4-Methylmethamphetamine (4-MMA) acts at the human serotonin

transporter (hSERT) as an inward current-inducing, depolarizing substrate

Joshua Chiu1, Iwona Ruchala2, Ernesto Solis, Jr2., Jose M. Eltit2, Louis J. De Felice2
1VCU School of Engineering, Virginia Commonwealth University, Richmond, VA 23298, United States
2Department of Physiology and Biophysics, School of Medicine, Virginia Commonwealth University, Richmond, VA 23298, United States

Introduction Methods Results

Abstract: Expression of hSERT in Xenopus laevis oocytes
A variety of substituted amphetamines (AMPH) act as Plasmids for hSERT expression were linearized with Not1 in Buffer 5-HT
neurotransmitter releasers, acting synergistically at 3 and BSA, were PCR purified (Qiagen), and were finally used to 1.9 1.9

monoamine neurotransmitter transporters such as the generate RNA (Life Technologies). Sample concentrations were in

Scaled fluorescence F/F0

Scaled fluorescence F/F0
human serotonin transporter (hSERT) to increase release of excess of 2000ng/uL so 23nL cRNA was used to inject each oocyte. 1.7 1.7

serotonin (5-HT). In this study we explore the effect of 4- Oocytes were allowed to express for several days before TEVC was
MMA on the activity of induced electrical currents mediated performed. 1.5 1.5

by hSERT in Xenopus laevis oocytes, as well as its effects on Electrophysiology (TEVC) 1.3 1.3

voltage-gated Ca2+ channels co-expressed with hSERT in HEK- Two-electrode voltage clamp with Vhold = -60mV. 1-5 MΩ
293 cells. These data will characterize 4-MMA action on the resistance electrodes were filled with 3M KCl. Solution used 1.1 1.1

induced inward current and hSERT-Cav1.2 electrical coupling in recordings consisted of 120 NaCl, 7.5 HEPES, 5.4
and further our understanding of this unscheduled KGluconate, 1.2 CaGluconate, pH 7.4, in mM. All 0.9
5-HT FLX FLX+5-HT K+ 0.9 5-HT FLX FLX+5-HT K+
0 20 40 60 80 100 120 140 0 20 40 60 80 100 120 140
Figure 4. Currents induced by 4-MMA and 5-HT in voltage-clamped (--60mV) Xenopus laevis oocytes expressing
substituted amphetamine. experiments were performed at room temperature (23-25°C). time (s) time (s) hSERT with the TEVC technique. (A) Initial exposure to 5-HT yields an hSERT-mediated inwards current.
Protocol consisted of perfusion with 5-HT (5uM), followed by 4-MMA Exposure to FLX concurrently with 5-HT diminishes the response. (B) Initial exposure to 5-HT yields an hSERT-
Key results and conclusions: 4-MMA (5uM) and FLX (1uM) + 4-MMA (5 uM). 1.9
2.3
mediated inwards current. Subsequent exposure to 4-MMA yields a comparable inwards current. Exposure to
FLX concurrently with 4-MMA diminishes the response.
The observed 4-MMA-induced inward current seen in TEVC Data courtesy of Ernesto Solis, Jr.
Expression of hSERT, Cav1.2 in HEK-293 cells
2.1

is greater than that of that of 5-HT. 4-MMA induces a

Scaled fluorescence F/F0

Scaled fluorescence F/F0

Discussion
1.7

quantitatively greater hSERT-linked Cav1.2 activity than that HEK-293 cells expressing hSERT were maintained in appropriately 1.9

of 5-HT; additionally, it is similarly blocked by fluoxetine, a
selective serotonin reuptake inhibitor. Overall, these data
supplemented DMEM. 72 hours before the assay the cells were 1.5
1.7
The 4-MMA-induced inward current seen in the TEVC is larger than
incubated with doxycycline to induce hSERT expression, and were
the 5-HT-induced inward current, while both are similarly blocked by
transfected with Cav1.2 plasmids using FuGENE, as well as 1.5

strongly suggest that 4-MMA acts at the human serotonin 1.3

transporter as a inward current-inducing, depolarizing
substrate with effects comparable in size to that of 5HT.
FLX. Here we have identified hSERT-induced Cav1.2 activity induced
eGFP to identify Cav1.2 expressing cells.
1.3
by a novel substituted amphetamine. The calcium fluorescence
Calcium fluorescence assay 1.1

1.1 patterns of 5-HT and 4-MMA were qualitatively similar in terms of

Background and Objective
Background:
Monoamine transporters including hSERT, hNET
and hDAT have been found to release their
associated neurotransmitter in response to abused
drugs such as methamphetamine (METH), among
with other substituted amphetamines. The abuse of
HEK-293 cells in cell culture plates were incubated with Fura-2AM responses to the drug, FLX and FLX + drug. Critically, the maximal
4-MMA FLX FLX+4-MMA K+ 4-MMA FLX FLX+4-MMA K+
for 20 minutes at 37C, and were then washed in imaging solution 0.9
0 20 40 60 80 100 120 140
0.9
0 20 40 60 80 100 120 140
calcium signal of 4-MMA was quantitatively greater than that of 5-HT.
time (s) time (s)
(IS, 130 NaCl, 4KCl, 2 CaCl2, 1 MgCl2, 10 Hepes, 10 glucose, pH 7.4 From both the TEVC and calcium fluorescence data, we conclude that
in mM). Concentration of both 4-MMA and 5-HT was 10uM; all Figure 2. HEK-293 stably expressing hSERT and Cav1.2 were loaded
solutes were dissolved in IS. The setup consists of a Olympus IX70 with Fura and were observed for calcium activity. Top panel: Calcium
5-HT is possibly saturated at a lower concentration than 4-MMA and
fluorescence ratios of serotonin using the protocol described; Bottom that 4-MMA is more efficacious in causing an hSERT-mediated
microscope using a Polycrome V light source, a Luca S digital panel: Calcium fluorescence ratios of 4-MMA using the protocol inwards current and membrane depolarization, which is implicated in
camera (exposure time 50ms) and an automatic perfusion system. described. The depolarization induced by hSERT activation by both 5-HT neurotransmitter release and stimulant effects.
The experiment was performed with constant perfusion at RT (23- and 4-MMA was electrically coupled to Cav1.2 activation. Additionally, the
25C). The protocol was as follows: 10 s IS, 5 s 4-MMA or 5-HT hSERT activation activity of both compounds were nearly completed The investigation of other substituted amphetamines which replace
attenuated by the addition of fluoxetine for 20 s (t = 45-65 s) before the N-methyl with other alkyl chains such as ethyl, propyl and butyl
(10uM), 30 s IS, 20 s 1uM fluoxetine (FLX), 5s FLX + 10uM 4- reloading the compound around t = 65-70 s.
merits further attention, as these are highly structurally similar and
MMA/5-HT, 30 s IS, 5s K+ and 30 s IS. Maximal fluorescent response
2
may have similar effects in terms of hSERT-mediated inward current
1.863192534
1.901721221

1.8 1.772714307
and Cav1.2 electrical activity.
these drugs therefore exerts profound effects on 1.6

mental function and behavior. hSERT is a sodium- 1.4 *

1.438744748

-dependent symporter and mediates an inward Figure 1. From top left to

Scaled fluorescence averages

5-HT AMPH 1.2

current. This substrate-induced inward current has bottom right, the chemical
1
References
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0.6
dopamine transporter. British Journal of Pharmacology, 168(7), 1750–1757. doi:10.1111/bph.12061

through fluorescence is indicative of hSERT activity. (amphetamine), METH

0.4
Ernesto Solis, Jr., Renata Kolanos, Farhana Sakloth, Krasnodara N. Cameron, Richard A. Glennon,
Louis J. De Felice. (2013, April). Methylenedioxypyrovalerone (MDPV) is a potent inhibitor of hDAT
Objective: METH 4-MMA (methamphetamine), 4-MMA 0.2
and hNET. Poster presented at Experimental Biology Annual Meeting, Boston, MA.

Compare induced inward current of 4-MMA to (4-methylmethamphetamine) 0

that of 5-HT under two electrode voltage clamp. with oxygen and nitrogen color
coded and hydrogens noted on
Measure the calcium response to 4-MMA induced
tertiary carbons. Adapted from NCBI
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5-HT max drug response
Figure 3. Quantitative analysis of 4-MMA-induced calcium activity compared to that of
5-HT. Included standard error bars for drug response and K+ response of 5-HT and 4-
MMA (n=25 for 5-HT, n=19 for 4-MMA, from left to right standard error = 0.021022,
0.043718, 0.023626 and 0.029437). * indicates statistically significant results, t=9.43,
df = 42 and P < .0001.
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Iwona Ruchala, Vanessa Cabra, Ernesto Solis Jr., Richard A. Glennon, Louis J. De Felice, Jose M.
Eltit, Electrical coupling between the human serotonin transporter and voltage-gated Ca2+ channels,
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http://dx.doi.org/10.1016/j.ceca.2014.04.003.