In order to identify functionally important regions of a neuropeptide gene in D. melanogaster (DM), the occurrence of FMRFamid in related species was studied (Taghert, 1990) and the structure of a homologous gene in D. virilis (DV) was characterized. The DM gene encodes a precursor that contains 13 neuropeptides related to the molluscan tetrapeptide FMRFamide. Using the DM gene as a probe in Southern blot analysis, related sequences were detected in DNA from each of 7 species tested. DV, which is estimated to have diverged from DM between 60 and 80 million years ago, was chosen for more detailed study. Immunocytochemical staining using an antibody to authentic FMRFamide reveals a similar set of immunoreactive neurons in the CNS of larvae from the 2 Drosophila species. Using a DM gene probe, overlapping clones were isolated from a DV genomic library; DNA sequence analysis indicates the presence of a homologous gene. A comparison of the genes and deduced proteins between the two species finds the following:

1. Both genes are divided into 2 exons: in DM the exons are 106 and 1352 bp long; in DV, they are 169 and at least 1232 bp long; in both species, the intron is approximately 2.5 kb long.
2. The sequence of exon I has largely diverged, and in neither species are exon I sequences translated. In this vicinity of the gene, sequence conservation is limited to a 67 bp region that spans the TATA box and the RNA start site.
3. The deduced neuropeptide precursors have very similar sizes (347 vs 339 amino acids) and the presumed signal sequences are perfectly conserved.
4. While the DM precursor contains 13 FMRFamide-related peptides, the DV precursor contains only 10.
5. The sequences of some but not all of the FMRFamide-like peptides are perfectly conserved.
6. In the rest of the precursor, significant sequence conservation is found only in the N-terminal portion; immediately downstream of the final FMRFamide-like peptide, the protein sequences are highly divergent.
7. 5' to the RNA start sites (1.2 kb of DM DNA and 1.8 kb of DV DNA), 17 small (9-52 base pairs) regions are evolutionarily conserved (greater than 80% sequence conservation) (Taghert, 1990).

The organization of the Drosophila gene contrasts in several ways with that of the homologous gene in the marine mollusc, Aplysia. First, the structure of the two genes are different: the Aplysia precursor is encoded by at least two exons, while the entire Drosophila precursor is encoded within a single exon. Second, the splicing strategy of the Aplysia gene appears more complex. Sequencing of different Aplysia cDNAs indicates that the primary transcript can be spliced within the peptide-encoding exon to produce varying numbers of FMRFamide peptides in different mature transcripts. Furthermore, multipe transcripts that have different sizes can be detected in Aplysia neural tissue. Finally, the two genes encode different members of the FMRF amide family of neuropeptides. The Aplysia gene encodes 28 copies of the sequence FMRFG, and a single copy of FLRFG. The Drosophila gene encodes 13 hepta- and nonapeptides that represent amino-terminal extensions of the FMRFamide sequence and the copy number of each individual peptide sequence varies from 1 to 5 (Schneider, 1990).

An extract of adult Drosophila melanogaster was separated by gel exclusion, ion exchange, and reversed-phase chromatography. Four peptides, each with an -ArgPheNH2 C-terminal sequence, were identified by radioimmunoassay. The primary sequences were determined by Edman degradation and confirmed by mass spectrometry and sequence-specific radioimmunoassay. Three of the peptides are encoded by Drosophila proFMRFamide:

• AspProLysGlnAspPheMetArgPheNH2 (DPKQDFMRFamide)

• ThrProAlaGluAspPheMetArgPheNH2 (TPAEDFMRFamide)

• SerAspAsnPheMetArgPheNH2 (SDNFMRFamide)

A novel Drosophila peptide ThrAspValAspHisValPheLeuArgPheNH2 (TDVDHVFLRFamide) was also isolated. TDVDHVFLRFamide is structurally related to peptides isolated from chicken, cockroach, locust, and snail; the cockroach, fruitfly, and locust peptides differ only by the N-terminal amino acid residue. Two Drosophila neural genes, dsk and FMRFamide, are known to encode -ArgPheNH2-containing peptides; however, neither encodes TDVDHVFLRFamide, indicating that Drosophila contains another precursor encoding -ArgPheNH2 peptides (Nichols, 1992a).

FMRFamide genes

Numerous FMRFamide-related peptides (FaRPs) have been isolated and sequenced from extracts of free-living and parasitic nematodes. KHEYLRFamide (AF2) is the most abundant FaRP identified in the parasitic forms Ascaris suum and Haemonchus contortus, as well as in the free-living nematode, Panagrellus redivivus. Analyses of the nucleotide sequences of cloned FaRP-precursor genes from C. elegans and, more recently, Caenorhabditis vulgaris, have identified a series of related FaRPs that do not include AF2. An acid-ethanol extract of C. elegans was screened radioimmunometrically for the presence of FaRPs using a C-terminally directed FaRP antiserum. The most abundant immunoreactive peptide was purified to homogeneity. The primary structure of the heptapeptide is Lys-His-Glu-Tyr-Leu-Arg-Phe-NH2 (AF2). These results indicate that C. elegans possesses more than one FaRP gene (Marks, 1995).

To date, 9 FMRFamide-related peptides (FaRPs) have been identified in C. elegans. Eight of these peptides are encoded by the flp-1 gene. However, AF2 (KHEYLRFamide), which was not co-encoded, is the most abundant FaRP identified in ethanolic extracts. Further radioimmunometrical screening of acidified ethanol extracts of C. elegans has revealed the presence of other novel FaRPs, that are not encoded on the flp-1 gene. One of these peptides has been isolated by sequential rpHPLC and subjected to Edman degradation analysis and gas-phase sequencing. The unequivocal primary structure of the decapeptide Ala-Pro-Glu-Ala-Ser-Pro-Phe-Ile-Arg-Phe-NH2 was determined following a single gas-phase sequencing run. The molecular mass of the peptide is 1133.7 Da. Synthetic replicates of this peptide induce a profound relaxation of both dorsal and ventral somatic muscle-strip preparations of Ascaris suum with a threshold for activity of 10 nM. The inhibitory response is not dependent on the presence of nerve cords, indicating a post-synaptic site-of-action. The relaxation is Ca(+2)- and Cl(-)-independent but is abolished in high-K+ medium and can be distinguished from those of other inhibitory nematode FaRPs, including PF1 (SDPNFLRFamide) and PF4 (KPNFIRFamide).

To date, seven FMRFamide-related peptides (FaRPs) have been structurally characterized from C. elegans, of which one is structurally identical to the parasitic nematode peptide AF2 (KHEYLRFamide). The other six FaRPs have so far been identified in free-living forms only. In the present study an additional FaRP was isolated and structurally characterized from an ethanolic extract of C. elegans. The extract was screened using a C-terminally directed FaRP antiserum, and the FMRFamide-immunoreactive peptide purified to homogeneity using HPLC. Approximately 80 pmol of the peptide was subjected to Edman degradation and the unequivocal primary structure of the K7-amide, KSAYMRFamide (PF3/AF8) was determined following a single gas-phase sequencing run. The molecular mass of the peptide was found to be 919 (MH+), which is in agreement with the theoretical mass of C-terminally amidated PF3. A new flp-gene, designated flp-6, has recently been identified that encodes six copies of KSAYMRFamide (PF3/AF8) (Marks, 1998).

Neuropeptides serve as important signaling molecules in the nervous system. The FMRFamide (Phe-Met-Arg-Phe-amide)-related neuropeptide gene family in the nematode Caenorhabditis elegans is composed of at least 18 genes that may encode 53 distinct FMRFamide-related peptides. Disruption of one of these genes, flp-1, causes numerous behavioral defects, including uncoordination, hyperactivity, and insensitivity to high osmolarity. Conversely, overexpression of flp-1 results in the reciprocal phenotypes. On the basis of epistasis analysis, flp-1 gene products appear to signal upstream of a G protein-coupled second messenger system. These results demonstrate that varying the levels of FLP-1 neuropeptides can profoundly affect behavior and that members of this large neuropeptide gene family are not functionally redundant in C. elegans (Nelson, 1998).

A gene encoding multiple FMRFamide-like peptides has been idenified in the necromenic nematode Caenorhabditis vulgaris. This gene, Cv-flp-1, shares strong sequence homology in the coding regions with the flp-1 gene from the related free-living soil nematode C. elegans. The predicted neuropeptide precursor proteins differ by only four conservative amino acid changes, none of which affects sequences of the predicted neuropeptides. DNA sequences in the non-coding areas are less conserved, but areas of sequence homology are found in introns and in 3' and 5' non-translated regions, suggesting some functional significance for these conserved regions. In C. vulgaris, as was found in C. elegans, two transcripts are presumably produced as a result of use of an alternative 3' splice acceptor site. Lastly, an antibody specific for the RF-moiety of FMRFamide stains a similar subset of cells in C. elegans and C. vulgaris. These results indicate that the function and regulation of the peptides are likely to be conserved in both species (Schinkmann, 1994).

A gene, afp-1, has been identified that encodes a new subfamily of six FMRFamide-like neuropeptides in the nematode Ascaris suum. The predicted peptides share the C-terminal sequence PGVLRF-NH2 but have different N-terminal extensions. Possible functional roles of these different peptides are discussed based upon experiments with Ascaris as well as results from other organisms. Three of the peptides have been isolated from extracts of A. suum and three other are novel sequences. The translated product of afp-1 is a precursor protein containing two main regions: a C-terminal region containing a series of putative peptides separated by characteristic processing sites and a relatively hydrophobic N-terminal region with no obvious peptides. Although the overall structure of the translated product of afp-1 is similar to flp-1 from C. elegans, there is little evidence for homology between the two nematode neuropeptide genes. At least four different transcripts of afp-1 have been identified. These transcripts differ in their 3' and 5' untranslated regions, and one of the transcripts predicts a truncated precursor protein that contains only the C-terminal peptide-containing region (Edison, 1997).

In the CNS of the snail Lymnaea stagnalis, Phe-Met-Arg-Phe-amide (FMRFamide)-like and additional novel neuropeptides are encoded by a common, multi-exon gene. This complex locus, comprising at least five exons, is subject to post-transcriptional regulation at the level of alternative RNA splicing. Exons III, IV and V are always coexpressed and colocalized whereas the expression of exon II is always differential and mutually exclusive. Both sets of exons are, however, coexpressed with exon I: the total number of exon I-expressing neurons is equal to the combined number of neurons expressing exon III/IV/V and neurons expressing exon II. In addition, the extreme 5' of exon II, encoding a potential hydrophobic leader signal, is not expressed in the CNS of Lymnaea but is apparently spliced out during RNA processing. Both mRNA transcripts of the FMRFamide locus, type 1 (exons I/II) and type 2 (exons I/III/IV/V), are translated in the CNS and the resulting protein precursors are also expressed in a mutually exclusive fashion, as are their respective transcripts. The expression of alternative transcripts within identified networks or neuronal clusters is heterogeneous, as exemplified by the cardiorespiratory network (Santama, 1996).

FMFR amide developmental expression

Hydra magnipapillata has three distinct genes coding for preprohormones A, B, and C, each yielding a characteristic set of Hydra-RFamide (Arg-Phe-NH2) neuropeptides, and a fourth gene coding for a preprohormone that yields various Hydra-LWamide (Leu-Trp-NH2) neuropeptides. Using a whole-mount double-labeling in situ hybridization technique, it was found that each of the four genes is specifically expressed in a different subset of neurons in the ectoderm of adult Hydra. The preprohormone A gene is expressed in neurons of the tentacles, hypostome (a region between tentacles and mouth opening), upper gastric region, and peduncle (an area just above the foot). The preprohormone B gene is exclusively expressed in neurons of the hypostome, whereas the preprohormone C gene is exclusively expressed in neurons of the tentacles. The Hydra-LWamide preprohormone gene is expressed in neurons located in all parts of Hydra with maxima in tentacles, hypostome, and basal disk (foot). Studies on animals regenerating a head show that the prepro-Hydra-LWamide gene is expressed first, followed by the preprohormone A and subsequently the preprohormone C and the preprohormone B genes. This sequence of events could be explained by a model based on positional values in a morphogen gradient. These head-regeneration experiments also give support for transient phases of head formation: first tentacle-specific preprohormone C neurons (frequently associated with a small tentacle bud) appear at the center of the regenerating tip, where they are then replaced by hypostome-specific preprohormone B neurons. Thus, the regenerating tip first attains a tentacle-like appearance and only later this tip develops into a hypostome. In a developing bud of Hydra, tentacle-specific preprohormone C neurons and hypostome-specific preprohormone B neurons appear about simultaneously in their correct positions, but during a later phase of head development, additional tentacle-specific preprohormone C neurons appear as a ring at the center of the hypostome and then disappear again. Nerve-free Hydra consisting of only epithelial cells do not express the preprohormone A, B, or C or the LWamide preprohormone genes. These animals, however, have a normal phenotype, showing that the preprohormone A, B, and C and the LWamide genes are not essential for the basic pattern formation of Hydra (Mitgutsch, 1999).

Developmental changes in the expression of a FMRFamide-like (Phe-Met-Arg-Phe-NH2) peptide or peptides in motoneurons of the tobacco hornworm, Manduca sexta were demonstrated using immunohistochemical techniques. The onset of FMRFamide-like immunoreactivity (FLI) is gradual during larval growth but by the final larval stage, immunoreactivity is present in the majority of motoneurons. FLI then declines during metamorphosis and is absent in all identified adult motoneurons. A novel in vivo culture system was used demonstrate that the steroid hormone, 20-hydroxyecdysone, regulates the loss of FLI in motoneurons during metamorphosis. The small commitment peak of ecdysteroid appears to shut off the program of neuropeptide accumulation that is characteristic of the larval state of the motoneurons. The prepupal peak of steroid release then causes the rapid loss of stored FLI. This steroid-induced change in the neuropeptide content of motoneurons may reflect major changes in neuromuscular functions between the larval and adult stages (Witten 1996).

Both allatostatin immunoreactivity (AS-IR) and FMRFamide immunoreactivity (FMRFa-IR) have been demonstrated light-microscopically in the lateral heart nerve of the cockroach, Periplaneta americana. The identical labeling of some fibers suggests the coexistence of the two antigens. Electron-microscopically, six granule types in the peripheral part of the lateral heart nerve can be distinguished according to their size and density (types 1-6). These granule types can be subdivided immunocytochemically by means of a new mirror-section technique. Granules of types 4 and 5 always show FMRFa-IR exclusively. In the populations of fibers containing granules of types 1 and 6, axon profiles can be found that contain granules colocalizing FMRFa-IR and AS-IR. Other axon profiles of these populations only contain immunonegative granules of the same ultrastructure. Granules of type 2 can be differentiated immunocytochemically in three forms in the same section: in some fibers, they are nonreactive; in other fibers of the same section, they show FMRFa - IR, whereas in a third fiber type, granules show AS-IR. Finally, granules of type 3 can be observed with FMRFa-IR. In other fibers, they occur with the same ultrastructure but exhibit no immunoreactivity. Two soma types occur in the lateral heart nerve. Soma type I is characterized by the production of electron-dense granules that show FMRFa-IR. Type II is in close contact with various fibers, forming different types of axosomatic synapses, hitherto unknown in Insecta (Ude, 1995).

Immunoreactivity against RFamide-like peptides of the medicinal leech Hirudo medicinalis reveal elaborate neuronal aborizations of a neuron in the nephridium, around the urinary bladder sphincter and in the central nervous system. The processes arise from the nephridial nerve cell (NNC), a previously identified receptor neuron. Authentic FMRFamide has been identified as the major peptide of the NNC. Sensory and neurosecretory innervation of the nephridia is thus accomplished by a single neuron, which is thought to modulate nephridial performance (Wenning, 1993).

The FMRFamide receptor

Numerous peptides are structurally related to the cardioexcitatory tetrapeptide FMRFamide. One subgroup of FMRFamide-related peptides (FaRPs) contains an FMRFamide C terminus. Searches of the Drosophila melanogaster genome database identified the first invertebrate FMRFamide G-protein coupled receptor (GPCR), DrmFMRFa-R. In order to explore molecular mechanisms involved in FMRFamide signal transduction, a receptor was cloned from the malaria mosquito Anopheles gambiae genome, AngFMRFa-R, and its structure was compared to that of DrmFMRFa-R. The cytoplasmic loops, extracellular loops, and transmembrane regions are highly conserved between these two FMRFamide receptors. Another subgroup of FaRPs is the sulfakinins, which are represented by the consensus structure -XDYGHMRFamide, where X is D or E. AngFMRFa-R and DrmFMRFa-R were compared to the A. gambiae sulfakinin receptors, ASK-R1 and ASK-R2, and the D. melanogaster sulfakinin receptors, DSK-R1 and DSK-R2, were compared. The cytoplasmic loops, extracellular loops, and the transmembrane regions are not highly conserved between the FMRFamide and sulfakinin receptors. In order to explore the role of FMRFamide in mosquito biology the effect of the tetrapeptide on in vivo heart rate was measured. The tetrapeptide increases the frequency of spontaneous contractions of the larval mosquito heart and, thus, increases heart rate. These data support the conclusion that the structure of the FMRFamide receptor and activity of the cardioexcitatory FMRFamide neuropeptide are conserved in mosquito (Duttlinger, 2003).

FMRFamide, receptors and channels

SKPYMRFamide, a novel FMRFamide-like endogenous peptide reversibly decreases excitatory responses (depolarization and inward current) evoked by local ionophoretic application of acetylcholine (ACh) onto the soma of identified neurons F1, F2, F4 and F5/6 of the land snail, Helix aspersa. SKPYMRFamide inhibits ACh receptors through activation of specific binding sites on the plasma membrane. The possible role of different second messengers in the modulatory influence of SKPYMRFamide on ACh receptors was tested using 13 modulators of different second messenger systems. The results indicate that SKPYMRFamide may inhibit ACh receptors through activation of one or more of the following systems: phospholipases C, A2, NO-synthase, soluble guanylate cyclase and lipoxygenases that elevate basal intracellular levels of NO. Additional systems might include cGMP, arachidonic acid, acyclic eicosanoids, inositol-1,4,5-trisphosphate (I(1,4,5)P3), I(1,4,5)P3-dependent Ca(2+)-mobilization followed by activation of calmodulin and Ca2+/calmodulin-dependent protein kinase II. Protein kinases A, C and cyclic eicosanoids do not appear to participate in modulatory action of SKPYMRFamide (Pivovarov, 1995).

The peptide neurotransmitter Phe-Met-Arg-PheNH2 (FMRFamide) increases outward K+ currents and promotes dephosphorylation of many phosphoproteins in Aplysia sensory neurons. FMRFamide-induced current responses were examined in sensory neurons injected with thiophosphorylated protein phosphate inhibitor-1 and inhibitor-2 (I-1 and I-2), two structurally different vertebrate protein phosphatase-1 (PP1) inhibitors, to define a role for PP1 in the physiological actions of FMRFamide. Thiophosphorylated I-1 and I-2 both reduce the amplitude of outward currents elicited by FMRFamide by 50-60%, and are as effective as microcystin-LR, which inhibited both PP1 and protein phosphatase-2A in Aplysia neuronal extracts. These data suggest that of the two major neuronal protein serine/threonine phosphatases, FMRFamide utilizes primarily PP1 to open serotonin-sensitive K+ (S-K+) channels. Earlier studies have shown that a membrane-associated phosphatase regulates S-K+ channels in cell-free patches from sensory neurons. Utilizing its unique substrate specificity and inhibitor sensitivity, PP1 has been characterized as the principal protein phosphatase associated with neuronal plasma membranes. Two protein phosphatase activities (apparent M[r] values of 170,000 and 38,000) extracted from crude membrane preparations from the Aplysia nervous system are shown to be isoforms of PP1. These biochemical and physiological studies suggest that PP1 is preferentially associated with neuronal membranes and that its activity may be required for the induction of outward K+ currents by FMRFamide in the Aplysia sensory neurons (Endo, 1995).

The optic lobe of squid (Loligo pealei) contains FMRFamide receptors that can bind an iodinated FMRFamide analog: [125I]-desaminoTyr-Phe- norLeu-Arg-Phe-amide ([125I]-daYFnLRFa). Squid FMRFamide receptors are specific, saturable, high affinity sites densely concentrated in optic lobe membranes. The receptors appear to be coupled to Gs because guanine nucleotides inhibit receptor binding and the stimulation of adenylate cyclase by FMRFamide is GTP-dependent. Both the binding and cyclase data show that FMRFamide, but not FMRF-OH, interacts at FMRFamide receptors; thus the C-terminal Arg-Phe-amide is critical for binding. The high binding affinity of FMRFamide is specific for FMRFamide-like peptides. The structure-activity relations of many FMRFamide analogs are defined in detail and are nearly identical for both the membrane-bound and detergent-solubilized receptors. Squid optic lobe contains FMRFamide-like reactivity as measured with both a radioimmunoassay and a radioreceptor assay. Moreover, a fragment of genomic DNA that encodes a FMRFamide precursor has been sequenced. These findings suggest that FMRFamide is a neurotransmitter in squid optic lobe, and that this tissue is a good source from which to purify FMRFamide receptors (Chin, 1994).

A nonpeptide mimetic analog of an invertebrate peptide receptor has been identified. Benzethonium chloride (Bztc) is an agonist of the SchistoFLRFamide (PDVDHVFLRFamide) receptors found on locust oviducts. Bztc competitively displaces SchistoFLRFamide binding to both high- and low-affinity receptors of membrane preparations. Bztc mimics the physiological effects of SchistoFLRFamide on locust oviduct, by inhibiting myogenic and induced contractions in a dose-dependent manner. Bztc is therefore recognized by the binding and activation regions of the SchistoFLRFamide receptors (Lange, 1995).

Ion channels with characteristics of Ca2+ channels have been found in isolated heart ventricle cells of the snail Lymnaea stagnalis. Although spontaneous Ca2+ or Ba2+ currents are seen only occasionally, spontaneous inward Na+ currents are readily observed in the absence of patch pipette Ca2+ between membrane potentials of -100 mV and +20 mV. When currents are blocked by Ni2+, Co2+ and Ca2+, the channels usually cease conducting within a few minutes after seal formation with the patch pipette and cannot be re-activated with depolarizing voltage steps. However, at the cell's resting potential, the molluscan cardioactive peptide FMRFamide or its analog, FLRFamide2+, applied to the cell membrane away from the patch pipette, induces unitary Ba2+ currents or, in the absence of Ca2+ in the patch pipette, Na+ currents. This suggests that a secondary messenger is involved in the FMRFamide-induced activation of these channels rather than a direct activation of a channel-receptor complex by the peptide (Brezden, 1992).

The effects were determined of D-Ala2-Leu-enkephalin (DALEU), D-Ala2-Met-enkephalin (DAMET), and FMRFamide on the metacerebral cell (MCC) of Aplysia. Distinct receptors exist on this neuron for the three substances. DALEU elicits a depolarizing response due to an inward current, notably unaccompanied by a significant change in membrane conductance. In contrast, DAMET elicits a hyperpolarizing response due to an outward current, also not associated with a significant change in membrane conductance. Both the DALEU and the DAMET responses increase with hyperpolarization, decrease with depolarization, but do not reverse at potentials less than -30 mV. Neither response is sensitive to naloxone. FMRFamide induces a voltage-dependent outward current that reverses at about -76 mV. This neuron is responsive to much lower concentrations of FMRFamide than either of the enkephalins, and the response to FMRFamide appears to be a conductance increase to K+. These results suggest that the MCC neuron has distinct receptors for Leu- and Met-enkephalin that activate unusual responses of opposite polarity, as well as more usual inhibitory responses to FMRFamide (Kemenes, 1992).

The Helix aspersa Phe-Met-Arg-Phe-amide (FMRFamide)-gated sodium channel is formed by homomultimerization of several FMRFamide-activated Na+ channel (FaNaCh) proteins. FaNaCh is homologous to the subunits that compose the amiloride-sensitive epithelial sodium channel; to C. elegans degenerins, and to acid-sensing ionic channels. FaNaCh properties were analyzed in stably transfected human embryonic kidney cells (HEK-293). The channel is functional with an EC50 for FMRFamide of 1 microM and an IC50 (25°C) for amiloride of 6.5 microM as assessed by 22Na+ uptake measurements. The channel activity is associated with the presence of a protein at the cell surface with an apparent molecular mass of 82 kDa. The 82-kDa form was derived from an incompletely glycosylated form of 74 kDa found in the endoplasmic reticulum. Formation of covalent bonds between subunits of the same complex were observed either after formation of intersubunit disulfide bonds following cell homogenization and solubilization with Triton X-100 or after the use of bifunctional cross-linkers. This results in the formation of covalent multimers that contain up to four subunits. Hydrodynamic properties of the solubilized FaNaCh complex also indicate a maximal stoichiometry of four subunits per complex. It is likely that epithelial Na+ channels, acid-sensing ionic channels, degenerins, and the other proteins belonging to the same ion channel superfamily also associate within tetrameric complexes (Coscoy, 1998).

Acidosis is associated with inflammation and ischemia and activates cation channels in sensory neurons. Inflammation also induces expression of FMRFamide-like neuropeptides, which modulate pain. Neuropeptide FF (Phe-Leu-Phe-Gln-Pro-Gln-Arg-Phe amide) and FMRFamide (Phe-Met-Arg-Phe amide) generate no current on their own but potentiate H+-gated currents from cultured sensory neurons and heterologously expressed ASIC and DRASIC channels. The neuropeptides slow inactivation and induce sustained currents during acidification. The effects are specific; different channels show distinct responses to the various peptides. These results suggest that acid-sensing ion channels may integrate multiple extracellular signals to modify sensory perception (Askwith, 2000).

Some effects of FMRFamide and neuropeptide FF appear to be mediated through opioid receptors; these effects are blocked by the opioid antagonist naloxone. Yet, other effects of FMRFamide and FMRFamide-related peptides are independent of opioid receptors and are insensitive to naloxone. In mammals, the nonopioid receptor(s) for FMRFamide and related peptides has not been identified, and it is not known how these peptides modulate pain sensation. However, the discovery of a FMRFamide-activated Na+ channel (FaNaCh) in the mollusc Helix aspersa (Lingueglia, 1995) has provided a clue that similar receptors might exist in mammals (Askwith, 2000 and references therein).

Unlike many neuropeptide receptors, FaNaCh is an ion channel gated directly by its peptide ligand, FMRFamide (Lingueglia, 1995). The neuropeptide receptor FaNaCh is a member of the DEG/ENaC family of channels. DEG/ENaC channels are homo- or hetero-multimers composed of multiple subunits. Each subunit contains two transmembrane domains separated by a large, extracellular, cysteine-rich domain and cytosolic N and C termini. DEG/ENaC channels are not voltage gated and are cation selective (usually Na+ greater than K+). FaNaCh is the only known DEG/ENaC channel that acts as a neuropeptide receptor. Other members of this family are involved in mechanosensation, salt taste, and epithelial Na+ absorption. Although a mammalian FaNaCh has not yet been isolated, mammals do possess multiple DEG/ENaC family members. Interestingly, one subset of this channel family, the acid-sensing ion channels, has been postulated to play a role in sensory perception and may, like FMRFamide, play a role in pain perception. The acid-sensing DEG/ENaC channels respond to protons and generate a voltage-insensitive cation current when the extracellular solution is acidified (Askwith, 2000 and references therein).

The tissue acidosis associated with inflammation, infection, and ischemia causes pain. Acidosis also generates proton-dependent transient and sustained Na+ currents in cultured sensory neurons. Although the molecular identity of the channels responsible for these currents is unknown, they have been hypothesized to be acid-sensing members of the DEG/ENaC protein family based on their ion selectivity, voltage insensitivity, and expression pattern. The acid-sensing ion channels include the brain Na+ channel 1 (BNC1, also known as MDEG, BNaC1, and ASIC2) and its differentially spliced isoform, MDEG2; the acid-sensing ion channel (ASICalpha, also known as BNaC2 and ASIC1) and its differentially spliced isoform, ASICbeta; and the dorsal root acid-sensing ion channel (DRASIC, also known as ASIC3). BNC1, MDEG2, ASICalpha, and DRASIC are expressed in the central nervous system. ASICalpha, ASICbeta, DRASIC, and MDEG2 are expressed in sensory neurons of the dorsal root ganglia (DRG) (Askwith, 2000 and references therein).

It is hypothesized that FMRFamide or FMRFamide like-neuropeptides might modulate pH-dependent currents in DRG neurons. This hypothesis was based on the ability of FMRFamide and related peptides to modulate pain perception, the potential connections between painful stimuli and the acid-gated currents in DRG neurons, and the sequence similarity between FaNaCh and the acid-sensing ion channels expressed in sensory ganglia (Askwith, 2000).

The ability of neuropeptide FF and FMRFamide-related peptides to induce sustained currents suggests that such peptides and the acid-gated channels play a role in nociception. Interestingly, these peptides have been previously linked to pain perception in the spinal cord and brain. For example, chronic inflammation induces neuropeptide FF expression in the spinal cord. FMRFamide-related peptides may also contribute to opiate tolerance, in which increasing amounts of opiates are required to achieve the same analgesic effect. This may in part be explained by opiate-induced secretion of FMRFamide-related peptides from spinal cord neurons possibly inducing hypersensitivity of the nociceptive neurons (Askwith, 2000 and references therein).

These data may also have implications for DEG/ENaC function in the brain. For example, intracerebroventricular injection of FMRFamide-related peptides induces a variety of physiologic responses. Recently, it has been demonstrated that an amiloride analog inhibits FMRFamide-induced regulation of the brain renin-angiotensin system and hypertension. This suggests that these channels are a target of FMRFamide in the brain (Askwith, 2000 and references therein).

Proton-gated DEG/ENaC channels may function to integrate the response to acid and neuropeptides in the nervous system. Interestingly, another channel thought to be involved in nociception, the capsaicin receptor, also integrates multiple stimuli, heat and acidosis. Thus, H+-gated currents in neurons could vary, depending upon the type and combinations of DEG/ENaC subunits expressed and on the presence of different FMRFamide-like neuropeptides. The diversity of channel subunits and neuropeptides offers rich opportunities for interactions and new targets for pharmacotherapy (Askwith, 2000 and references therein).

Physiological effect of FMRFamides