Biophysical Society Thematic Meeting| Padova 2019

Quantitative Aspects of Membrane Fusion and Fission

Poster Abstracts

59-POS Board 59 ENDOCYTOSIS AND DEGRADATION OF K V 1.5 CHANNELS INDUCED BY ACTIVATION OF PROTEIN KINASE C Shetuan Zhang 1 ; Tingzhong Wang 1 ; 1 Queen’s University, Biomedical and Molecular Sciences, Kingston, Ontario, Canada The voltage-gated potassium channel Kv1.5 plays important roles in the rhythmic beating of the atria, vascular tone of the pulmonary artery, and insulin secretion of pancreas. The activity of ion channels in a cell is determined by the function of single channels and the total number of channels in the plasma membrane. While the function and modulation of ion channels have been extensively studied, much less is known about how the number of ion channels in the plasma membrane is regulated. Using the patch clamp recording technique and biochemical methods in human embryonic kidney cells stably expressing Kv1.5 channels, we discovered that activation of protein kinase C (PKC) with PMA (phorbol 12-myristate 13-acetate, 10 nM) abolished Kv1.5 channel expression and current within 3 hours. Biochemical experiments revealed that PKC activation enhanced ubiquitination of Kv1.5 proteins. Vps24 (also known as charged multivesicular body protein 3, CHMP3) is a protein that sorts transmembrane proteins into lysosomes via the multivesicular body (MVB) pathway. It contributes to the endosomal sorting complexes required for transport (ESCRT)-III protein complex for protein sorting into the MVB. STAMBP (STAM-binding protein, also known as AMSH, associated molecule with the SH3 domain of STAM) is a deubiquitination isopeptidase that participates in the endosomal sorting of several cell-surface molecules. Our results showed that overexpression of Vps24 or STAMBP accelerated, whereas knockdown of Vps24 or STAMBP by siRNA transfection impeded PKC- mediated Kv1.5 degradation. Lysosome inhibitor bafilomycin A1 (1 µM) prevented PKC- mediated Kv1.5 degradation. Truncation of the N-terminus of Kv1.5 up to residue 209 (deltaN209) abolished PKC-mediated reduction in Kv1.5 current and expression. We conclude that PKC activation targets N-terminus of Kv1.5, leading to channel ubiquitination which triggers endocytosis and degradation of Kv1.5 channels through ESCRT machinery (Supported by the Canadian Institutes of Health Research Grant MOP-72911).

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