Trafficking of an endogenous potassium channel in adult ventricular myocytes.
The roles of several small GTPases in the expression of an endogenous potassium current, I(to,f), in adult rat ventricular myocytes have been investigated. The results indicate that forward trafficking of newly synthesized Kv4.2, which underlies I(to,f) in these cells, requires both Rab1 and Sar1 function. Expression of a Rab1 dominant negative (DN) reduced I(to,f) current density by roughly one-half relative to control, mCherry-transfected myocytes. Similarly, expression of a Sar1DN nearly halved I(to,f) current density. Rab11 is not essential to trafficking of Kv4.2, as expression of a Rab11DN had no effect on I(to,f) over the time frames investigated here. In a process dependent on intact endoplasmic reticulum (ER)-to-Golgi transport, however, overexpression of wild-type Rab11 resulted in a doubling of I(to,f) density; block of ER-to-Golgi traffic by Brefeldin A completely abrogated the effect. Also implicated in the trafficking of Kv4.2 are Rab5 and Rab4. Rab5DN expression increased endogenous I(to,f) by two- to threefold, nonadditively with inhibition of dynamin-dependent endocytosis. And, in a phenomenon similar to that previously reported for myoblast-expressed Kv1.5, Rab4DN expression roughly doubled endogenous peak transient currents. Colocalization experiments confirmed the involvement of Rab4 in postinternalization trafficking of Kv4.2. There was little role evident for the lysosome in the degradation of internalized Kv4.2, as overexpression of neither wild-type nor DN isoforms of Rab7 had any effect on I(to,f). Instead, degradation may depend largely on the proteasome; the proteasome inhibitor MG132 significantly increased I(to,f) density.
Dept. of Anesthesiology, Pharmacology and Therapeutics, Univ. of British Columbia, 2350 Health Sciences Mall, Vancouver, BC, Canada V6T 1Z3. dfedida@exchange.ubc.ca
Tiantian
T
Wang
Yvonne
Y
Cheng
Ying
Y
Dou
Charitha
C
Goonesekara
Jens-Peter
JP
David
David F
DF
Steele
Chen
C
Huang
David
D
Fedida
D000818:Animals; D020126:Brefeldin A / Q000494:pharmacology; D002478:Cells, Cultured; D004721:Endoplasmic Reticulum / Q000378:metabolism; D019204:GTP-Binding Proteins / Q000378:metabolism; D006056:Golgi Apparatus / Q000378:metabolism; D006352:Heart Ventricles / Q000187:drug effects / Q000378:metabolism*; D007976:Leupeptins / Q000494:pharmacology; D008297:Male; D020559:Monomeric GTP-Binding Proteins / Q000378:metabolism; D032383:Myocytes, Cardiac / Q000187:drug effects / Q000378:metabolism*; D061988:Proteasome Inhibitors / Q000494:pharmacology; D021381:Protein Transport / Q000187:drug effects / Q000502:physiology*; D051381:Rats; D017208:Rats, Wistar; D051672:Shal Potassium Channels / Q000378:metabolism*; D020761:rab1 GTP-Binding Proteins / Q000378:metabolism; D020695:rab4 GTP-Binding Proteins / Q000378:metabolism; D020696:rab5 GTP-Binding Proteins / Q000378:metabolism
D007976:Leupeptins; D061988:Proteasome Inhibitors; D051672:Shal Potassium Channels; C492252:Wdr44 protein, rat; D020126:Brefeldin A; D019204:GTP-Binding Proteins; C487879:SAR1 protein, rat; D020559:Monomeric GTP-Binding Proteins; D020761:rab1 GTP-Binding Proteins; D020695:rab4 GTP-Binding Proteins; D020696:rab5 GTP-Binding Proteins; C072553:benzyloxycarbonylleucyl-leucyl-leucine aldehyde