Functional properties and modulation of extracellular epitope-tagged Ca(V)2.1 voltage-gated calcium channels.
Depolarisation-induced Ca2+ influx into electrically excitable cells is determined by the density of voltage-gated Ca2+ channels at the cell surface. Surface expression is modulated by physiological stimuli as well as by drugs and can be altered under pathological conditions. Extracellular epitope-tagging of channel subunits allows to quantify their surface expression and to distinguish surface channels from those in intracellular compartments. Here we report the first systematic characterisation of extracellularly epitope-tagged Ca(V)2.1 channels. We identified a permissive region in the pore-loop of repeat IV within the Ca(V)2.1 alpha(1) subunit, which allowed integration of several different tags (hemagluttinine [HA], double HA; 6-histidine tag [His], 9-His, bungarotoxin-binding site) without compromising alpha(1) subunit protein expression (in transfected tsA-201 cells) and function (after expression in X. laevis oocytes). Immunofluorescence studies revealed that the double-HA tagged construct (1722-HAGHA) was targeted to presynaptic sites in transfected cultured hippocampal neurons as expected for Ca(V)2.1 channels. We also demonstrate that introduction of tags into this permissive position creates artificial sites for channel modulation. This was demonstrated by partial inhibition of 1722-HA channel currents with anti-HA antibodies and the concentration-dependent stimulation or partial inhibition by Ni-nitrilo triacetic acid (NTA) and novel bulkier derivatives (Ni-trisNTA, Ni-tetrakisNTA, Ni-nitro-o-phenyl-bisNTA, Ni-nitro-p-phenyl-bisNTA). Therefore our data also provide evidence for the concept that artificial modulatory sites for small ligands can be introduced into voltage-gated Ca2+ channel for their selective modulation.
Watschinger
Katrin
K
Pharmacology and Toxicology Unit, Institute of Pharmacy and Centre for Molecular Biosciences Innsbruck, University of Innsbruck, Innsbruck, Austria. katrin.watschinger@i-med.ac.at
Horak
Silja B
SB
Schulze
Katrin
K
Obermair
Gerald J
GJ
Wild
Claudia
C
Koschak
Alexandra
A
Sinnegger-Brauns
Martina J
MJ
Tampé
Robert
R
Striessnig
Jörg
J
D020864:Calcium Channels, N-Type / Q000032:analysis / Q000187:drug effects*; D004594:Electrophysiology; D006624:Hippocampus / Q000166:cytology; D006801:Humans; D015640:Ion Channel Gating; D015336:Molecular Probe Techniques; D009474:Neurons / Q000502:physiology; D017729:Presynaptic Terminals / Q000737:chemistry*; D021122:Protein Subunits
D020864:Calcium Channels, N-Type; D021122:Protein Subunits
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