Calcium channel antagonists. Omega-conotoxin defines a new high affinity site

The omega-conotoxins, a class of Ca2+ channel antagonists from fish-hunting marine snails, have recently been described (Olivera, B. M., McIntosh, J. M., Zeikus, R., Gray, W. R., Varga, J., Rivier, J., de Santos, V., and Cruz, L. J. (1985) Science, 230, 1338-1343). One of these peptide neurotoxins, omega-conotoxin GVIA, was radiolabeled with iodine, and the 125I-labeled toxin was shown to bind specifically to high affinity sites on chick brain synaptosomes. The toxin-receptor complex was extremely stable; addition of an excess of unlabeled toxin did not cause significant displacement of the labeled toxin after 2 h. Binding competition data suggest that omega-conotoxin defines a new high affinity receptor site affecting voltage-activated Ca2+ channels, distinct from both the verapamil and dihydropyridine target sites.     

Modulation of neuronal voltage-activated calcium and sodium channels by polyamines and pH

The endogenous polyamines spermine, spermidine and putrescine are present at high concentrations inside neurons and can be released into the extracellular space where they have been shown to modulate ion channels. Here, we have examined polyamine modulation of voltage-activated Ca(2+) channels (VACCs) and voltage-activated Na(+) channels (VANCs) in rat superior cervical ganglion neurons using whole-cell voltage-clamp at physiological divalent concentrations. Polyamines inhibited VACCs in a concentration-dependent manner with IC(50)s for spermine, spermidine, and putrescine of 4.7 +/- 0.7, 11.2 +/- 1.4 and 90 +/- 36 mM, respectively. Polyamines caused inhibition by shifting the VACC half-activation voltage (V(0.5)) to depolarized potentials and by reducing total VACC permeability. The shift was described by Gouy-Chapman-Stern theory with a surface charge density of 0.120 +/- 0.005 e(-) nm(-2) and a surface potential of -19 mV. Attenuation of spermidine and spermine inhibition of VACC at decreased pH was explained by H(+) titration of surface charge. Polyamine-mediated effects also decreased at elevated pH due to the inhibitors having lower valence and being less effective at screening surface charge. Polyamines affected VANC currents indirectly by reducing TTX inhibition of VANCs at high pH. This may reflect surface charge induced decreases in the local TTX concentration or polyamine-TTX interactions. In conclusion, polyamines inhibit neuronal VACCs via complex interactions with extracellular H(+) and Ca. Many of the observed effects can be explained by a model incorporating polyamine binding, H(+) binding and surface charge screening.     

Effects of the blockade of high voltage-activated calcium channels on in vitro pineal melatonin synthesis

The presence of high voltage-activated calcium channels in the rat pineal gland is well known. However, their role in pineal metabolism is not completely understood and is even controversial. Better to understand this matter, we investigated the effects of L-, N- or P/Q-type calcium channel blockers (nifedipine, omega-conotoxin GVIA, omega-agatoxin IVA, respectively) on melatonin content and arylalkylamine-N-acetyltransferase activity of denervated rat pineal glands kept for 48 h in culture and stimulated with norepinephrine. Melatonin was measured by high performance liquid chromatography with electrochemical detection and arylalkylamine-N-acetyltransferase activity was quantified by radiometric assay. Pre-incubation with any of these high voltage-activated calcium channel blockers reduced the melatonin production induced by norepinephrine although arylalkylamine-N-acetyltransferase activity was reduced only by the N-type calcium channel antagonist, omega-conotoxin GVIA. The results indicate that calcium influx through L-, N- or P/Q-type of high voltage-activated calcium channels is necessary for the full expression of the metabolic process leading to melatonin synthesis in the rat pineal glands. However, the mechanisms involved in this process are different for the L- or P/Q- and N-type calcium channels.     

Generating a high affinity scorpion toxin receptor in KcsA-Kv1.3 chimeric potassium channels

The crystal structure of the bacterial K(+) channel, KcsA (Doyle, D. A., Morais, C. J., Pfuetzner, R. A., Kuo, A., Gulbis, J. M., Cohen, S. L., Chait, B. T., and MacKinnon, R. (1998) Science 280, 69-77), and subsequent mutagenesis have revealed a high structural conservation from bacteria to human (MacKinnon, R., Cohen, S. L., Kuo, A., Lee, A., and Chait, B. T. (1998) Science 280, 106-109). We have explored this conservation by swapping subregions of the M1-M2 linker of KcsA with those of the S5-S6 linker of the human Kv-channel Kv1.3. The chimeric K(+) channel constructs were expressed in Escherichia coli, and their multimeric state was analyzed after purification. We used two scorpion toxins, kaliotoxin and hongotoxin 1, which bind specifically to Kv1.3, to analyze the pharmacological properties of the KcsA-Kv1.3 chimeras. The results demonstrate that the high affinity scorpion toxin receptor of Kv1.3 could be transferred to KcsA. Our biochemical studies with purified KcsA-Kv1.3 chimeras provide direct chemical evidence that a tetrameric channel structure is necessary for forming a functional scorpion toxin receptor. We have obtained KcsA-Kv1.3 chimeras with kaliotoxin affinities (IC(50) values of approximately 4 pm) like native Kv1.3 channels. Furthermore, we show that a subregion of the S5-S6 linker may be an important determinant of the pharmacological profile of K(+) channels. Using available structural information on KcsA and kaliotoxin, we have developed a structural model for the complex between KcsA-Kv1.3 chimeras and kaliotoxin to aid future pharmacological studies of K(+) channels.     

Bovine myeloperoxidase and lactoperoxidase each contain a high affinity site for calcium

Both bovine myeloperoxidase and lactoperoxidase contain one calcium per iron with no other metal present in significant amount. Calcium is bound with high affinity and is removed upon exposure to 6 M guanidine hydrochloride/EGTA which results in precipitation of protein. Computer amino acid sequence analyses of human myeloperoxidase reveal two plausible calcium binding sites. This is the first evidence for the presence of calcium in these peroxidases.     

The high affinity binding site for calcium on the oxidizing side of photosystem II

Preparations of photosystem II complex from spinach chloroplasts with Triton X-100 were treated with 1 M KCl to release 17 KDa and 23 KDa polypeptides. The inhibited oxygen evolution activity could be reactivated by adding high concentration (mM) of Ca++ or by reconstituting 17 KDa and 23 KDa polypeptides which were found to promote high affinity binding of Ca++ to the reconstituted membranes (Ghanotakis et al. FEBS (1984) 170, 169-173). Inclusion of 50 mM Ca++ during KCl treatment did not prevent the release of 17 KDa and 23 KDa polypeptides but protected oxygen evolution from being inactivated. It is explained by preservation of the high affinity binding site for Ca++ if, Ca++ is present during the salt treatment even though depletion of 17 KDa and 23 KDa polypeptides usually results in replacement by a low affinity (mM) binding site for Ca++. It also implies that the high affinity binding site is not located on 17 KDa and 23 KDa polypeptides.     

Identification of inactivation determinants in the domain IIS6 region of high voltage-activated calcium channels

We have recently reported that transfer of the domain IIS6 region from rapidly inactivating R-type (alpha(1E)) calcium channels to slowly inactivating L-type (alpha(1C)) calcium channel confers rapid inactivation (Stotz, S. C., Hamid, J., Spaetgens, R. L., Jarvis, S. E., and Zamponi, G. W. (2000) J. Biol. Chem. 275, 24575-24582). Here we have identified individual amino acid residues in the IIS6 regions that are responsible for these effects. In this region, alpha(1C) and alpha(1E) channels differ in seven residues, and exchanging five of those residues individually or in combination did not significantly affect inactivation kinetics. By contrast, replacement of residues Phe-823 or Ile-829 of alpha(1C) with the corresponding alpha(1E) residues significantly accelerated inactivation rates and, when substituted concomitantly, approached the rapid inactivation kinetics of R-type channels. A systematic substitution of these residues with a series of other amino acids revealed that decreasing side chain size at position 823 accelerates inactivation, whereas a dependence of the inactivation kinetics on the degree of hydrophobicity could be observed at position 829. Although these point mutations facilitated rapid entry into the inactivated state of the channel, they had little to no effect on the rate of recovery from inactivation. This suggests that the development of and recovery from inactivation are governed by separate structural determinants. Finally, the effects of mutations that accelerated alpha(1C) inactivation could still be antagonized following coexpression of the rat beta(2a) subunit or by domain I-II linker substitutions that produce ultra slow inactivation of wild type channels, indicating that the inactivation kinetics seen with the mutants remain subject to regulation by the domain I-II linker. Overall, our results provide novel insights into a complex process underlying calcium channel inactivation.     

Action potential waveform voltage-clamp commands reveal striking differences in calcium entry via low and high voltage-activated calcium channels.

Calcium channels transduce natural voltage transients, like action potentials, into functionally important intracellular calcium transients. We have used digitally constructed waveforms that simulate natural action potentials as voltage-clamp commands to study channel function in transduction. Whole-cell calcium currents elicited by several action potential waveforms (APWs) were studied. The currents were subdivided into T (or low voltage-activated) and high voltage-activated components. Calcium entry through T channels constituted a disproportionately large fraction of the total during normal, brief APWs. Entry through high voltage-activated channels was much more responsive to APW, increasing more significantly as APW duration increased. Thus the results indicate that differences in the gating properties of these two channel classes combine to endow them with strikingly different transducer properties.     

A new scorpion toxin with a very high affinity for sodium channels. An electrophysiological study

The neurotoxic action of toxin gamma from the venom of the Brazilian scorpion Tityus serrulatus (TiTx gamma) has been investigated in cultured mouse neuroblastoma cells (N1E115) using the suction pipette technique. Addition of 14 to 53 nM TiTx gamma to the external solution causes nerve cell membrane depolarization, membrane potential oscillations and spontaneous action potentials within 10 min. None of these effects were observed within 15 min after application of 1 microM toxin IV from Centruroides sculpturatus venom. Under voltage clamp the amplitude of the sodium current evoked by test pulses to potentials more positive than -30 mV is reversibly reduced by 50% after 17 to 105 nM TiTx gamma. On the other hand, a sodium current component appears after TiTx gamma at test pulse potentials between -70 and -40 mV, for which no sodium current is observed in the control experiment. The outward potassium current is not significantly affected by the highest TiTx gamma concentrations used. The potential-dependence of inactivation of the sodium current component that is induced by TiTx gamma is shifted by -30 mV with respect to control values. The local anaesthetic procain at 1 mM discriminates between the two populations of sodium channels observed in the presence of TiTx gamma.     

Differential regulation of low and high voltage-activated calcium channels in neonatal rat myocytes following chronic PKA modulation

The biophysical properties of voltage-dependent cardiac calcium channels (VDCC) can be modulated by protein kinases. In this study, we investigate whether long-term treatment with protein kinase A (PKA) modulators alters the VDCC activity in neonatal ventricular myocytes. Using whole-cell patch-clamp recordings, we found an increase in high-voltage activated (HVA) current density and a corresponding decrease in low-voltage activated (LVA) current density in neonatal rat ventricular myocytes up to 6 days in culture. Long-term exposure to 8Br-cAMP, a PKA stimulator, increased the HVA current density at 7 and 24 hours. In contrast, H89, a PKA inhibitor, caused a biphasic change in the HVA, an initial reduction at 7 hours exposure followed by an increase up to 4 days. In addition, H89 caused a sustained increase in LVA currents from 7 hours to 4 days. These findings suggest that chronic exposure to H89 changes LVA and HVA calcium current activities in cardiac myocytes. PKA is a key target of β-adrenoceptor activiation, thus, our findings suggest long-term repeated use of β-adrenergic drugs may induce unexpected functional alteration of VDCCs.     

Forced-exercise delays neuropathic pain in experimental diabetes: effects on voltage-activated calcium channels

Physical exercise produces a variety of psychophysical effects, including altered pain perception. Elevated levels of centrally produced endorphins or endocannabinoids are implicated as mediators of exercise-induced analgesia. The effect of exercise on the development and persistence of disease-associated acute/chronic pain remains unclear. In this study, we quantified the physiological consequence of forced-exercise on the development of diabetes-associated neuropathic pain. Euglycemic control or streptozotocin (STZ)-induced diabetic adult male rats were subdivided into sedentary or forced-exercised (2-10 weeks, treadmill) subgroups and assessed for changes in tactile responsiveness. Two weeks following STZ-treatment, sedentary rats developed a marked and sustained hypersensitivity to von Frey tactile stimulation. By comparison, STZ-treated diabetic rats undergoing forced-exercise exhibited a 4-week delay in the onset of tactile hypersensitivity that was independent of glucose control. Exercise-facilitated analgesia in diabetic rats was reversed, in a dose-dependent manner, by naloxone. Small-diameter (< 30 μm) DRG neurons harvested from STZ-treated tactile hypersensitive diabetic rats exhibited an enhanced (2.5-fold) rightward (depolarizing) shift in peak high-voltage activated (HVA) Ca(2+) current density with a concomitant appearance of a low-voltage activated (LVA) Ca(2+) current component. LVA Ca(2+) currents present in DRG neurons from hypersensitive diabetic rats exhibited a marked depolarizing shift in steady-state inactivation. Forced-exercise attenuated diabetes-associated changes in HVA Ca(2+) current density while preventing the depolarizing shift in steady-state inactivation of LVA Ca(2+) currents. Forced-exercise markedly delays the onset of diabetes-associated neuropathic pain, in part, by attenuating associated changes in HVA and LVA Ca(2+) channel function within small-diameter DRG neurons possibly by altering opioidergic tone.     

Differential regulation of low and high voltage-activated calcium channels in neonatal rat myocytes following chronic PKA modulation

The biophysical properties of voltage-dependent cardiac calcium channels (VDCC) can be modulated by protein kinases. In this study, we investigate whether long-term treatment with protein kinase A (PKA) modulators alters the VDCC activity in neonatal ventricular myocytes. Using whole-cell patch-clamp recordings, we found an increase in high-voltage activated (HVA) current density and a corresponding decrease in low-voltage activated (LVA) current density in neonatal rat ventricular myocytes up to 6 days in culture. Long-term exposure to 8Br-cAMP, a PKA stimulator, increased the HVA current density at 7 and 24 hours. In contrast, H89, a PKA inhibitor, caused a biphasic change in the HVA, an initial reduction at 7 hours exposure followed by an increase up to 4 days. In addition, H89 caused a sustained increase in LVA currents from 7 hours to 4 days. These findings suggest that chronic exposure to H89 changes LVA and HVA calcium current activities in cardiac myocytes. PKA is a key target of beta-adrenoceptor activiation, thus, our findings suggest long-term repeated use of beta-adrenergic drugs may induce unexpected functional alteration of VDCCs.     

The effect of dihydropyridine calcium agonists and antagonists on neuronal voltage sensitive calcium channels

The effect of dihydropyridine agonists and antagonists on neuronal voltage sensitive calcium channels was investigated. The resting intracellular calcium concentration of synaptosomes prepared from whole brain was 110 +/- 9 nM, as assayed by the indicator quin 2. Depolarisation of the synaptosomes with K+ produced an immediate increase in [Ca2+]i. The calcium agonist Bay K 8644 and antagonist nifedipine did not affect [Ca2+]i under resting or depolarising conditions. In addition, K+ stimulated 45Ca2+ uptake into synaptosomes prepared from the hippocampus was insensitive to Bay K 8644 and PY 108-068 in normal or Na+ free conditions. In neuronally derived NG108-15 cells the enantiomers of the dihydropyridine derivative 202-791 showed opposite effects in modulating K+ stimulated 45Ca2+ uptake. (-)-R-202-791 inhibited K+ induced 45Ca2+ uptake with an IC50 of 100 nM and (+)-S-202-791 enhanced K+ stimulated uptake with an EC50 of 80 nM. These results suggest that synaptosomal voltage sensitive calcium channels either are of a different type to those found in peripheral tissues and cells of neural origin or that expression of functional effects of dihydropyridines requires different experimental conditions to those used here.     

Substituted diphenylbutylpiperidines bind to a unique high affinity site on the L-type calcium channel. Evidence for a fourth site in the cardiac calcium entry blocker receptor complex

Fluspirilene binds with high affinity to a single class of sites in purified porcine cardiac sarcolemmal membrane vesicles at a Kd of 0.6 nM and a Bmax that is in approximately 1:1 stoichiometry with other Ca2+ entry blocker receptors. Fluspirilene binding is modulated by various classes of L-type Ca2+ channel effectors. Metal ion channel inhibitors (e.g. Cd2+) stimulate binding primarily by increasing ligand affinity, whereas channel substrates (e.g. Ca2+) inhibit binding. Dihydropyridine, aralkylamine, and benzothiazepine Ca2+ entry blockers partially inhibit binding with Ki values equivalent to their respective Kd values, indicating close coupling between binding sites for the former agents and the diphenylbutylpiperidine site. All of these agents function as mixed inhibitors and affect both Kd and Bmax of fluspirilene binding. Only other substituted diphenylbutylpiperidines (e.g. pimozide) inhibit binding competitively. Diphenylbutylpiperidines, on the other hand, block nitrendipine, D-600, and diltiazem binding through a noncompetitive mechanism with Ki values much reduced from their measured Kd values, suggesting that coupling between the diphenylbutylpiperidine site and receptors for diverse Ca2+ entry blockers is more indirect. In addition, high affinity sites have been detected for fluspirilene in bovine aortic sarcolemmal vesicles, rat brain synaptic membranes, and GH3 rat anterior pituitary cell plasma membranes. Fluspirilene also effectively blocks Ca2+ flux through L-type Ca2+ channels in GH3 cells. Together, these results suggest that fluspirilene binds with high affinity to a unique fourth site in the Ca2+ entry blocker receptor complex and that substituted diphenylbutylpiperidines represent a new structural class of potent L-type Ca2+ channel inhibitors.     

The tandem beta-zipper model defines high affinity fibronectin-binding repeats within Staphylococcus aureus FnBPA

Binding of the fibronectin-binding protein FnBPA from Staphylococcus aureus to the human protein fibronectin has previously been implicated in the development of infective endocarditis, specifically in the processes of platelet activation and invasion of the endothelium. We recently proposed a model for binding of fibronectin to FnBPA in which the bacterial protein contains 11 potential binding sites (FnBPA-1 to FnBPA-11), each composed of motifs that bind to consecutive fibronectin type 1 modules in the N-terminal domain of fibronectin. Here we show that six of the 11 sites bind with dissociation constants in the nanomolar range; other sites bind more weakly. The high affinity binding sites include FnBPA-1, the sequence of which had previously been thought to be encompassed by the fibrinogen-binding A domain of FnBPA. Both the number and sequence conservation of the type-1 module binding motifs appears to be important for high affinity binding. The in vivo relevance of the in vitro binding studies is confirmed by the presence of antibodies in patients with S. aureus infections that specifically recognize complexes of these six high affinity repeats with fibronectin.     

Omega Conus geographus toxin: a peptide that blocks calcium channels

We previously reported that omega Conus geographus toxin (omega CgTX), blocks evoked-release of transmitter at synapses in frog and attenuates the Ca2+ component of the action potential of chick dorsal root ganglion neurons. We report here voltage-clamp experiments on cultured chick dorsal root ganglion neurons which demonstrate that omega CgTX produces a persistent block of voltage-gated Ca2+ currents. Thus, we conclude that omega CgTX inhibits synaptic transmission by blocking Ca2+ channels in the presynaptic nerve terminal. The toxin had no effect on K+ currents; however, in some but not all neurons, omega CgTX reduced Na+ currents by 10-25%. These findings suggest that omega CgTX should be useful as a probe to examine synaptic Ca2+ channels.     

The high affinity receptor for omega-conotoxin represents calcium channels different from those sensitive to dihydropyridines in mammalian brain

A monoclonal antibody recognizing the alpha 2 delta complex of the dihydropyridine (DHP)-sensitive calcium channel of skeletal muscle immunoprecipitated most of the DHP receptor solubilized from bovine and rabbit brains, and bovine cardiac muscle. However, it did not significantly immunoprecipitate the high affinity omega-conotoxin receptor solubilized from these brains. These results indicate that the DHP receptor and the high affinity omega-conotoxin receptor are different molecules in mammalian brain.