Development of an Automated Patch Clamp Assay for recording STIM1/Orai1 – mediated currents using Qube 384

著者: Daniel Sauter, Rasmus Jacobsen

Calcium (Ca2+) is an important second messenger that is involved in critical processes in cell homeostasis and signal transduction. The ratio between extra- and intracellular Ca2+ contributes to setting the plasma membrane potential. Intracellularly, Ca2+ is stored in organelles such as Mitochondria and the Endoplasmic reticulum (ER). Depletion of these stores triggers the opening of Ca2+ – permeable channels that allow entry of Ca2+ into the cell along the electrochemical gradient. This process is called store-operated calcium entry (SOCE). Activation of SOCE happens as a secondary result of ER depletion (ref). ER is being depleted by an inositol-triphosphate (IP3) pathway and phospholipase C cascade, via the IP3 receptor. SOCE can experimentally be activated by elevating intracellular levels of IP3 in the presence of a Ca2+ chelator.

The activation of SOCE can directly be measured as a change in clamp current when performing patch-clamp experiments. In this study the automated patch clamp system, Qube 384 was used to record Icrac. Qube 384 is a second-generation automated patch clamp device capable of testing thousands of compounds per day whilst providing true giga-ohm seal quality data. Using the Qube 384 in a drug discovery cascade enables acquisition of mode of action data simultaneous with hit detection during the primary screen, thereby minimizing the need for extended follow-up validations studies.

In this study, we present an assay for STIM1/Orai1. Firstly, we present that Orai1 current can be activated either by exchange of the intracellular solution or alternative by de facto caging an agonist in the IC in the on-cell configuration. Secondly, we pharmacologically validate the assay using the trivalent lanthanide cations blocker lanthanum (La). With this, we demonstrate that Qube 384 also is a versatile patch clamp system, even though it is fully automated and can be running unattended.