Molecular mechanisms regulating HERG channel synthesis and trafficking
MetadataShow full item record
The HERG potassium channel is responsible for cardiac repolarization. Studies of HERG synthesis and trafficking reveal that the protein is sensitive to perturbations, both intrinsic to the protein and environmental.;I first postulated a link between increased adrenergic signaling, as seen in heart failure patients, and regulation of HERG synthesis and trafficking. We observed protein kinase A or C stimulation led to a four-fold increase in HERG protein abundance, which was not transcriptionally mediated. I have now determined: 1) channel phosphorylation is necessary but not sufficient for increased HERG abundance; 2) a cAMP/PKA responsive cellular co-factor is required; 3) cAMP does not significantly alter HERG mRNA stability; 4) the association of HERG mRNA with monosomes or polysomes does not measurably change and 5) changes in degradation via the proteasome or lysosome do not account for increased HERG abundance.;Secondly, I studied the effects of sequence-specific elements encoded in the HERG mRNA on channel processing. I used an artificial, re-synthesized HERG coding sequence where the codons were mutated synonymously, preserving the amino acid sequence. Codon-modified HERG (HERG-CM) channels produced a near-identical current. HERG-CM had reduced total protein expression but enhanced surface expression. Chimera studies showed the N-terminal sequence affected expression level. In vitro translation showed HERG-CM had attenuated synthesis. Polysome profiling showed HERG-CM mRNA is more abundant in the monosome fraction, indicating low translation levels. These data show synonymous codon-modification alters synthesis and processing of HERG by reducing total expression while independently enhancing surface expression.;Lastly, I characterized a clinically relevant HERG mutation G816V, which was found in an adult female who succumbed to sudden death with suspected hypokalemia. Heterologous expression showed both total protein level and surface expression were reduced for mutant channels. Electrophysiology showed no current for the mutant however, reduced temperature partially rescued this phenotype. Co-expression studies showed the mutant was partially dominant over the wild type in current generation. Low extracellular potassium reduced wild-type HERG surface expression thereby further compromising repolarizing currents in a heterozygous situation. Low potassium induced degradation of wild-type HERG combined with haplo-insufficiency of G816V HERG likely contributed to the patient's lethal arrhythmia.
- Theses and Dissertations