Down-regulation of endogenous KLHL1 decreases voltage-gated calcium current density.
The actin-binding protein Kelch-like 1 (KLHL1) can modulate voltage-gated calcium channels in vitro. KLHL1 interacts with actin and with the pore-forming subunits of Cav2.1 and CaV3.2 calcium channels, resulting in up-regulation of P/Q and T-type current density. Here we tested whether endogenous KLHL1 modulates voltage gated calcium currents in cultured hippocampal neurons by down-regulating the expression of KLHL1 via adenoviral delivery of shRNA targeted against KLHL1 (shKLHL1). Control adenoviruses did not affect any of the neuronal properties measured, yet down-regulation of KLHL1 resulted in HVA current densities ~68% smaller and LVA current densities 44% smaller than uninfected controls, with a concomitant reduction in α(1A) and α(1H) protein levels. Biophysical analysis and western blot experiments suggest Ca(V)3.1 and 3.3 currents are also present in shKLHL1-infected neurons. Synapsin I levels, miniature postsynaptic current frequency, and excitatory and inhibitory synapse number were reduced in KLHL1 knockdown. This study corroborates the physiological role of KLHL1 as a calcium channel modulator and demonstrates a novel, presynaptic role.
Cell and Molecular Physiology Department, Loyola University Chicago, Stritch School of Medicine, 2160 S. First Avenue, Maywood, IL 60153, USA.
Paula P
PP
Perissinotti
Cell and Molecular Physiology Department, Loyola University Chicago, Stritch School of Medicine, 2160 S. First Avenue, Maywood, IL 60153, USA.
Elizabeth G
EG
Ethington
Office of Research Services, Loyola University Chicago, Stritch School of Medicine, 2160 S. First Avenue, Maywood, IL 60153, USA.
Leanne
L
Cribbs
Institute for Translational Neuroscience, Department of Lab Medicine & Pathology, University of Minnesota, Minneapolis, MN 55455, USA.
Michael D
MD
Koob
Cell and Molecular Physiology Department, Loyola University Chicago, Stritch School of Medicine, 2160 S. First Avenue, Maywood, IL 60153, USA; Neuroscience Institute, Loyola University Chicago, Stritch School of Medicine, 2160 S. First Avenue, Maywood, IL 60153, USA.
Jody
J
Martin
Cell and Molecular Physiology Department, Loyola University Chicago, Stritch School of Medicine, 2160 S. First Avenue, Maywood, IL 60153, USA; Neuroscience Institute, Loyola University Chicago, Stritch School of Medicine, 2160 S. First Avenue, Maywood, IL 60153, USA. Electronic address: epiedra@lumc.edu.
Erika S
ES
Piedras-Rentería
D000818:Animals; D001640:Bicuculline / Q000494:pharmacology; D002118:Calcium / Q000378:metabolism*; D020864:Calcium Channels, N-Type / Q000378:metabolism*; D020747:Calcium Channels, T-Type / Q000378:metabolism; D002478:Cells, Cultured; D015536:Down-Regulation; D055724:Electrophysiological Phenomena / Q000187:drug effects; D006624:Hippocampus / Q000166:cytology / Q000378:metabolism*; D006801:Humans; D008840:Microfilament Proteins / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D011810:Quinoxalines / Q000494:pharmacology; D034622:RNA Interference; D034741:RNA, Small Interfering / Q000378:metabolism; D051381:Rats; D055366:Synaptic Potentials / Q000187:drug effects
D001640:Bicuculline; D002118:Calcium; D020864:Calcium Channels, N-Type; D020747:Calcium Channels, T-Type; D008840:Microfilament Proteins; D011810:Quinoxalines; D034741:RNA, Small Interfering; C412617:KLHL1 protein, human; C062865:2,3-dioxo-6-nitro-7-sulfamoylbenzo(f)quinoxaline
ATXN8OS; Actin-binding protein; HVA calcium current; LVA calcium currents; P/Q-type channel; Spinocerebellar ataxia type 8; Synaptic; T-type channel