Voltage-gated sodium channel expression in rat spiral ganglion neurons.

The spiral ganglion neurons (SGN) provide the afferent innervation of the hair cells in the organ of Corti and relay auditory information from the inner ear to the brain. Voltage-gated sodium channels (Na(V)) initiate and propagate action potentials that encode this sensory information but little is known regarding the subtypes expressed in these cells. We have used RT-PCR and immunohistochemistry to study the compliment and anatomical distribution of Na(V) channels in rodent SGN. Na(V)1.1, Na(V)1.6 and Na(V)1.7 were all detected at the mRNA level. Fluorescence or streptavidin-horseradish peroxidase immunohistochemistry extended these findings, demonstrating predominant localisation of Na(V)1.6 and Na(V)1.7 on SGN cell bodies and Na(V)1.1 on axonal processes. Dual labelling with peripherin demonstrated higher Na(V)1.6 and Na(V)1.7 expression on Type I rather than Type II neurons. These results provide evidence for selective expression and variations in the distribution of VGSC in the rodent SGN, which may guide further studies into afferent function in the auditory pathway and therapeutic approaches for diseases such as hearing loss and tinnitus.

TRPA1 receptor localisation in the human peripheral nervous system and functional studies in cultured human and rat sensory neurons.

TRPA1 is a receptor expressed by sensory neurons, that is activated by low temperature (<17 degrees C) and plant derivatives such as cinnamaldehyde and isoeugenol, to elicit sensations including pain. Using immunohistochemistry, we have, for the first time, localised TRPA1 in human DRG neurons, spinal cord motoneurones and nerve roots, peripheral nerves, intestinal myenteric plexus neurones, and skin basal keratinocytes. TRPA1 co-localised with a subset of hDRG neurons positive for TRPV1, the heat and capsaicin receptor. The number of small/medium TRPA1 positive neurons (< or =50 microm) was increased after hDRG avulsion injury [percentage of cells, median (range): controls 16.5 (7-23); injured 46 (34-55); P<0.005], but the number of large TRPA1 neurons was unchanged [control 19.5 (13-31); injured 21 (11-35)]. Similar TRPA1 changes were observed in cultured hDRG neurons, after exposure to a combination of key neurotrophic factors NGF, GDNF and NT-3 (NTFs) in vitro. We used calcium imaging to examine responses of HEK cells transfected with hTRPA1 cDNA, and of human and rat DRG neurons cultured with or without added NTFs, to cinnamaldehyde (CA) and isoeugenol (IE). Exposure to NTFs in vitro sensitized cultured human sensory neuronal responses to CA; repeated CA exposure produced desensitisation. In rDRG neurons, low (225 microM) CA preincubation enhanced capsaicin responses, while high (450 microM and 2mM) CA caused inhibition which was partially reversed in the presence of 8 bromo cAMP, indicating receptor dephosphorylation. While TRPA1 localisation is more widespread than TRPV1, it represents a promising novel drug target for the treatment of chronic pain and hypersensitivity.

Identification and characterisation of SB-366791, a potent and selective vanilloid receptor (VR1/TRPV1) antagonist.

Vanilloid receptor-1 (TRPV1) is a non-selective cation channel, predominantly expressed by peripheral sensory neurones, which is known to play a key role in the detection of noxious painful stimuli, such as capsaicin, acid and heat. To date, a number of antagonists have been used to study the physiological role of TRPV1; however, antagonists such as capsazepine are somewhat compromised by non-selective actions at other receptors and apparent modality-specific properties. SB-366791 is a novel, potent, and selective, cinnamide TRPV1 antagonist isolated via high-throughput screening of a large chemical library. In a FLIPR-based Ca(2+)-assay, SB-366791 produced a concentration-dependent inhibition of the response to capsaicin with an apparent pK(b) of 7.74 +/- 0.08. Schild analysis indicated a competitive mechanism of action with a pA2 of 7.71. In electrophysiological experiments, SB-366791 was demonstrated to be an effective antagonist of hTRPV1 when activated by different modalities, such as capsaicin, acid or noxious heat (50 degrees C). Unlike capsazepine, SB-366791 was also an effective antagonist vs. the acid-mediated activation of rTRPV1. With the aim of defining a useful tool compound, we also profiled SB-366791 in a wide range of selectivity assays. SB-366791 had a good selectivity profile exhibiting little or no effect in a panel of 47 binding assays (containing a wide range of G-protein-coupled receptors and ion channels) and a number of electrophysiological assays including hippocampal synaptic transmission and action potential firing of locus coeruleus or dorsal raphe neurones. Furthermore, unlike capsazepine, SB-366791 had no effect on either the hyperpolarisation-activated current (I(h)) or Voltage-gated Ca(2+)-channels (VGCC) in cultured rodent sensory neurones. In summary, SB-366791 is a new TRPV1 antagonist with high potency and an improved selectivity profile with respect to other commonly used TRPV1 antagonists. SB-366791 may therefore prove to be a useful tool to further study the biology of TRPV1.

TRPV3 is a temperature-sensitive vanilloid receptor-like protein.

Vanilloid receptor-1 (VR1, also known as TRPV1) is a thermosensitive, nonselective cation channel that is expressed by capsaicin-sensitive sensory afferents and is activated by noxious heat, acidic pH and the alkaloid irritant capsaicin. Although VR1 gene disruption results in a loss of capsaicin responses, it has minimal effects on thermal nociception. This and other experiments--such as those showing the existence of capsaicin-insensitive heat sensors in sensory neurons--suggest the existence of thermosensitive receptors distinct from VR1. Here we identify a member of the vanilloid receptor/TRP gene family, vanilloid receptor-like protein 3 (VRL3, also known as TRPV3), which is heat-sensitive but capsaicin-insensitive. VRL3 is coded for by a 2,370-base-pair open reading frame, transcribed from a gene adjacent to VR1, and is structurally homologous to VR1. VRL3 responds to noxious heat with a threshold of about 39 degrees C and is co-expressed in dorsal root ganglion neurons with VR1. Furthermore, when heterologously expressed, VRL3 is able to associate with VR1 and may modulate its responses. Hence, not only is VRL3 a thermosensitive ion channel but it may represent an additional vanilloid receptor subunit involved in the formation of heteromeric vanilloid receptor channels.

Ethanol elicits and potentiates nociceptor responses via the vanilloid receptor-1.

The vanilloid receptor-1 (VR1) is a heat-gated ion channel that is responsible for the burning sensation elicited by capsaicin. A similar sensation is reported by patients with esophagitis when they consume alcoholic beverages or are administered alcohol by injection as a medical treatment. We report here that ethanol activates primary sensory neurons, resulting in neuropeptide release or plasma extravasation in the esophagus, spinal cord or skin. Sensory neurons from trigeminal or dorsal root ganglia as well as VR1-expressing HEK293 cells responded to ethanol in a concentration-dependent and capsazepine-sensitive fashion. Ethanol potentiated the response of VR1 to capsaicin, protons and heat and lowered the threshold for heat activation of VR1 from approximately 42 degrees C to approximately 34 degrees C. This provides a likely mechanistic explanation for the ethanol-induced sensory responses that occur at body temperature and for the sensitivity of inflamed tissues to ethanol, such as might be found in esophagitis, neuralgia or wounds.

Characterisation of a human acid-sensing ion channel (hASIC1a) endogenously expressed in HEK293 cells.

Acid-sensing ion channels (ASICs) are a new and expanding family of proton-gated cation (Na+/Ca2+) channels that are widely expressed in sensory neurons and the central nervous system. Their distribution suggests that they may play a critical role in the sensation of the pain that accompanies tissue acidosis and may also be important in detecting the subtle pH variations that occur during neuronal signalling. Here, using whole-cell patch-clamp electrophysiology and reverse transcriptase-polymerase chain reaction (RT-PCR), we show that HEK293 cells, a commonly used cell line for the expression and characterisation of many ion channels, functionally express an endogenous proton-gated conductance attributable to the activity of human ASIC1a. These data therefore represent the first functional characterisation of hASIC1 and have many important implications for the use of HEK293 cells as a host cell system for the study of ASICs, vanilloid receptor-1 and any other proton-gated channel. With this latter point in mind we have devised a simple desensitisation strategy to selectively remove the contribution of hASIC1a from proton-gated currents recorded from HEK293 cells expressing vanilloid receptor-1.

Conversion of the ion selectivity of the 5-HT(3a) receptor from cationic to anionic reveals a conserved feature of the ligand-gated ion channel superfamily.

The 5-hydroxytryptamine(3) (5-HT(3)) receptor is a member of a superfamily of ligand-gated ion channels, which includes nicotinic acetylcholine, gamma-aminobutyric acid, and glycine receptors. The receptors are either cation or anion selective, leading to their distinctive involvement in either excitatory or inhibitory neurotransmission. Using a combination of site-directed mutagenesis and electrophysiological characterization of homomeric 5-HT(3A) receptors expressed in HEK293 cells, we have identified a set of mutations that convert the ion selectivity of the 5-HT(3A) receptor from cationic to anionic; these were substitution of V13'T in M2 together with neutralization of glutamate residues (E-1'A) and the adjacent insertion of a proline residue (P-1') in the M1-M2 loop. Mutant receptors showed significant chloride permeability (P(Cl)/P(Na) = 12.3, P(Na)/P(Cl) = 0.08), whereas WT receptors are predominantly permeable to sodium (P(Na)/P(Cl) > 20, P(Cl)/P(Na) < 0.05). Since the equivalent mutations have previously been shown to convert alpha7 nicotinic acetylcholine receptors from cationic to anionic (Galzi J.-L., Devillers-Thiery, A, Hussy, N., Bertrand, S. Changeux, J. P., and Bertrand, D. (1992) Nature 359, 500-505) and, recently, the converse mutations have allowed the construction of a cation selective glycine receptor (Keramidas, A., Moorhouse, A. J., French, C. R., Schofield, P. R., and Barry, P. H. (2000) Biophys. J. 78, 247-259), it appears that the determinants of ion selectivity represent a conserved feature of the ligand-gated ion channel superfamily.

Characterisation using FLIPR of human vanilloid VR1 receptor pharmacology.

A full pharmacological characterisation of the recently cloned human vanilloid VR1 receptor was undertaken. In whole-cell patch clamp studies, capsaicin (10 microM) elicited a slowly activating/deactivating inward current in human embryonic kidney (HEK293) cells stably expressing human vanilloid VR1 receptor, which exhibited pronounced outward rectification (reversal potential -2.1+/-0.2 mV) and was abolished by capsazepine (10 microM). In FLIPR-based Ca(2+) imaging studies the rank order of potency was resiniferatoxin>olvanil>capsaicin>anandamide, and all were full agonists. Isovelleral and scutigeral were inactive (1 nM-30 microM). The potencies of capsaicin, olvanil and resiniferatoxin, but not anandamide, were enhanced 2- to 7-fold at pH 6.4. Capsazepine, isovelleral and ruthenium red inhibited the capsaicin (100 nM)-induced Ca(2+) response (pK(B)=6.58+/-0.02, 5.33+/-0.03 and 7.64+/-0.03, respectively). In conclusion, the recombinant human vanilloid VR1 receptor stably expressed in HEK293 cells acted as a ligand-gated, Ca(2+)-permeable channel with similar agonist and antagonist pharmacology to rat vanilloid VR1 receptor, although there were some subtle differences.

Conversion of the ion selectivity of the 5-HT(3a) receptor from cationic to anionic reveals a conserved feature of the ligand-gated ion channel superfamily.

The 5-hydroxytryptamine(3) (5-HT(3)) receptor is a member of a superfamily of ligand-gated ion channels, which includes nicotinic acetylcholine, gamma-aminobutyric acid, and glycine receptors. The receptors are either cation or anion selective, leading to their distinctive involvement in either excitatory or inhibitory neurotransmission. Using a combination of site-directed mutagenesis and electrophysiological characterization of homomeric 5-HT(3A) receptors expressed in HEK293 cells, we have identified a set of mutations that convert the ion selectivity of the 5-HT(3A) receptor from cationic to anionic; these were substitution of V13'T in M2 together with neutralization of glutamate residues (E-1'A) and the adjacent insertion of a proline residue (P-1') in the M1-M2 loop. Mutant receptors showed significant chloride permeability (P(Cl)/P(Na) = 12.3, P(Na)/P(Cl) = 0.08), whereas WT receptors are predominantly permeable to sodium (P(Na)/P(Cl) > 20, P(Cl)/P(Na) < 0.05). Since the equivalent mutations have previously been shown to convert alpha7 nicotinic acetylcholine receptors from cationic to anionic (Galzi J.-L., Devillers-Thiery, A, Hussy, N., Bertrand, S. Changeux, J. P., and Bertrand, D. (1992) Nature 359, 500-505) and, recently, the converse mutations have allowed the construction of a cation selective glycine receptor (Keramidas, A., Moorhouse, A. J., French, C. R., Schofield, P. R., and Barry, P. H. (2000) Biophys. J. 78, 247-259), it appears that the determinants of ion selectivity represent a conserved feature of the ligand-gated ion channel superfamily.

Voltage- and time-dependent properties of the recombinant rat vanilloid receptor (rVR1).

Whole-cell voltage-clamp techniques were used to investigate the capsaicin-, voltage- and time-dependent properties of the rat vanilloid receptor (rVR1) stably expressed in human embryonic kidney (HEK) 293 cells. At a holding potential of -70 mV, application of capsaicin (0.03-30 microM) to HEK 293 cells expressing the rVR1 receptor led to the appearance of inward currents (EC50, 497 nM; Hill coefficient, nH, 2.85) which were reversibly antagonized by 10 microM capsazepine. Current-voltage relationships, determined using depolarizing or hyperpolarizing voltage ramps, had reversal potentials close to 0 mV, exhibited substantial outward rectification and possessed a region of negative slope conductance at holding potentials negative to around -70 mV. Further experiments indicated that the outward rectification and the region of negative slope conductance did not result from external block of the channel by either Ba2+, Ca2+ or Mg2+. During our characterization of rVR1, it became apparent that the rectification behaviour of this receptor was not entirely instantaneous as might be expected for a ligand-gated ion channel, but rather displayed clear time-dependent components. We characterized the kinetics of these novel gating properties in a series of additional voltage-step experiments. The time-dependent changes in rVR1-mediated conductance due to membrane depolarization or repolarization occurred with bi-exponential kinetics. On depolarization to +70 mV the time-dependent increase in outward current developed with mean time constants of 6.7 +/- 0.7 and 51.8 +/- 18.4 ms, with the faster time constant playing a dominant role (64.4 +/- 3.8 %). Similar kinetics also described the decay of 'tail currents' observed on repolarization. Furthermore, these time-dependent changes appeared to be unaffected by the removal of extracellular divalent cations and were not significantly voltage dependent. Our data reveal that rVR1 exhibits substantial time- and voltage-dependent gating properties that may have significance for the physiology of sensory transduction of nociceptive signals.

Vanilloid receptor-1 is essential for inflammatory thermal hyperalgesia.

The vanilloid receptor-1 (VR1) is a ligand-gated, non-selective cation channel expressed predominantly by sensory neurons. VR1 responds to noxious stimuli including capsaicin, the pungent component of chilli peppers, heat and extracellular acidification, and it is able to integrate simultaneous exposure to these stimuli. These findings and research linking capsaicin with nociceptive behaviours (that is, responses to painful stimuli in animals have led to VR1 being considered as important for pain sensation. Here we have disrupted the mouse VR1 gene using standard gene targeting techniques. Small diameter dorsal root ganglion neurons isolated from VR1-null mice lacked many of the capsaicin-, acid- and heat-gated responses that have been previously well characterized in small diameter dorsal root ganglion neurons from various species. Furthermore, although the VR1-null mice appeared normal in a wide range of behavioural tests, including responses to acute noxious thermal stimuli, their ability to develop carrageenan-induced thermal hyperalgesia was completely absent. We conclude that VR1 is required for inflammatory sensitization to noxious thermal stimuli but also that alternative mechanisms are sufficient for normal sensation of noxious heat.

The taurine uptake inhibitor guanidinoethyl sulphonate is an agonist at gamma-aminobutyric acid(A) receptors in cultured murine cerebellar granule cells.

In patch clamp experiments the beta-amino acid uptake inhibitor guanidinoethyl sulphonate (GES) activated currents in intact cultured murine cerebellar granule neurones. These responses could be attenuated by the gamma-aminobutyric acid(A) (GABA(A)) receptor antagonists bicuculline and picrotoxin. With intracellular chloride concentrations of either 20 or 130 mM, GES-induced current responses reversed polarity near the chloride equilibrium potential. When fast applications of agonist were made to excised granule cell macropatches GES responses were dose-dependent and exhibited significant outward rectification. Like taurine (but unlike GABA and beta-alanine) responses, macroscopic desensitisation of GES-induced currents was slow. Our data indicate that care should be exercised when using GES as a taurine uptake inhibitor in systems that also contain GABA(A) receptors.

The endogenous lipid anandamide is a full agonist at the human vanilloid receptor (hVR1).

The endogenous cannabinoid anandamide was identified as an agonist for the recombinant human VR1 (hVR1) by screening a large array of bioactive substances using a FLIPR-based calcium assay. Further electrophysiological studies showed that anandamide (10 or 100 microM) and capsaicin (1 microM) produced similar inward currents in hVR1 transfected, but not in parental, HEK293 cells. These currents were abolished by capsazepine (1 microM). In the FLIPR anandamide and capsaicin were full agonists at hVR1, with pEC(50) values of 5. 94+/-0.06 (n=5) and 7.13+/-0.11 (n=8) respectively. The response to anandamide was inhibited by capsazepine (pK(B) of 7.40+/-0.02, n=6), but not by the cannabinoid receptor antagonists AM630 or AM281. Furthermore, pretreatment with capsaicin desensitized the anandamide-induced calcium response and vice versa. In conclusion, this study has demonstrated for the first time that anandamide acts as a full agonist at the human VR1.

The 4'lysine in the putative channel lining domain affects desensitization but not the single-channel conductance of recombinant homomeric 5-HT3A receptors.

The 5-HT3 receptor is a transmitter-gated ion channel of the Cys-loop superfamily. Uniquely, 5-HT3 receptor subunits (5-HT3A and 5-HT3B) possess a positively charged lysine residue within the putative channel lining M2 domain (4' position). Using whole cell recording techniques, we examined the role of this residue in receptor function using wild-type (WT) and mutant 5-HT3A receptor subunits of murine origin transiently expressed in human embryonic kidney (HEK 293) cells. WT 5-HT3A receptors mediated rapidly activating currents in response to 5-HT (10-90 % rise time, 103 ms; EC50, 2.34 microM; Hill coefficient, nH, 2.87). The currents rectified inwardly, reversed in sign at a potential of -9 mV and desensitized in the continuous presence of agonist (half-time of desensitization, t(1/2), 2.13 s). 5-HT3A receptor subunits in which the 4'lysine was mutated to arginine, glutamine, serine or glycine formed functional receptors. 5-HT EC50 values were approximately 2-fold lower than for WT 5-HT3A receptors, but Hill coefficients, kinetics of current activation, rectification, and reversal potentials were unaltered. Each of the mutants desensitized more slowly than the WT 5-HT3A receptor, with the arginine and glycine mutations exhibiting the greatest effect (5-fold reduction). The rank order of effect was arginine > glycine > serine > glutamine. The single-channel conductance of the WT 5-HT3A receptor, as assessed by fluctuation analysis of macroscopic currents, was 390 fS. A similar value was obtained for the 4'lysine mutant receptors. Thus it appears unlikely that 4'lysine is exposed to the channel lumen. Mutation of residues immediately adjacent to 4'lysine to glutamate or lysine resulted in lack of receptor expression or function. We conclude that 4'lysine does not form part of the channel lining, but may play an important role in 5-HT3 receptor desensitization.

Design strategies and performance of custom DNA sequencing primers.

This study surveyed strategies of sequencing primer selection and evaluated primer performance in automated DNA sequencing. We asked participants to relate their preferred primer design strategies to identify primer characteristics that are considered most important in sequencing primer design. The participants preferred primers of 18-24 nucleotides (nt), 39%-58% G + C, a melting temperature (Tm) of 53 degrees-65 degrees C with a 1-2 nt 3' GC clamp, hairpin stems of less than 2-3 bp, homopolymeric runs of less than 4-5 nt, primer dimers of less than 3-4 bp and secondary priming sites of less than 3-4 bp. We provided a 300-bp test sequence and asked participants to submit sequences of 1-3 optimal sequencing primers. Submitted primers ranged from 17-24 nt and largely conformed to the preferred parameters. Submitted primers were distributed across the test sequence, although some sites were disfavored. Surprisingly, approximately 45% of the primers were selected "manually", more than by any software package. Each of 69 submitted and 95 control primers, distributed at 3-bp intervals across the test sequence, were synthesized, purified and tested using a Model 377 PRISM DNA Sequencer with dichlororhodamine dye terminator reagents (dRhodamine dye terminators). Approximately half of the control primers were also tested using rhodamine dye terminator reagents ("old" rhodamine dye terminators). The results indicated that primer physico-chemical characteristics thought to have a strong impact on sequencing performance had surprisingly little effect. Thus, primers with high or low percent G + C or Tm, strong secondary priming scores or long 3' homopolymeric stretches yielded excellent sequences with the dRhodamine dye terminator reagents, although these characteristics had a stronger effect when the old rhodamine reagents were used. The old rhodamine reagents gave sequences with a similar average read length, but the number of errors and ambiguities or "N's" was consistently higher. Moreover, the effects of the primer physico-chemical characteristics were also more evident with the old rhodamine dyes. We conclude that under optimal sequencing conditions with highly pure template and primer, many of the commonly applied primer design parameters are dispensable, particularly when using one of the new generation of sequencing reagents such as the dichlororhodamine dye terminators.

Diltiazem causes open channel block of recombinant 5-HT3 receptors.

1. To extend our knowledge of the site and mechanism of action of L-type Ca2+ channel antagonists on 5-HT3 receptors, whole-cell voltage clamp electrophysiology was used to investigate the action of one of these compounds, diltiazem, on the recombinant receptor expressed in human embryonic kidney (HEK) 293 cells. 2. Application of diltiazem with 5-HT (30 microM) caused an increase in the rate of receptor current decay, but did not significantly affect peak current (Ip), the EC50 or the Hill coefficient, indicating a non-competitive mechanism of action. Pre-application of the antagonist had no effect indicating that diltiazem mediates its effects by binding preferentially to the open state of the 5-HT3 receptor. 3. To examine the effects of diltiazem on the open state of the receptor in more detail we used 10 mM 5-hydroxyindole (5-OHi) to reduce receptor desensitisation. These experiments showed that diltiazem causes a rapid, reversible, block in the presence of agonist but can become trapped in the unliganded state of the receptor by prior washout of agonist. Dose-inhibition data yielded an IC50 of 5.5 microM and a Hill coefficient of 0.96; inhibition was slightly voltage dependent as the degree of blockade at +60 mV was reduced. 4. The Hill coefficient of near unity suggests a single molecule of diltiazem mediates inhibition and, indeed, kinetic analysis verified that the interaction of diltiazem with the 5-HT3 receptor was well described by a bimolecular reaction scheme. The results suggest that diltiazem acts by causing open-channel block of the 5-HT3 receptor.

Survey of current trends in DNA synthesis core facilities.

The Nucleic Acids Research Group of the Association of Biomolecular Resource Facilities (ABRF) last surveyed DNA synthesis core facilities in April 1995. Because of the introduction of new technologies and dramatic changes in the market, we sought to update survey information and to determine how academic facilities responded to the challenge presented by commercial counterparts. The online survey was opened in January 1999 by notifying members and subscribers to the ABRF electronic discussion group. The survey consisted of five parts: general facility information, oligonucleotide production profile, oligonucleotide charges, synthesis protocols, and trends in DNA synthesis (including individual comments). All submitted data were anonymously coded. Respondents from DNA synthesis facilities were primarily from the academic category and were established between 1984 and 1991. Typically, a facility provides additional services such as DNA sequencing and has upgraded to electronic ordering. There is stability in staffing profiles for these facilities in that the total number of employees is relatively unchanged, the tenure for staff averages 5.9 years, and experience is extensive. On average, academic facilities annually produce approximately 1/16 the number of oligonucleotides produced by the average commercial facilities, but all facilities report an increase in demand. Charges for standard oligonucleotides from academic facilities are relatively higher than from commercial companies; however, the opposite is true for modified phosphoramidites. Subsidized facilities charge less than nonsubsidized facilities. Synthesis protocols and reagents are standard across the categories. Most facilities offer typical modifications such as biotinylation. Despite the competition by large commercial facilities that have reduced costs dramatically, academic facilities remain a stable entity. Academic facilities enhance the quality of service by focusing on nonstandard oligonucleotides and valuable services such as personal consultations, electronic ordering, and diversifying into other services.

Direct inhibition of 5-hydroxytryptamine3 receptors by antagonists of L-type Ca2+ channels.

Homopentameric complexes of either the A or As subunit of the 5-hydroxytryptamine3 receptor form Ca(2+)-permeable channels that can be activated by the selective agonist 1-(m-chlorophenyl)-biguanide (mCPBG). In both N1E-115 neuroblastoma cells and human embryonic kidney 293 cells stably expressing the 5-HT3 receptor As subunit, (+)-verapamil, (-)-verapamil, diltiazem, and nimodipine caused reversible and concentration-dependent (IC50 = 2.5-6.5 microM) inhibition of the increases in cytosolic [Ca2+] evoked by mCPBG. In voltage-clamped human embryonic kidney 293 cells stably expressing the 5-HT3 receptor As subunit, similar concentrations of the Ca2+ channel antagonists (IC50 = 3.0-6.8 microM) accelerated the rate at which 5-HT-evoked currents decayed without affecting the amplitude of the peak current. In equilibrium competition binding assays to membranes from Sf9 cells infected with the 5-HT3 receptor As subunit, [3H]mCPBG and [3H]granisetron were displaced by (+)-verapamil, (-)-verapamil, and diltiazem; (+)-verapamil was approximately 10-fold more potent than (-)-verapamil and approximately-30-fold more potent than diltiazem. Nimodipine neither displaced [3H]granisetron binding nor affected its displacement by diltiazem and (+)-verapamil. The stereoselectivity of verapamil binding, which contrasts with the similar potency of each isomer in functional assays, was maintained when the incubations were performed at 20 degrees or when an antagonist of the 5-HT3 receptor, [3H]granisetron, was used as the radioligand. The interaction between verapamil and either [3H]mCPBG or [3H]granisetron binding was not competitive. We conclude that the inhibition of [3H]mCPBG binding by diltiazem and verapamil is mediated by a site that is distinct from both the agonist-binding site and from the site through which nimodipine inhibits 5-HT3 receptor function. Our results provide evidence for allosteric regulation of agonist binding to 5-HT3 receptors and the first example of a ligandgated ion channel whose function is directly inhibited by members of all three major classes of L-type Ca2+ channel antagonists.

Bradykinin analogs as inhibitors of angiotensin-converting enzyme.

A series of peptide analogs and fragments of bradykinin were designed and synthesized on solid supports using Boc and Fmoc strategies, and on polyethylene pins using Fmoc strategy. The peptides were purified, characterized and tested for their inhibitory effects on angiotensin-converting enzyme. The inhibition of the converting enzyme. The inhibition of the enzyme was measured spectrophotometrically using Furylacryloyl-Phe-Gly-Gly as the substrate. Apparent Ki's were determined for the substrates, which exhibited significant inhibition in the initial screening assay using 10 microM of the peptide inhibitor. Short peptides corresponding to the carboxyl terminus of bradykinin were found to be poor inhibitors of angiotensin-converting enzyme. However, bradykinin-like peptides with modifications at their amino terminus are effective inhibitors. The best inhibitor found in this study, Ala2,6-des-Pro3-bradykinin, has an apparent Ki of 30.2 nM, compared to an apparent Ki of 94 nM for des-Pro3-bradykinin, which was reported to be a better inhibitor of angiotensin-converting enzyme than captopril.