Dissociation of the vesicular acetylcholine transporter domains important for high-affinity transport recognition, binding of vesamicol and targeting to synaptic vesicles

J Physiology (Paris) (1998) 92, 141-144 © Elsevier, Paris

Dissociation of the vesicular acetylcholine transporter domains important for high-affinity transport recognition, binding of vesamicol and targeting to synaptic vesicles H. Varoqui,

J.D. Erickson

Neuroscience Center and Department of Pharmacology, Louisiana State University Medical School, 2020 Gravier Street, Suite D, New Orleans, Louisiana 70112, USA

Abstract w Chimeras between the human vesicular acetylcholine transporter (hVAChT) and the neuronal isoform of the human

vesicular monoamine transporter (hVMAT2) have been constructed and stably expressed in a rat pheochromocytoma cell line (PC12) in an effort to identify cholinergic-specific domains of VAChT. Examination of the transport properties of a chimera in which the N-terminal portion (up to putative transmembrane domain II and including the lumenal glycosylated loop) of hVAChT was replaced with hVMAT2 sequences (2/V@NheI) revealed that its apparent affinity for acetylcholine (ACh) was reduced approximately seven-fold compared to wild-type. However, the affinity of this chimera for vesamicol did not significantly differ from hVAChT. Similarly, the 21V@NheI chimera retained its preferential targeting to the small synaptic-like vesicles found in PC12 cells in agreement with our recently reported observations that the synaptic vesicle targeting domain resides in the cytoplasmic tail of VAChT. (©Elsevier, Paris)

R~sum~ n Dissociation des domaines du transporteur v~siculaire d'ac~tyicholine impliqu~s dans la reconnaissance du substrat, ia liaison du vesamicol, et I'adressage vers les vesicules synaptiques. Nous avons cherch~ ~ identifier les r6gions sp~cifiquement

cholinergiques du transporteur v~siculaJre d'ac~tylcholine (VAChT) ~tl'aide de proteines chim~riques humaines composdes de fragments de hVAChT et de l'isoforme neuronal du tranporteur vdsiculaire de monoamines (hVMAT2), exprimdes de fa~on stable dans des cellules PC I2 (pheochromocytomes de rat). L'affinit6 apparente pour l'acdtylcholine de la chim~re 21V@Nhel (hVMAT2 jusqu'au ddbut du domaine transmembranaire II, puis hVAChT) est rdduite 7 fois par rapport/~ hVAChT. L'affinit6 de cette chim~re pour le vdsamicol est quant ~ elle inchangde. De plus, cette chim&e 2/V@NheI, comme hVAChT, est adressde prdfdrentiellement vers les petites vdsicules de type synaptiques dans les cellules PC12. Ce resultat est en accord avec notre rdcente observation que le signal d'adressage vers les vdsicules synaptiques se situe h l'extrdmit6 C-terminale cytoplasmique de hVAChT. (©Elsevier, Pads) vesicular acetylcholine transporter (VAChT) / neuronal isoform of the vesicular monoamine transporter (VMAT2) / acetylcholine / vesamicol / synaptic vesicles / targeting

1. I n t r o d u c t i o n The vesicular acetylcholine transporter (VAChT) is responsible for packaging cytoplasmic acetylcholine (ACh) into the small synaptic vesicles of cholinergic neurons where it is available for regulated neurosecretion. Vesamicol, a neuromuscular blocking agent, disrupts this process by specifically inhibiting vesicular accumulation o f ACh. Recently, the m o l e c u l a r cloning and functional identification of VAChT has revealed that vesicular A C h transport and vesamicol binding properties are encoded by a single polypeptide [11]. VAChT exhibits approximately 40% identity with the two other members of this gene family, the endocrine-specific (VMAT1) and neuronal (VMAT2) isoforms o f the vesicular monoamine transporter [8]. Since these proteins all utilize a c o m m o n H+/antiport mechanism for vesicular substrate accumulation, chimeric transporters offer an opportunity to identify regions of these proteins which are important in de-

termining substrate and inhibitor specificity as well as specific targeting to cholinergic versus monoaminergic secretory organelles. Rat p h e o c h r o m o c y t o m a cells (PC12) synthesize both dopamine and ACh with expression o f rVMAT1 on large dense core vesicles (LDCV) and rVAChT on the small synaptic vesicle clusters which can be observed following treatment with nerve growth factor [13]. Recently, we have shown that structural information resides within the terminal c y t o p l a s m i c domain of hVAChT, which specifically targets it to the PC12 synaptic vesicles [12]. Overexpression of hVAChT in PC12 cells results in approximately a 20-fold increase in vesamicol-sensitive vesicular acc u m u l a t i o n of A C h [10]. This expression system should prove useful for the structure/function analysis o f VAChT and help to further establish a relationship between the activity of vesicular neurotransmitter transporters and vesicular storage and levels o f neurotransmitter available for regulated neurosecretion.

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2. Materials and methods

3. Results

Polyclonal antipeptide antibodies against the C-terminal amino acids of hVAChT [6] were affinity purified and previously characterized [10, 12]. Polyclonal antibodies against chromogranin B (CgB) were a generous gift of Reiner Fischer-Colbrie (Innsbrtick, Austria). Monoclonal antibodies directed against synaptophysin (p38) were purchased from Sigma.

The hVAChT and 2/V@NheI chimera could be d i s t i n g u i s h e d from the e n d o g e n o u s l y expressed rVAChT in stably transfected PC I2 cells lines by immunocytochemistry and Western blotting using a human-specific antibody. Furthermore, the binding of [3H]vesamicol and transport of [3H]ACh in postnuclear supernatants of PC12 cells expressing the human proteins was significantly higher than levels detected in PC I2 cells containing only the endogenous rVAChT. Uptake mediated by the endogenous rVAChT protein was less than t w o - f o l d greater than that observed in the presence of vesamicol or at 4 oC.

2.1.2/V@Nhel chimeric construction An NheI restriction site was introduced into both hVAChT and hVMAT2 cDNAs at an equilavent site within the putative 2nd transmembrane domain of these proteins (conserved amino acids Ala ]49 and Ser 15°) by site-directed mutagenesis [4]. Mutagenesis was performed using the full-length hVMAT2 cDNA in pCDM7amp. Since hVAChT has two internal NheI sites, the 5' HindlII/Sse8387I fragment of hVAChT was first subcloned into pUC18 where mutagenesis was performed. Transformants were screened by restriction analysis and verified by cDNA double-stranded sequencing using Sequenase II (U.S. Biochemical Corp.). The 5' region of hVMAT2 was then subcloned into the hVAChT fragment (at HindlII/NheI in pUC18) and the 2/V chimeric fragment (HindlII/Sse8387I) was subcloned back into hVAChT in the mammalian expression vector Rc/CMV (Invitrogen). 2.2. Transfection and selection of stable PCI2 cell lines Rat PC 12 cells were transfected with the 2/V @NheI construct using Lipofectin (10 mg/mL: Life Technologies, Inc.), and stable transformants were selected with 0.5 mg/mL Geneticin (G418; Life Technologies, Inc.) and screened by immunocytochemistry as previously described [10, 12]. 2.3. Vesicular acetylcholine transport and vesamicol binding assays ATP-dependent vesicular transport of [3H]-ACh (55.2 mCi/mmol, DuPont NEN) and the binding of [3H]-vesamicol (AH5183, L-[piperidinyl-3,4-3H]vesamicol; 31 Ci/mmol, DuPont NEN) were measured using postnuclear supernatants (800 g for 10 min) prepared from control, hVAChT-expressing or 2/V@NheI-expressing PCI 2 cells as previously described [9, 10]. Uptake of 3H-acetylcholine by control PC I2 cells was subtracted. Non-specific binding of 3H-vesamicol was determined in the presence of 30 laM L-vescamicol and was subtracted from the total binding. 2.4. Subcellula r localization Postnuclear supernatants were centrifuged at 10 000 g for 10 min, and the resulting supernatant (about 2 mg of protein) was loaded onto a 4.6 mL 5-25% glycerol gradient prepared in homogenization buffer for a 45-rain-long centrifugation at 55 000 rpm (SW55 rotor), which enables separation of endosomal membranes from synaptic vesicles [2]. Successive 350~tL fractions were collected and processed for Western blot analysis as previously described [12].

3.1. ACh transport and vesamicol binding properties of 2/V@Nhel chimera Specific vesicular uptake of [3H]ACh was observed in homogenates from the 2/V@Nhel chimera. Uptake of [3H]ACh was completely inhibited by 2 mM L-vesamicol, bafilomycin Al (1 raM), a specific inhibitor of the vesicular H + ATPase, and the proton ionophore FCCP (2.5 p.M). Furthermore, [3H]ACh transport was reduced approximately 90% in the absence of exogenous ATE The kinetic analysis of the uptake of [3H]ACh by the 2/V@Nhel chimera is shown in figure 1A. The initial rate of [3H]ACh uptake was measured during the linear portion of the time course (6 rain) and was saturable and displayed an apparent Km of 7.4 raM. The apparent affinity of hVAChT for ACh (Kin = 0.9 raM) is shown for comparison. Thus, the presence of hVMAT2 sequences in the N-terminal portion of hVAChT significantly reduces the affinity of this chimeric transporter for ACh. The 2/V@NheI chimera does not however transport monoamines when expressed in digotonin-permeabilized CV-1 fibroblasts (data not shown). The affinity of [3H]vesamicol for the 2/V@NheI chimera was not significantly different from that observed with hVAChT and exhibited a Kd of approximately 5 nM (figure 1B). 3.2. Subcellular localization of 2/V@Nhel chimera Western blot analysis of fractions obtained following velocity sedimentation of high speed supernatants o f stably transfected P C I 2 cells through glycerol is shown in .figure 2. The 2/V@NheI immunoreactivity sedimented in synaptic vesicles fractions containing synaptophysin (p38), similarly to that observed with hVAChT-expressing PC I2 homogenates. Thus, the presence of hVMAT2 sequences in the N-terminal portion of hVAChT does not in-

Xth International Symposium on Cholinergic Mechanisms

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Figure 1.2/V@NheI chimera dissociates high-affinity ACh recognition from vesamicol binding. A. Lineweaver-Burk analysis

of initial [3H]ACh (0.03-10 mM) uptake velocity. B. Saturation isotherm of binding of [3H]vesamicol (0.08-116 nM). Data are from a representative experiment performed in duplicate.

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terfer with its targeting to the small synaptic vesicles in PCI2 cells.

4. D i s c u s s i o n

The results presented here indicate that high-affinity vesicular transport of ACh requires cholinergic-specific amino acids within the N-terminal portion of hVAChT. It is likely that this specificity lies within the first putative transmembrane domain

(TMD) as the amino terminus and large lumenal glycosylated loop between TMDs I and II are poorly conserved. The conserved aspartic acid residue (Asp 33) in TMD I of VMATs has been implicated in high-affinity monoamine recognition [5]. Our data suggest that other amino acids within TMD 1 of VAChT, in addition to the aspartic acid residue (Asp46), are also important for ACh transport function. The binding of vesamicol does not require these cholinergic-specific amino acids in the N-terminal portion of hVAChT. Vesamicol is a non-competitive inhibitor of active ACh transport in synaptic vesicles isolated from Torpedo [1, 3]. Our results are consistent with earlier work suggesting that ACh and vesamicol do not bind to the same site in hVAChT. However, preliminary evidence suggests the presence of a second, lower affinity ACh binding site in hVAChT which may interact with vesamicol. It has recently been observed that the VAChT-specific aspartic acid residue (Asp 193) in TMD IV and the conserved aspartic acid residue (Asp 39~) in TMD X are required for ACh transport function [7]. We are currently examining the role these residues play in high- and low-affinity ACh recognition and in the binding of vesamicol. The intramembrane aspartic acid residues as well as other polar amino acids (e.g., serine, glycine, cysteine) of VAChT are likely to be part of a 'pore' and be important for the binding and exchange of H+ and ACh.

144

H. Varoqui, J. D. E r i c k s o n

W h e n e x p r e s s e d in P C I 2 cells, h V A C h T is pref e r e n t i a l l y t a r g e t e d to t h e s m a l l s y n a p t i c v e s i c l e s w h i l e h V M A T 2 is c o n f i n e d to the L D C V s [12]. H e r e w e s h o w t h a t t h e p r e s e n c e o f the l u m e n a l g l y c o s y l a t e d l o o p o f h V M A T 2 d o e s n o t i n t e r f e r e w i t h the t a r g e t i n g o f t h e 2lV@NheI c h i m e r a to t h e s m a l l synaptic vesicles. The synaptic vesicle targeting information instead resides within the terminal c y t o p l a s m i c d o m a i n o f V A C h T [12]. F u t u r e w o r k will d e t e r m i n e w h a t c o n s e q u e n c e a r e d u c e d a f f i n i t y o f h V A C h T for A C h h a s on r e g u l a t e d A C h s e c r e t i o n f r o m the s m a l l s y n a p t i c v e s i c l e s o f P C 1 2 cells.

[4]

[5]

[6]

[7]

[8]

Acknowledgment This work was supported by a grant from the National Institutes of Health (NS36936).

[9]

References

[10]

[1]

Bahr B.A., Parsons S.M., Acetylcholine transport and drug inhibition kinetics in Torpedo synaptic vesicles, J. Neurochem. 46 (1986) 1214-1218.

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[2]

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[3]

Diebler M.F., Morot Gaudry-Talarmain Y., AH5183 and cetiedil: two potent inhibitors of acetylcholine uptake into synaptic vesicles from Torpedo marmorata, J. Neurochem. 52 (1989) 813-821.

[13]

Deng W.P., Nickoloff J.A., Site-directed mutagenesis of virtually any plasmid by eliminating a unique site, Anal. Biochem. 20(1 (1992) 81-85. Merickel A., Rosandich E, Peter D., Edwards R.H., Identification of residues involved in substrate recognition by a vesicular monoamine transporter, J. Biol. Chem. 270 (1995) 25798-25804. Sch~iferM.K.-H., Weihe E., Erickson J.D., Eiden L.E., Human and monkey cholinergic neurons visualized in paraffinembedded tissues by immunoreactivity for VAChT, the vesicular acetylcholine transporter, J. Mol. Neurosci. 6 0995) 225-235. Song H.-J., Ming G.-L., Fon E., Bellocchio E., Edwards R.H., Poo M.-M., Expression of a putative vesicular acetylcholine transporter facilitates quantal transmitter packaging, Neuron 18 (1997) 815-826. Usdin "EB., Eiden L.E., Bonner T.I., Erickson J.D., Molecular biology of the vesicular ACh transporter, Trends Neurosci. 18 (1995) 218-224. Varoqui H., Diebler M.-F., Meunier F.-M., Rand J.B., Usdin T.B., Bonner T.I., Eiden L.E., Erickson J.D., Cloning and expression of the vesamicol binding protein from the marine ray Torpedo: homology with the putative vesicular acetylcholine transporter UNC-17 from Caenorhabditis elegans, FEBS Lett. 342 (1994) 97-102. Varoqui H., Erickson J.D., Active transport of acetylcholine by the human vesicular acetylcholine transporter, J. Biol. Chem. 271 (19961 27229-27232. Varoqui H., Erickson J.D., Vesicular neurotransmitter transporters: potential sites for the regulation of synaptic function, Mol. Neurobiol. 15 (1997) 165-191. Varoqui H., Erickson J.D., The cytoplasmic tail of the vesicular acetylcholine transporter contains a synaptic vesicle targeting signal, J. Biol. Chem. 273 (1998) 9094-9098. Weihe E., Tao-Cheng J.-H., Sch~ifer M.K.-H., Erickson J.D., Eiden L.E., Visualization of the vesicular acetylcholine transporter in cholinergic nerve terminals and its targeting to a specific population of small synaptic vesicles, Proc. Natl. Acad. Sci. USA 93 (1996) 3547-3552.