release in sensory neurons. 3. Although maximal facilitation occurs only at unphysiologically high membrane potentials (above +100 mV), some facilitation is seen after prepulses to voltages reached during action potentials. After return to the holding potential, facilitation persists for hundreds of milliseconds, considerably longer than in other neurons. Brief trains of pulses designed to mimic action potentials caused small facilitation (19% of maximal) in a fraction (8 of 24) of opioid-inhibited neurons. 4. We conclude that 1) prepulses to extremely positive voltages can cause partial recovery of Ca2+ channels inhibited by opioids; and 2) small, but detectable, facilitation is also seen after physiological stimulation in some DRG neurons. Facilitation, largely considered a biophysical epiphenomenon because of the extreme voltages used to induce it, appears to be physiologically relevant during opioid inhibition of Ca2+ channels in DRG neurons.
We used patch-clamp methods to describe signal transduction between the mu opioid receptor, the binding site for morphine, and high-threshold Ca2+ channels in dorsal root ganglion (DRG) sensory neurons from adult rats. Opioid signaling persists in excised membrane patches, and an activated opioid receptor can only inhibit nearby Ca2+ channels; thus, no readily diffusible second-messenger molecule mediates between the mu receptor and Ca2+ channels. Inhibition of Ca2+ channels begins several hundred msec after application of opioid and it is maximal by 5 sec; this is faster than typical phosphorylation cascades. Blockade of the known serine-threonine kinases and phosphatases does not affect this opioid signaling and, as shown previously by Seward et al. (1991) and Moises et al. (1994a), pertussis toxin eliminates virtually all of the effect. Inhibited channels can open, but their half-activation voltage is unphysiologically positive. The link between the mu receptor and Ca2+ channels is clearly unlike the protein kinase C-dependent paths that couple mu receptors to NMDA channels in dorsal horn neurons (Chen and Huang, 1991) and alpha-adrenergic receptors to Ca2+ channels in DRG neurons (Diversé-Pierluissi and Dunlap, 1993). The rapid kinetics and tight localization of the signaling path are properties expected if receptor and channel are linked directly by a G-protein, but these properties do not constitute proof of such a pathway.
The effect of Panax ginseng root extract on Ca2+ current of adult rat trigeminal ganglion neurons was investigated using whole-cell patch-clamp methods. The application of P. ginseng root extract (100 micrograms/ml) produced rapid, reversible reduction of the Ca2+ current by 22 +/- 4%. Treatment with pertussis toxin (250 ng/ml) for 16 h reduced the inhibition to 4 +/- 1%. The continual presence of 1 microM DAGO, a selective mu-opioid agonist that inhibits Ca2+ channels, occluded further inhibition of Ca2+ current by P. ginseng root extract. Yohimbine, phaclofen, atropine, and naloxone--antagonists of alpha 2-adrenergic, GABAB, muscarinic, and opiate receptors, respectively--did not block the inhibitory effect on Ca2+ current of P. ginseng root extract. Thus, P. ginseng root extract acts on sensory neurons through a similar pathway as mu-type opioids: both inhibit Ca2+ channels through pertussis toxin-sensitive GTP-binding proteins. However, the receptor for P. ginseng root extract is not an alpha 2-adrenergic, GABAB, muscarinic, or opioid receptor.
An unexpected variety of different types of Ca2+ channels have been identified using molecular cloning and selective Ca2+ channel toxins. A main focus of current research in this field is to characterize these different channel types and the molecular mechanisms by which Ca2+ channels perform their basic activities of gating, selectivity and modulation. Recent advances demonstrate the roles of different types of Ca2+ channels in muscle and nerve, and provide hints about the structures involved in selectivity and gating.
During its 48-hour cycle inside the red blood cell, the human malaria parasite, Plasmodium falciparum, increases its volume 25-fold and divides asexually. This rapid growth demands large amounts of nutrients, a problem exacerbated by the lower metabolic rate and relative ionic impermeability of the host red blood cell. Direct passage of small nutrients across the two membranes that separate the parasite from the erythrocyte cytosol may be important for parasite development and has been demonstrated for radiolabelled glucose, amino acids, and purine nucleosides. Flux studies on plasmodia are limited, however, to suspensions of erythrocyte-free parasites and so cannot be used to examine the individual transport properties of the two membranes involved. Here we use the cell-attached patch clamp method to overcome this limitation. After removing the intervening red blood cell membrane and forming gigaohm seals on the small (3-5 microns) parasite, we studied transport across the parasitophorous vacuole membrane (PVM), the outer of the two membranes that separate the parasite from the erythrocyte cytosol. A 140-pS channel which is permeable to both cations and anions was identified on the PVM. This channel is present at high density, is open more than 98 per cent of the time at the resting potential of the PVM, and is permeable to lysine and glucuronate. The channel can readily transport amino acids and monosaccharides across the PVM and may be essential for fulfilling the parasite's metabolic demands.
Using the whole-cell patch-clamp technique on acutely dissociated and cultured adult rat sensory neurons, we characterized the K+ currents by voltage dependence, kinetics, calcium dependence, and pharmacology. In the presence of Ca channel blockers, the cells heterogeneously expressed transient and sustained outward K+ currents. The transient current was a high-threshold A-current which activated at potentials greater than -30 mV and was blocked by 4-aminopyridine. Some of the sustained current was classified as a delayed rectifier. It demonstrated shallow voltage-dependent inactivation and was blocked by tetraethylammonium. Capsaicin produced large reductions in both transient and sustained currents with an EC50 of 8 microM. Likewise, dendrotoxin partially blocked both currents but with an EC50 of 21 nM. In the absence of Ca channel blockers, a prominent Ca-dependent K+ current was observed. The kinetics of whole-cell potassium currents varied widely among cells, perhaps reflecting the different functional properties of sensory neurons. We also investigated the effects of elevating intracellular cyclic AMP and applying opioids on K+ currents. Membrane-permanent analogs of cyclic AMP and phosphodiesterase inhibitors caused small reductions in voltage-dependent outward current. In contrast, forskolin produced a large reduction in outward current. This response was not solely mediated by cyclic AMP, since large responses were elicited with an inactive congener, 1,9-dideoxyforskolin, but not with the active, water-soluble congener, 7-deacetyl-6-[N-acetylglycyl]-forskolin. Surprisingly, opioids had no effect on resting or voltage-dependent K+ conductances. However, opioid inhibition of Ca2+ currents and Ca-dependent K+ currents was observed. The failure to demonstrate opioid modulation of resting or voltage dependent K+ currents suggests that modulation of Ca2+ currents is the principal mechanism for the inhibitory effect of opioids on sensory neurons.
1. Whole-cell and cell-attached patch-clamp recordings were made from enzymatically isolated type I cells from the carotid body of adult rats. Voltage-dependent K+ and Ca2+ channels were observed, but there was no detectable Na+ current. In this respect, rat carotid body cells are unlike those from rabbit, which have Na+ currents and Na(+)-dependent action potentials. 2. The observed Ca2+ channels had the following properties: 1) activation requires voltage steps above -20 mV; 2) little inactivation occurred with holding voltages below -40 mV; 3) one single-channel conductance of 21 pS was found with 90 or 110 mM Ba2+ in the cell-attached pipette and this was the only conductance observed; 4) open probability was increased by the dihydropyridine Ca2+ channel agonist Bay K 8644 and was decreased by the antagonist nifedipine; and 5) omega-conotoxin had little or no effect on the channels. These are properties expected of L-type Ca2+ channels. 3. To investigate whether these voltage-dependent channels would be available for opening on membrane depolarization, we measured the type I cell resting membrane potential noninvasively using unitary openings of the L-type Ca2+ channel with Bay K 8644 in the cell-attached pipette. Resting potentials ranged from -62 to -13 mV, with a mean of -32 mV in 12 cells. 4. Judging from single-channel conductance and pharmacology, the Ca2+ current is mostly, if not solely, carried by L channels. Thus it should be possible to use modulators of L channel activity to determine the role of Ca2+ channels in stimulus-secretion coupling in the rat carotid body.
Activation of the endogenous opioid system can suppress pain without affecting other sensations, but the cellular mechanism of this selectivity is unclear. The analgesia might be due to inhibitory synapses arranged only on neurons whose activity leads to pain sensations. Alternatively, opioids might be released broadly, with neurons involved in pain sensation being especially sensitive. Therefore, we asked whether different subsets of rat dorsal root ganglion (DRG) sensory neurons vary in their sensitivity to opioids. Dissociated neurons were subdivided according to the spinal laminae to which they likely had projected, and whether they had innervated muscle. Using the patch-clamp method, we measured the inhibition of Ca2+ current by DAGO (Tyr-D-Ala-Gly-MePhe-Gly-ol), a peptide that selectively activates the mu (morphine) receptor. We also investigated the presence of different types of Ca2+ channels. In DRG neurons chosen at random, Ca2+ currents were inhibited by DAGO to widely varying degrees, with an average inhibition of 38%. Ca2+ currents in neurons in a subset that projects to laminae I and II had a lower average inhibition, and unlike the randomly selected cells, the responses were predictable and tightly distributed about the mean. This indicates that the variability of opioid sensitivity among DRG neurons reflects the presence of different subsets of cells. Since neurons projecting to laminae I and II, the projection site of nociceptive neurons, did not show high opioid sensitivity, there is no evidence that nociceptive neurons have stronger responses to opioids. But a firm conclusion is impossible because projection site does not strictly define sensory modality.
Following purification using Ca2+ channel drugs as ligands, the skeletal muscle Ca2+ channel was shown to be a five-subunit structure containing one large (175 kDa) protein that is the pore and four auxiliary subunits. Each subunit has been cloned and expression studies are proceeding rapidly. Particular success has been made in structure-function studies of excitation-contraction coupling using a Ca2+ channel-free mutant muscle. The work confirmed the suggestion made from physiological studies that muscle Ca2+ channels serve dual roles: passing Ca2+ and triggering Ca2+ release from an intracellular organelle. A variety of other predictions about the structure of Ca2+ channels have been reviewed here and these may soon be possible to test. Such concrete predictions along with analogies to studies on other voltage-dependent ion channels should speed progress in structure-function studies of Ca2+ channels.
Opioids and opiates decrease the duration of action potentials and the amount of neurotransmitter released from sensory neurons. The mu-type opioid receptor, the binding site for morphine, is thought to act exclusively on K+ channels. Here, we show that activation of the mu receptor inhibits Ca2+ channels in rat sensory neurons; the effect is blocked by a mu antagonist and is not mimicked by kappa or delta receptor agonists. Both low-threshold (T-type) and high-threshold Ca2+ currents are partially suppressed. omega-Conotoxin-sensitive and omega-conotoxin-insensitive, high-threshold Ca2+ currents are inhibited. The kinetic effect on high-threshold current is like that caused by diminished rest potential: the transient component is selectively lost, whereas the sustained component is spared.