Authors
A Obergrussberger2; N Becker2; M Rapedius2; T A Goetze2; M G Rotordam2; N Brinkwirth2; I Rinke-Weiß2; S Stoelzle-Feix2; C Haarmann2; M George2; A Brueggemann1; N Fertig2;
1 Nanion Technologies, Germany; 2 Nanion Technologies GmbH, Germany
Abstract
Chronic and
neuropathic pain is a significant problem affecting millions of people
worldwide each year. A variety
of different ion channels play significant roles in pain transmission including
TRP, P2X, NMDA, NaV and, more recently, HCN1 and 2. Among
ligand-gated ion channels, NMDA and P2X receptors have been implicated in
diverse chronic and neuropathic pain pathways. HCN1 and 2, primarily expressed
in dorsal root ganglion neurons, have received some attention recently as pain mediators.
The Na+/K+ inward current which flows due to activation
of HCN channels (Ih), appears to have a role in mediating neuropathic pain,
supported by gain-of-function mutations or over-expression of HCN in animal
models.
We have systematically tested these diverse ion
channels using automated patch clamp. Firstly, we have recorded NMDA receptor
combinations NR1/NR2A and NR1/NR2B to investigate both positive and negative
modulation. P2X receptors, particularly P2X3 homo- or P2X2/3 heteromers,
are thought to be involved in pain conditions such as allodynia and
hyperalgesia. P2X2/3 and P2X3 receptors expressed in CHO
or 1321N1 cells were activated by ATP and aß-methylene ATP in a
concentration-dependent manner and blocked by suramin or A317491. Secondly, we
have focused on the newly identified contribution of Ih to neuropathic pain, by
measuring HCN2 currents in HEK cells. We were able to record the current on a
high throughput patch clamp instrument. HCN2 was blocked by ivabradine and
ZD-7288 in a concentration-dependent manner with an IC50 of 5.8 µM
(n = 88) and 17.3 µM (n = 93), respectively. Finally, we focused on human induced
pluripotent stem cell-derived neurons (hiPSC-neurons) as important models to
study neurodegenerative diseases such as motor neuron disease, Alzheimer’s and
Parkinson’s disease. We have successfully recorded NaV
currents and ligand-gated currents mediated by GABAA, AMPA and
nAChα7 receptors from these neurons using the automated patch clamp approach.
Our results
demonstrate that pain pathways can be successfully studied on automated patch
clamp systems, facilitating the discovery of novel pain therapeutics.
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