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2020 - A novel approach to detect electrogenic transporter activity in intact cells applied to investigate iPSC derived cardiomyocytes and neurons

 Icon N1   SURFE²R N1 and   Icon 96SE   SURFE²R 96SE poster, 64th Annual Meeting of the Biophysical Society   logo pdf   (1.6 MB)

Abstract:

SSM-based electrophysiology is a capacitive sensor-based method to detect membrane currents generated by low turnover proteins such as transporters and membrane pumps. To date, this method has been successfully used to study approximately 100 electrogenic membrane proteins. All these studies were performed using purified protein reconstituted in artificial liposomes or purified membranes from cell culture or tissue.

Here we introduce a novel method for the use of intact cells instead of purified membranes or liposomes for SSM-based electrophysiology. This supersedes laborious and potentially destructive purification of membranes or proteins, enables the use of cell material available only in limited amounts, such as patient cells or iPSC derived cells and finally represents a more physiological sample with homogeneously oriented proteins.

We established a protocol for the coating of the sensor with intact cells and validated reproducibility, robustness and amplitude of the generated signals in comparison to purified plasma membranes. Cell lines expressing the SLC transporters NCX1, GAT1, SGLT1 and PepT1 were tested. NCX1 signals triggered by calcium (35 sensors) were sensitive to 500 nM SEA0400 and 10 µM KB-R9743 blockade; In GAT-1 cells, application of 10 mM GABA evoked an outward current, blocked by 10 µM . No wash out of the internal salts was observed, an evidence for an intact cell configuration.

Finally, we applied the method to iPSC derived cardiomyocytes and neurons and were able to record transporter currents from these cells for the first time. In cardiomyocytes we could detect calcium induced NCX activity, and in neurons glutamate and GABA transport. This contributes to the understanding of properties and possibilities of these model cells and indicates that the use of intact cells opens a new field of applications for SSM-based electrophysiology.

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