Kv1.5 surface expression is modulated by retrograde trafficking of newly endocytosed channels by the dynein motor.
In this article we have investigated the mechanisms by which retrograde trafficking regulates the surface expression of the voltage-gated potassium channel, Kv1.5. Overexpression of p50/dynamitin, known to disrupt the dynein-dynactin complex responsible for carrying vesicle cargo, substantially increased outward K+ currents in HEK293 cells stably expressing Kv1.5 (0.57+/-0.07 nA/pF, n=12; to 1.18+/-0.2 nA/pF, n=12, P<0.01), as did treatment of the cells with a dynamin inhibitory peptide, which blocks endocytosis. Nocodazole pretreatment, which depolymerizes the microtubule cytoskeleton along which dynein tracks, also doubled Kv1.5 currents in HEK cells and sustained K+ currents in isolated rat atrial myocytes. These increased currents were blocked by 1 mmol/L 4-aminopyridine, and the specific Kv1.5 antagonist, DMM (100 nM). Confocal imaging of both HEK cells and myocytes, as well as experiments testing the sensitivity of the channel in living cells to external Proteinase K, showed that this increase of K+ current density was caused by a redistribution of channels toward the plasma membrane. Coimmunoprecipitation experiments demonstrated a direct interaction between Kv1.5 and the dynein motor complex in both heterologous cells and rat cardiac myocytes, supporting the role of this complex in Kv1.5 trafficking, which required an intact SH3-binding domain in the Kv1.5 N terminus to occur. These experiments highlight a pathway for Kv1.5 internalization from the cell surface involving early endosomes, followed by later trafficking by the dynein motor along microtubules. This work has significant implications for understanding the way Kv channel surface expression is regulated.
Choi
Woo Sung
WS
Department of Cellular and Physiological Sciences, University of British Columbia, Vancouver, BC V6T 1Z3, Canada.
Khurana
Anu
A
Mathur
Rajesh
R
Viswanathan
Vijay
V
Steele
David F
DF
Fedida
David
D
D000818:Animals; D001665:Binding Sites; D016466:CHO Cells; D002460:Cell Line; D006224:Cricetinae; D004398:Dyneins / Q000502:physiology*; D004705:Endocytosis; D011992:Endosomes / Q000378:metabolism; D006801:Humans; D047468:Immunoprecipitation; D051666:Kv1.5 Potassium Channel; D032383:Myocytes, Cardiac / Q000378:metabolism*; D015739:Nocodazole / Q000494:pharmacology; D024642:Potassium Channels, Voltage-Gated / Q000737:chemistry / Q000378:metabolism*; D021381:Protein Transport; D051381:Rats; D018909:src Homology Domains
C496981:KCNA5 protein, human; C496983:Kcna5 protein, rat; D051666:Kv1.5 Potassium Channel; D024642:Potassium Channels, Voltage-Gated; D004398:Dyneins; D015739:Nocodazole