Choline uptake by cultured neurones from the central nervous system of embryonic cockroaches

Molecular cloning and functional characterization of a neuronal choline transporter from

“…Infection of Sf9 cells with TrnCHTrecombinant baculovirus, however, doubled the rate of [ 3 H]-choline accumulation, with the saturable Na + -dependent component of [ 3 H]-choline being sufficiently above the endogenous rate in intact cells to make a kinetic analysis of TrnCHT possible. The affinity of TrnCHT for choline ðK m ¼ 8:4 mMÞ is lower than that reported for high-affinity choline uptake described in synaptosomes isolated from the locust CNS (K m ¼ 0:98 mM; Breer, 1982) and cockroach nerve cells in vitro (K m ¼ 0:5 mM; Bermudez et al, 1985) but close to the range reported for high-affinity mammalian CHTs ðK m ¼ 125 mMÞ Haga, 2000, 2003;Apparsundaram et al, 2000). The affinity of TrnCHT for choline is considerably higher than that of Na + -independent choline transporters reported from the locust (K m ¼ 25 mM; Breer, 1982) and mammalian CNS (K m ¼ 302100 mM; Lockman and Allen, 2002).…”

“…We suspect that the rapid binding kinetics of choline to its binding site on the TrnCHT protein, relative to those of HC3 and the other structural analogues tested, partially obscured the stronger inhibition that longer term incubation may have revealed, although this remains to be shown experimentally. Choline uptake by cultured cockroach neurons is also relatively insensitive to inhibition by HC3 (Bermudez et al, 1985). The apparent weak inhibition of some insect CHTs by HC3 conflicts with published data on the binding kinetics of [ 3 H]-HC3 to insect synaptosomes.…”

“…The TrnCHT protein, on the other hand, is most likely localized to cholinergic axons and synapses in the ganglionic neuropile. Bermudez et al (1985) showed by autoradiography that the majority of the axons of cockroach neurons cultured in vitro were labelled after incubation in [ 3 H]-choline, but their somata remained unlabelled. The antibody raised by Knipper et al (1989c) against the locust choline transporter failed to stain the cortex and perineurial sheath of the mesothoracic ganglion or the ganglionic connectives.…”

“…The uptake of [ 3 H]-choline by TrnCHT occurred at Na + levels above 20 mM and failed to saturate at levels as high as 140 mM, suggesting that the extra-axonal synaptic space in caterpillars has a Na + concentration in excess of this. Li + is a poor substitute for Na + in the cation requirement of insect choline transporters (Breer, 1983;Bermudez et al, 1985). The uptake of [ 3 H]-choline by locust synaptosomes peaks in about 150 mM NaCl, corresponding to a K m for Na + of between 40 and 60 mM (Breer, 1982).…”

“…Synaptic choline transport in the insect CNS was first studied by Breer (1982) and Bermudez et al (1985). A high-affinity choline transport protein was successfully isolated from locust synaptosomes and purified with the use of monoclonal antibodies (Knipper et al, 1989a(Knipper et al, , 1991.…”