Abbreviations: HEK, human embryonic kidney; hERG, human ether ago go related gene; IC 50 , half maximal inhibitory concentration; I hERG , hERG current; I Kr , rapidly activating delayed rectifier potassium current; I Ks , slowly activating delayed rectifier K + current; LQTS, long QT syndrome; MEM, minimum essential medium; QT c , corrected QT interval; V 1/2 , voltage of half maximal activation. CQ, chloroquine; HCQ, hydroxychloroquine; AZ, azithromycin; RDV, remdesivir.
Inwardly rectifying potassium (Kir) channels are essential for numerous physiological processes, including neuronal and cardiac excitability. Recently, the very rare disease Keppen-Lubinsky syndrome (KPLBS), caused by de novo heterozygous mutations in the Kir3.2 channel has been described. KPLBS leads to severe developmental and intellectual problems, microcephaly, tightly adherent skin and severe generalized lipodystrophy. Recent breakthroughs in x-ray crystallography and cryo-electron microscopy provide insights into the structure of inward rectifier channels, and enable detailed characterization of the structural effects of disease-causing mutations. Multi-ms time scale molecular dynamics simulations on the WT Kir3.2 and disease mutant channel provide insights into the structural changes, caused by the point mutation, and the molecular mechanisms, leading to loss of K þ selectivity.
The voltage-gated potassium channel Kv1.5 plays important roles in the repolarization of atrial action potentials and regulation of the vascular tone. While the modulation of Kv1.5 function has been well studied, less is known about how the protein levels of Kv1.5 on the cell membrane are regulated. Here, through electrophysiological and biochemical analyses of Kv1.5 channels heterologously expressed in HEK293 cells and neonatal rat ventricular myocytes, as well as native Kv1.5 in human induced pluripotent stem cell (iPSC)-derived atrial cardiomyocytes, we found that activation of protein kinase C (PKC) with phorbol 12-myristate 13-acetate (PMA, 10 nM) diminished Kv1.5 current (I
Kv1.5
) and protein levels of Kv1.5 in the plasma membrane. Mechanistically, PKC activation led to monoubiquitination and degradation of the mature Kv1.5 proteins. Overexpression of Vps24, a protein that sorts transmembrane proteins into lysosomes
via
the multivesicular body (MVB) pathway, accelerated, whereas the lysosome inhibitor bafilomycin A1 completely prevented PKC-mediated Kv1.5 degradation. Kv1.5, but not Kv1.1, Kv1.2, Kv1.3, or Kv1.4, was uniquely sensitive to PMA treatment. Sequence alignments suggested that residues within the N terminus of Kv1.5 are essential for PKC-mediated Kv1.5 reduction. Using N-terminal truncation as well as site-directed mutagenesis, we identified that Thr15 is the target site for PKC that mediates endocytic degradation of Kv1.5 channels. These findings indicate that alteration of protein levels in the plasma membrane represents an important regulatory mechanism of Kv1.5 channel function under PKC activation conditions.
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