PrecisION™ hGlyRA1-HEK Recombinant Cell Line - Millipore

PrecisION hGlyRA1-HEK 

Recombinant Cell Line 

Catalog Number: CYL3056 

PRODUCT DESCRIPTION: 

Recombinant HEK293 cell line expressing the human GlyRA1 (glycine receptor, alpha 1) chloride ion channel (accession number 

NM_000171). 

ASSOCIATED PRODUCTS: 

The PrecisION hGlyRA1-HEK Recombinant Cell Line is provided to customers on the purchase of an appropriate license. 

The available licenses are: 

CYL3056SS PrecisION hGlyRA1-HEK Single Site License 

CYL3056TS PrecisION hGlyRA1-HEK Two Site License 

CYL3056MS PrecisION hGlyRA1-HEK Multiple Site License 

CONTENTS: 

2 x 1 mL aliquots containing 1.04 x 10 6 cells/mL in 10% DMSO at passage 15. 

STORAGE: 

Vials are to be stored in liquid N2. 

WARNINGS: 

For Research Use Only; Not for Use in Diagnostic Procedures 

Not for Animal or Human Consumption 

GMO 

This product contains genetically modified organisms. 

Este producto contiene organismos genéticamente modificados. 

Questo prodotto contiene degli organismi geneticamente modificati. 

Dieses Produkt enthält genetisch modifizierte Organismen. 

Ce produit contient organismes génétiquement des modifiés. 

Dit product bevat genetisch gewijzigde organismen. 

Tämä tuote sisältää geneettisesti muutettuja organismeja. 

Denna produkt innehåller genetiskt ändrade organismer. 

MYCOPLASMA TESTING: 

The cell line has been screened using the PCR Mycoplasma Test Kit (MDBiosciences) to confirm the absence of Mycoplasma 

species. 

FUNCTIONAL VALIDATION: 

HEK293 cells expressing GlyRA1 were characterised using IonWorks TM Quattro. Glycine dose-dependently activated a current with 

an EC50 value of around 39 µM and strychnine dose-dependently blocked the current evoked by 100 µM glycine with an IC50 value of 

29 nM. This is convincing evidence for the presence of glycine receptors. There was also good evidence to suggest the selective 

expression of homomeric channels since both picrotin and picrotoxin dose-dependently inhibited the currents with IC50 values of 36.9 

± 6.5 µM and 5.7 ± 1.9 µM respectively, close to published values and significantly lower than reported for heteromeric receptors 

containing β subunits. Zn 2+ dose-dependently blocked glycine (100 µM) responses with an IC50 value of 74.7 µM within the range 

reported for inhibition of homomeric α1 receptors. There were also indications that Zn 2+ potentiated the current at low micromolar 

concentrations. Channel expression was monitored over 30 cell passages and the mean glycine-evoked outward current was 

consistently >1.5 nA. 

RECOMMENDED CULTURE CONDITIONS: 

Recommended culture conditions and standard operating procedure are provided with the product. 

MM03-DJ-CYL3056 

DDServices@merckgroup.com ● www.millipore.com/DrugDiscovery




FUNCTIONAL VALIDATION: 

Introduction. 

Inhibitory neurotransmission in the adult mammalian CNS is primarily mediated by the amino acids, GABA and glycine, causing an 

increase in Cl - conductance by binding to specific receptors; GABAA and Glycine receptors (GlyR) respectively. Like GABAA 

receptors, GlyRs belong to Cys-loop family of ligand-gated receptors that also includes nAChRs and 5HT3Rs. 

Members of this family exist as pentamers where the binding site for the ligand and the ion selective pore are part of the same 

molecule. To date 4 different α subunits and 1 β subunit have been identified that in specific combinations make up the various 

subtypes of GlyRs. 

In adult mammalian CNS, the most abundant subtype consists of the α1 and β subunit in a 3:2 stochiometry (Jensen, 2008). Such α1 

containing receptors have been shown to mediate fast inhibitory synaptic transmission in motor reflex circuits of the spinal cord, 

responsible for regulating muscle tone. 

Based on the location of GlyRs and their fundamental role in modulating excitatory inputs, compounds that potentiate glycine 

responses may result in novel muscle relaxants, sedatives and analgesics (Laube et al., 2002). 

Pharmacological Properties of hGlyRA1. 

IonWorks TM Quattro Electrophysiology. 

All pharmacological characterization was performed on IonWorks Quattro (PPC mode). The voltage protocol used is shown in Figure 

5. Under these conditions the glycine evoked Cl - current is quantified by subtracting the outward current at 0 mV prior to addition 

from the outward current in the presence of glycine. Note that prior to glycine addition the outward current at 0 mV is negligible. 

Using this novel approach it was possible to obtain a dose-response curve for glycine (Figure 1) and examine the dose-dependent 

effects of strychnine (Figure 2), picrotoxin, picrotin (Figure 3) and Zn 2+ (Figure 4) on the glycine (100 µM) evoked outward current. 






Glycine Dose-Response Curve: 

The EC50 value for glycine obtained with IonWorks is very similar to values recorded with more conventional methods. For example, 

in patch clamp experiments using homomeric α1 in LtK cells an EC50 of 100 µM was obtained (Wick et al., 1999) whereas in HEK293 

cells value of around 50- 60 µM have been reported (Han et al., 2004, Slaughter, 2003 and Li and Slaughter, 2007). 

Figure 1. Glycine Dose-Response Curve. 

Increasing concentrations of glycine were added to separate patch-plate wells and the evoked outward current at 0 

mV was measured. Glycine dose-dependently increased the outward current with an EC50 of 39 µM. 

Effect of Strychnine on Glycine-evoked Current: 

Strychnine has been shown to competitively antagonise glycine receptors in the nanomolar range in both native 

tissues (McCool and Botting, 2000, Sergeeva, 1998 and Tapia et al., 1998) and in recombinant cell lines 

(Grudzinska, 2005 and Jensen, 2005). The IC50 value of 29 nM obtained here (Figure 2), assessing the effects of 

various concentrations of strychnine on the response evoked by 100 µM glycine, is consistent with these reports. 

Figure 2. Effect of Strychnine on Glycine-evoked Currents. 

Various concentrations of strychnine were incubated for 100 s and then in the presence of 100 µM glycine. 

Strychnine dose-dependently inhibited glycine evoked outward currents with an IC50 of 29 nM. 






Effect of Picrotin and Picrotoxin on Glycine-evoked Currents: 

The plant alkaloid picrotoxin, is an equimolar mixture of picrotin and picrotoxinin. However, although both these 

constituents inhibit glycine receptors only picrotoxinin blocks GABAA receptors. Furthermore, both picrotin and 

picrotixin have been reported to be much more effective as antagonists against homomeric rather than heteromeric 

receptors (Burzomato et al., 2007, Li and Slaughter, 2007). For example, using picrotin, Li and Slaughter obtained 

an IC50 value of 38 µM at homomeric α1 receptors whereas at heteromeric receptors, containing β subunits, the 

value was >100 µM. Similarly, using picrotoxin, Burzomato et al., 2007 obtained an IC50 value of 6 µM at 

homomeric receptors and around 150 µM at heteromeric receptors. Hence, these compounds are useful 

pharmacological tools, not only for establishing the presence of glycine receptors but also for gaining an 

understanding of subunit composition. The IC50 values obtained using IonWorks are almost identical to the 

published values described above for both picrotoxin and picrotin. This provides convincing evidence for the 

expression of homomeric glycine receptors in this cell line. 

Figure 3. Effect of Picrotoxin and Picrotin on Glycine-evoked Currents. 

Various concentrations of compound were incubated for 100 s and then in the presence of 100 µM glycine. 

Picrotoxin and picrotin dose-dependently inhibited glycine evoked outward currents with IC50 values of 5.7 µM and 

36.9 µM respectively. 






Effect of Zn 2+ on Glycine-evoked Currents: 

Zinc ions may have a physiological role modulating glycine responses as well as other ion channels located certain 

synaptic regions since they have been found to be packaged into synaptic vesicles and released upon nerve 

stimulation. Zn 2+ has been reported to have a biphasic effect – a potentiating effect at low concentrations (0.5–10 

µM) and an inhibitory effect at higher concentrations (50–100 µM, Bloomenthal et al., 1994, Harvey et al., 1999 

and Miller et al., 2005). Reported IC50 values for the inhibitory effect of homomeric α1 vary somewhat, 10-20 µM 

(Miller et al., 2005) to around 500 µM (Harvey et al., 1999). 

Using IonWorks, a slight potentiation by Zn 2+ could be detected at the lowest concentrations (0.14-10 µM). The 

mean glycine-evoked outward current amplitude in the presence of Zn 2+ was in excess of 1.8 nA, approximately 

200 pA greater than in the absence of Zn 2+ . Higher concentrations dose-dependently inhibited the current with an 

IC50 value of around 75 µM. Thus the biphasic effect within reported ranges could be detected. 

Figure 4. Effect of Zn 2+ on Glycine-evoked Currents. 

Various concentrations of Zn 2+ were incubated for 100 s and then in the presence of 100 µM glycine. Zn 2+ dosedependently 

inhibited glycine evoked outward currents with an estimated IC50 value of 74.7 µM. Between 1 and 10 

µM Zn 2+ the mean outward current evoked by 100 µM glycine was >1.8 nA whereas in the absence of Zn 2+ the 

mean current was around 1.6 nA. 






Stability of hGlyRA1-HEK293 Cell Line. 

IonWorks TM HT Electrophysiology. 

In order to monitor the stability of functional expression over time, IonWorks TM HT automated electrophysiology 

was used. Data from a representative cell is shown in Figure 5. Currents before and in the presence of Glycine 

(300 μM) were evoked using the voltage protocol shown below. Typically, glycine produces a large outward current 

on stepping to 0 mV (asterisk, red trace). 

Figure 5. Glycine evoked currents using IonWorks TM HT. 

Upper panel: typical current traces evoked before (black trace) and after (red trace) the application of 300 µM 

Glycine. The zero current level is shown by the black dotted line. 

Lower panel: Voltage protocol. 


� 

The amplitude of the Glycine (300 µM) evoked current at 0 mV (red asterisk) was measured and the percent of 

cells expressing currents >500 pA at each cell passage and having a seal resistance ≥50 MOhms prior to glycine 

addition were determined (Figure 6). Functional expression measured in this manner was consistently >85% and 

stable for >30 passages (Figure 6, blue squares). In addition the mean current amplitude for hGlyRA1 expressing 

cells was consistently >1.5 nA (Figure 6, red bars). 





Figure 6. Stability of expression over passage. 

The blue squares show the percentage of cells expressing mean current amplitude of ≥ 500 pA at 0 mV in 

response to 300 µM glycine at cell passage 10 through to 30. The lower panel shows the mean current amplitude 

(red bars) and adjacent to these the number of these cells (number of cells expressing >500 pA / number of cells 

sealing >50 MOhms). The maximum n-number was 24 cells for passages 10 through to 28 and 48 cells at passage 

30. 


Current Amplitude (nA) 

4.0 

3.5 

3.0 

2.5 

2.0 

1.5 

1.0 

0.5 

0.0 

18/19 

15/15 

16/16 

19/21 

14/15 

10 12 14 16 18 20 22 24 26 28 30 

DDServices@merckgroup.com ● www.millipore.com/DrugDiscovery 

19/20 

Passage Number 

10/10 12/13 20/21 

16/16 

36/37 

110 

100 

90 

80 

70 

60 

50 

40 

30 

20 

10 

0 

% Expression




Vector: 


AhdI 

PvuI 

Amp 

4000 

PCIN5_NEWC 

4904 bps 

Polylinker: CMV-BamHI-NotI-hGlyRA1-AscI-ClaI-HpaI-EcoRI-IRES-neo 

3000 

HincII 

XhoI 

AvaI 

SphI 

HincII 

SspI 

hGlyRA1‐pCIN5 

polyA 

XbaI 

BclI 

hGlyRA1 Sequence (Accession Number NM_000171): 

DDServices@merckgroup.com ● www.millipore.com/DrugDiscovery 

2000 

MluI 

HincII 

CMV promoter 

Neo 

1000 

BssHII 

SphI 

NdeI 

SnaBI 

IRES 

PstI 

BamHI 

ApaI 

NotI 

AvaI 

XhoI 

AscI 

BssHII 

NdeI 

ClaI 

HpaI 

HincII 

EcoRI




References: 

Bloomenthal, A.B., Goldwater, E., Pritchett, D.B. and Harrison, N.L. (1994). Biphasic modulation of the strychninesensitive 

glycine receptor by Zn 2+ . Mol Pharmacol 46, 1156-1159. 

Burzomato, V., Groot-Kormelink, P.J., Sivilotti, L.G. and Beato, M. (2003). Stoichiometry of recombinant 

heteromeric glycine receptors revealed by a pore-lining region point mutation. Receptors Channels 9, 353-361. 

Grudzinska, J., Schemm, R., Haeger, S., Nicke, A., Schmalzing, G., Betz, H. and Laube, B. (2005). The beta 

subunit determines the ligand binding properties of synaptic glycine receptors. Neuron 45, 727-739. 

Han, Y., Li, P. and Slaughter, M.M. (2004). Selective antagonism of rat inhibitory glycine receptor subunits. J 

Physiol 554, 649-658. 

Harvey, R.J., Thomas, P., James, C.H., Wilderspin, A. and Smart, T.G. (1999). Identification of an inhibitory Zn 2+ 

binding site on the human glycine receptor alpha1 subunit. J Physiol 520 Pt 1, 53-64. 

Jensen, A.A. (2005). Functional characterisation of human glycine receptors in a fluorescence-based high 

throughput screening assay. Eur J Pharmacol 521, 39-42. 

Jensen, A.A., Gharagozloo, P., Birdsall, N.J. and Zlotos, D.P. (2006). Pharmacological characterisation of 

strychnine and brucine analogues at glycine and alpha7 nicotinic acetylcholine receptors. Eur J Pharmacol 539, 

27-33. 

Laube, B., Maksay, G., Schemm, R. and Betz, H. (2002). Modulation of glycine receptor function: a novel approach 

for therapeutic intervention at inhibitory synapses? Trends Pharmacol Sci 23, 519-527. 

Li, P. and Slaughter, M. (2007). Glycine receptor subunit composition alters the action of GABA antagonists. Vis 

Neurosci 24, 513-521. 

McCool, B.A. and Botting, S.K. (2000). Characterization of strychnine sensitive glycine receptors in acutely isolated 

adult rat basolateral amygdala neurons. Brain Res 859, 341-351. 

Miller, P.S., Beato, M., Harvey, R.J. and Smart, T.G. (2005). Molecular determinants of glycine receptor alphabeta 

subunit sensitivities to Zn 2+ -mediated inhibition. J Physiol 566, 657-670. 

Sergeeva, O.A. (1998). Comparison of glycine- and GABA-evoked currents in two types of neurons isolated from 

the rat striatum. Neurosci Lett 243, 9-12. 

Tapia, J.C. and Aguayo, L.G. (1998). Changes in the properties of developing glycine receptors in cultured mouse 

spinal neurons. Synapse 28, 185-194. 

Webb, T.I. and Lynch, J.W. (2007). Molecular pharmacology of the glycine receptor chloride channel. Curr Pharm 

Des 13, 2350-2367. 

Wick, M.J., Bleck, V., Whatley, V.J., Brozowski, S.J., Nixon, K., Cardoso, R.A. and Valenzuela, C.F. (1999). Stable 

expression of human glycine alpha1 and alpha2 homomeric receptors in mouse L(tk-) cells. J Neurosci Methods 

87, 97-103. 







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PrecisION™ hGlyRA1-HEK Recombinant Cell Line - Millipore

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