Quantitative analysis of trafficking defects induced by heterozygous expression of hERG voltage sensor domain variants.

Long QT Syndrome Type 2 (LQT2) is a cardiac disorder caused by Loss of Function (LOF) mutations in the KCNH2 gene that encodes the K+ channel hERG (Kv11.1). Mistrafficking of hERG LOF variants is the dominant cause of LQT2. We recently characterized greatly attenuated cell surface trafficking in eight natural variants in a region of the hERG voltage sensor domain identified using evolutionary analysis. Here we have used quantitative On/In-Cell western assays to characterize trafficking of these variants under heterozygous conditions more relevant to the clinical circumstance. Dominant-negative effects of variant on wild-type (WT) cell surface expression and WT rescue of variant mistrafficking were separately assessed in co-expressions in which only WT or variant carried the HA-tag used to detect cell surface expression. Co-expression of all variants reduced cell surface expression and exhibited dominant-negative effects on trafficking. However, when compared to previous studies that utilized hERG glycosylation status as a measure of trafficking efficiency, the heterozygous effects on trafficking were smaller than expected. An identified relationship between pharmacological rescue of variant trafficking by the hERG blocker E-4031 and by co-expression with WT hERG indicated that inherent "rescuability" of variants might be characterized in advance of efforts to identify non-blocking trafficking rescuers.