Electrophysiological Recordings of LGIC and AA Transporters

Human neurons extracted from induced pluripotent stem cells (hiPSCs) are proving to be a vital component for studying basic neuronal physiology, offering ample models for studying neurological disorders.

Thus, hiPSC derived neurons offer the industry of drug discovery the potential to find innovative therapeutics to treat seizure-related and neurodegenerative disorders. Furthermore, hiPSC derived neurons are also an effective alternative to primary cells and animal models. As such, iCell® GlutaNeurons are high-quality glutamatergic-enriched cortical hiPSC-derived neurons: the presence of glutamate receptors AMPA, kainate and NMDA, as well as glutamate and GABA transporters1, have already been observed in single-cell gene transcription analysis.

Ionotropic glutamate receptors control most of the excitatory neurotransmission in the mammalian CNS and further the removal of glutamate from the synaptic cleft by reuptake via glutamate transporters; this plays a role in managing neuronal excitability. GABA is the major known inhibitory neurotransmitter in the brain and plays an important role in regulating excitability.

After release, GABA is withdrawn from the extracellular space by GABA transporters (GATs), thus breaking inhibitory synaptic transmission. Therefore, both GABA and glutamate transporters may provide novel therapeutic targets for, e.g. Parkinson’s disease2, Alzheimer’s disease3, and epilepsy4. Ligand-gated ion channel currents mediated by GABAA and AMPA receptors from iCell® GlutaNeurons were recorded on the Patchliner and SyncroPatch 384PE. Furthermore, using the SURFE2R N1 device it is possible to measure GABA and glutamate transporters in these neurons.

Results

Initiating transport on the SURFE2R N1 was carried out by inserting a sensor with attached iCell® GlutaNeurons into the device and introduced with a buffer containing NaCl and glutamate. When the substrate is present, the movement of Na+ and K+ can be observed across the membrane until an electrochemical equilibrium is reached.

To produce Na+ and K+ gradients as a driving force the sensor was exposed to KCl before and after the glutamate transporter activation. The traces and concentration-response curve for glutamate using an example sensor is illustrated in Figure 1. This confirms the presence of glutamate transporter in iCell® GlutaNeurons, although the exact identity was not investigated further.

Glutamate transport was activated in iCell® GlutaNeurons upon addition of glutamate. Peak amplitude increased with increasing glutamate concentration with an EC50 of 0.391 mM. Traces recorded from the cells on the sensor of the SURFE2R N1 are also shown.

Figure 1. Glutamate transport was activated in iCell® GlutaNeurons upon the addition of glutamate. Peak amplitude increased with increasing glutamate concentration with an EC50 of 0.391 mM. Traces recorded from the cells on the sensor of the SURFE2R N1 are also shown.

GATs transport GABA in exchange for Na+ and Cl- with the recommended stoichiometry 2 Na+: 1Cl- : 1 GABA2. Utilizing the SURFE2R N1, GABA transport was observable in the presence of Na+ and Cl- where the addition of GABA was concerned (Figure 2); thereby confirming the appearance of a GABA transporter in iCell® GlutaNeurons. However, once again the exact identity of the GAT was not subjected to further investigation.

A Addition of 10 mM GABA to sensors with attached iCell® GlutaNeurons resulted in the activation of a current mediated by a GABA transporter.

Figure 2. An Addition of 10 mM GABA to sensors with attached iCell® GlutaNeurons resulted in the activation of a current mediated by a GABA transporter.

iCell® GlutaNeurons were also put into place on the medium and high throughput patch clamp instruments; the Patchliner and SyncroPatch 384PE. As illustrated in figure 3A, the current response of an iCell® GlutaNeuron to glutamate can be seen in the presence and absence of the AMPA specific positive allosteric modulator (PAM) LY404187 on the Patchliner.

When applied to iCell® GlutaNeurons on the SyncroPatch 384PE and controlled by the GABAA receptor (Figure 3B) GABA induced an inward current. Conclusively, iCell® GlutaNeurons can be used effectively on the SURFE2R N1 to measure and record transporter activity. Furthermore, the Patchliner and SyncroPatch 384PE can be used to measure whole-cell currents utilizing the patch-clamp method for drug discovery and ion channel/transporter research.

(A) In the presence of the AMPA-specific PAM, LY404187, a response to glutamate in iCell® GlutaNeurons recorded on the Patchliner was seen. (B) GABAA receptor mediated response was recorded on the SyncroPatch 384PE.

Figure 3. (A) In the presence of the AMPA-specific PAM, LY404187, a response to glutamate in iCell® GlutaNeurons recorded on the Patchliner was seen. (B) GABAA receptor-mediated response was recorded on the SyncroPatch 384PE.

Methods

Cell culture

iCell® GlutaNeurons were supplied by FUJIFILM Cellular Dynamics, Inc. (Catalog # R1061 or R1034), and were cultured and harvested in line with Nanion’s standard procedures for hiPSCs.

Electrophysiology

iCell® GlutaNeurons were used on the Patchliner and SyncroPatch 384PE adhering to Nanion’s standard protocols. During  the glutamate experiments, cells were held at -80 mV, pre-incubated in LY404187 and later co-applied with glutamate. Throughout GABA experiments, cells were held at 80 mV and GABA was applied for ~5 s.

SURFE2R sensor preparation

According to the Nanion standard procedure “SURFE2R Sensor Preparation”, whole cells were used.

SURFE2R N1 measurement workflow

Glutamate and GABA transport are triggered by providing the respective substrate. In both cases a sodium gradient is established before substrate addition. During GAT experiments, an additional chloride gradient is established. Therefore, any 3-buffer Nanion standard protocol is acceptable. Buffers are HEPES based including NaCl with KCl for EAAT and K-Gluconate for GAT as the main salt.

References and Further Reading

  1. Chen, Y. 2018. In: Neurotoxins. DOI: 10.5772/intechopen.77043
  2. Jin, X-T. et al 2011. Frontiers in Systems Neuroscience. 5:Article 63
  3. Fuhrer, T.E., et al. 2017. Neuroscience. 351: 108-118
  4. Björn-Yoshimoto, W. & Underhill, S.M. 2016. Neurochem Int. 98: 4–18

About Nanion Technologies

Nanion Technologies is a spin-off from the Center of Nanoscience (CeNS) of the University of Munich (LMU).

Nanion combines bio- and microtechnology in a company serving the life sciences industry by offering products and services which will dramatically increase the speed and efficiency of the drug discovery process in an important segment of the pharmaceutical market.

Nanion bases its business on a proprietary chip technology and will design and develop High Throughput Screening (HTS) systems for ion channel active drugs (ICADs). Ion channels are prime targets for innovative medicines aimed at many important diseases.


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Last updated: Jul 22, 2020 at 5:52 AM

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