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Transportans 55
Indeed, galparan is a high affinity ligand for the galanin receptor present in the
Bowes melanoma cell line. 7 Unexpectedly, in vivo studies on acetylcholine release
in rat brain showed that galparan dose dependently increased acetylcholine release
in a PTX-dependent manner, 10 as could be expected from a mastoparan-based peptide,
but was opposite to the effects of galanin. In a further study, galparan, contrary
to mastoparan, was shown to stimulate the Na + /K + -ATPase in rat hippocampal
membranes where galanin had no effect. 7 Thus, it is obvious that galparan is a peptide
with different properties from those of the parent peptides.
Moreover, galparan was shown to be a strong stimulator of the release of insulin
from rat β-cells. 11 Again, contrary to the effects of the parent peptides, the effect is
reversible and not dependent on PTX- or CTX-sensitive G proteins, PKA, PKC, or
intracellular Ca 2+ levels.
In principle, the effects of galparan could be caused by extra- or intracellular
action of the peptide. In order to follow the mode of binding and cellular localization
of galparan, a labeled peptide was required. It was expected that the free N terminus
of galparan would be necessary for binding to galanin receptors in Bowes cells as
well as for accomplishing its effects in other assays. Consequently, it was necessary
to introduce a reporter group into the peptide chain.
Because a substitution of Pro 13 to a Lys in the galanin peptide did not influence
the affinity of the peptide for the galanin receptor, 12 a similar substitution of the Pro
to Lys was introduced in galparan. 5 The Lys residue possesses an amino group on
its side chain that is a convenient attachment point for a suitable reporter group.
Biotin was used as a reporter group because it was considered a less disturbing
modification compared to the widely used fluorophores, which possibly interact with
plasma membranes. Indirect detection of biotin is more time consuming than direct
visualization of a chromophore, but enables greater flexibility of methods. 5
Strikingly, whereas cells incubated with similarly substituted galanin showed
almost no intracellular staining in indirect immunofluorescence experiments, those
incubated with the biotinylated galparan analogue were heavily labeled both in the
cytoplasm and nucleus. In the hope that the new peptide could be used as a carrier
vector, the peptide was named transportan. 5
3.3 CELL PENETRATION OF TRANSPORTAN
Transportan is a 27-amino-acid-long peptide (Table 3.1) with properties of a typical
cell-penetrating peptide. It enters cells rapidly at both physiological and low temperature,
predominantly without using endocytosis or active transport. 5
Incubation of cells with biotinyl-transportan-containing culture medium for
1 min at 37°C is sufficient for insertion of the peptide into plasma membrane, where
it is easily detected by indirect immunofluorescence. In the following 5 to 10 min,
transportan distributes to the nuclear envelope and other intracellular membranous
structures. Subsequently, it concentrates in the nuclear envelope and inside the nuclei
(Figure 3.1A). Lowering the incubation temperature decelerates but does not abolish
translocation of transportan into cells (Figure 3.1D). The distribution of biotinyltransportan
in cells incubated at 37°C or 0 to 4°C is very similar, showing a
preference of the peptide for membrane structures.