High throughput phenotyping of hERG channel mutations

Ng CA1,2, Perry MD1,2 and Vandenberg JI1,2

  1. Victor Chang Cardiac Research Institute, 405 Liverpool St, Darlinghurst, NSW 2010.
  2. St Vincent’s Clinical School, UNSW Sydney, Darlinghurst, NSW 2010.

The expression of hERG potassium channels at the plasma membrane of cardiac myocytes is critical for the coordinated propagation of the electrical signals that regulate the rhythm of the heartbeat. Reduced hERG function due to mutations increases the risk of sudden cardiac arrest and death. Mutations may affect synthesis, assembly, trafficking and/or function of hERG channels. The majority of mutants affect channel trafficking and traditionally this has been assessed using Western blot assays. Manual patch clamping is the gold standard for assessing the gating phenotype of hERG mutants and can also be used to assess current density. However, manual patch clamp assays are far too labour intensive for them to have clinical utility in assessing large numbers of mutants. In the era of precision medicine, large numbers of hERG mutations with unknown significance are going to be identified. Therefore, there is a need to develop higher throughput methods that are amenable to automation to substitute for the current Western blot and manual patch clamp assays. In this study, wild-type and 25 clinical hERG mutations were expressed in HEK293 cells to quantify their expression and gating phenotypes using ELISA assay and automated patch clamp assays (Syncropatch-384), respectively. The expression levels determined using the ELISA assay versus traditional Western blot analysis had a correlation coefficient of 0.86. The expression levels determined by current density measurements using the syncropatch (more than 1000 successful recordings) showed good correlation with the ELISA assays (correlation coefficient, 0.85). Our results indicate that both ELISA and the syncropatch methods can be used to assess channel expression. These methods not only have a higher throughput than traditional methods but they can also be applied to any ion channel including channels where the lack of glycosylation precludes the use of simple Western blot analysis.