Co-expression of the A561V mutant with wild-type HERG reduced wild-type protein abundance due to decreased synthesis and increased turnover, establishing protein misfolding as a dominant effect mechanism.
Does the A561V mutation reduce wild-type HERG expression in mammalian cells?
The dominant negative effect of the A561V mutation in LQT2 results from assembly of wild-type subunits with mutant early in production, leading to rapid recognition and proteolysis.
HERG(1) K(+) channel mutations are responsible for one form of dominantly inherited long QT syndrome (LQT). Some LQT mutations exert a dominant negative effect on wild-type current expression. To investigate mechanisms of dominant-negative behavior, we co-expressed wild-type HERG with the A561V mutant in mammalian cells. Transfection with various cDNA ratios produced HERG K(+) current densities that approached a predicted binomial distribution where mutant and wild-type subunits co-assemble in a tetramer with nearly complete dominance. Using C terminus myc-tagged wild-type HERG we specifically followed the mutant's effect on full-length wild-type HERG protein expression. Co-expression with A561V reduced the abundance of full-length wild-type HERG protein comparable to the current reduction. Reduction of wild-type protein was due to decreased synthesis and increased turnover. Conditions facilitating protein folding (growth at 30 degrees C, or in 10% glycerol) resulted in partial rescue from the dominant effect, as did the 26 S proteosome inhibitor ALLN. Thus, for A561V, dominant negative effects result from assembly of wild-type subunits with mutant very early in production leading to rapid recognition of mutant channels and targeting for proteolysis. These results establish protein misfolding, cellular proofreading, and bystander involvement as contributing mechanisms for dominant effects in LQT2.
Kagan et al. (2000) studied Long QT syndrome (LQT2). Co-expression of wild-type HERG with the A561V mutant vs. Wild-type HERG alone was evaluated on HERG K(+) current densities and full-length wild-type HERG protein expression. Co-expression of the A561V mutant with wild-type HERG reduced wild-type protein abundance due to decreased synthesis and increased turnover, establishing protein misfolding as a dominant effect mechanism.