trans

382 NEUROTRANSMITTERS
C-terminals are essential for biological activity.
The analog peptides had a similar or reduced
level of potency to AF1, providing evidence for
the existence of different receptors for AF1
and AF2. In PF1 and PF2 the N-terminal serine
and aspartic acid residues are found to be
unnecessary for biological activity. On the
other hand, the phenylalanine and arginine
residues (positions 1, 2 and 4 from the C-
terminal) are essential. In PF4, leucine in place
of isoleucine (position 3 from the C-terminal)
did not affect the biological activity, and
the proline (position 6 from the C-terminal)
rendered the peptide resistant to enzymatic
degradation. This proline also appeared to
contribute to the biological activity of the molecule,
probably due to its influence on tertiary
structure.
For comparison, the structure–function relationship
of the mammalian FaRP, NPFF, is
presented. The two N-terminal amino acids
are not essential for biological activity or receptor
affinity. The remaining N-terminal amino
acids were found to control molecular conformation
and hence receptor affinity, while the
C-terminal amino acids are necessary for biological
activity. Hence, affinity for the receptor
does not correlate with potency. In fact, substituted
FMRFamides bound with lower affinity
than FMRFamide, but had greater biological
activity. These differences may be due to
the enzyme-resistance of the FMRFamide
analog, which could confer greater biological
availability.
In a study of the molecular structure of
related FaRPs from molluscs, it was observed
that the number of turns in the molecule was
inversely correlated with the receptor binding.
Molecules that tended towards ‘extended’
(linear) conformations bind to receptors with
higher affinity than FaRP molecules that tended
towards a ‘turn’ (twisted) conformation. The
turn conformation is most likely to be seen
when an aspartic acid residue formed hydrogen
bonds with the protonated amide groups
of phenylalanine and leucine residues. Hence,
the presence of aspartic acid conspicuously
reduced the ability of the FaRP to bind to a
receptor. This work demonstrates the important
contribution of single amino acids to FaRP
structure and, therefore, function.
FaRP mechanisms of action in nematodes
FaRPs can cause hyperpolarization or depolarization
of A. suum muscle cells, and in some
cases change the input conductance as well.
Some of the nematode FaRPs have been investigated
further to elucidate the ways in which
signals from stimulated receptors are relayed
to the effector mechanisms of the cell. The
effects of PF1 and PF2 appear to be mediated
by nitric oxide, and their muscle relaxant
effect is blocked by compounds preventing
nitric oxide synthesis. In contrast, AF1 and
AF2 may act by increasing intracellular cyclic
AMP, and AF3 by decreasing it. PF4 appears to
open non-GABA chloride ion channels in the
somatic muscle cell membrane.
Alternative splicing
Another feature of nematode genetics that is
particularly relevant to the production of
nematode FaRPs is alternative splicing. An
example of alternative splicing in a FaRPencoding
gene has been documented in the
nematode C. elegans. The gene flp-1 can be
transcribed in two different ways. Each transcript
gives rise to eight FaRPs in C. elegans.
Seven are the same in both sets; the transcripts
differ in the eighth FaRP. One set has an
extra copy of the FaRP SADPNFLRFamide
(PF2), and the other has a copy of AGSDPNFLR-
Famide. It is postulated that the second FaRP
may be age- or sex-specific.
BIOCHEMISTRY AND CELL BIOLOGY: HELMINTHS