Roles of PKC Isoforms in PMA-Induced Modulation of the hERG Channel (Kv11.1).
Protein kinases C (PKC) modulate the activity of the Kv11.1 ion channel current (hERG). However, the differential effects of specific PKC subtypes on the biophysics of the channel are unknown. The pharmaceutical tools to selectively modulate PKC subtypes are not membrane permeable and must be added directly to the intracellular solution in electrophysiology studies. Here, the PatchXpress electrophysiology robot was used to voltage clamp up to 16 cells simultaneously yet asynchronously across individual Sealchip chambers. The precision afforded by repeats of automation procedures minimized the experimental errors typical of these assays. Eight well-known PKC selective peptidomimmetics and general synthetic modulators were used to modulate the protein-protein interactions between hERG and the major PKC subtypes. We identified a specific role for the PKCε inhibitory peptidomimmetics in decreasing PKC-induced hERG τ activation (80%) and half-maximum activation voltage (90%) at steady state; a specific PKCε activator exhibited the opposite effect. Disruption of PKCβ, PKCα, and PKCη interactions also showed significant effects albeit of lower magnitudes. The effect of PKCδ inhibitor was only marginal. A significant correlation was observed between the shifts in τ activation and half-maximum voltage at steady state (R(2)= 0.85). Peak current amplitudes and time constant of deactivation remained unaffected in all conditions.
O.R.B.I.T. Bioassays Integration & Technologies, Montréal, Québec, Canada Laboratoire d'Electrophysiologie Cellulaire et de Canalopathies, Centre de Biomédecine, Hôpital du Sacré-Cœur de Montréal, Montréal, Quebec, Canada oradresa@hotmail.com.
Olivier
O
Radresa
AVIVA Biosciences Corporation, San Diego, CA, USA.
António
A
Guia
Laboratoire d'Electrophysiologie Cellulaire et de Canalopathies, Centre de Biomédecine, Hôpital du Sacré-Cœur de Montréal, Montréal, Quebec, Canada Département de Médecine & Département de Pharmacologie, Faculté de Médecine, Université de Montréal, Quebec, Canada.
Ghayath
G
Baroudi
D002462:Cell Membrane / Q000378:metabolism; D000072237:ERG1 Potassium Channel / Q000737:chemistry*; D004594:Electrophysiology; D057809:HEK293 Cells; D006801:Humans; D007700:Kinetics; D010455:Peptides / Q000737:chemistry; D057786:Peptidomimetics / Q000737:chemistry; D025941:Protein Interaction Mapping; D020033:Protein Isoforms / Q000737:chemistry; D011493:Protein Kinase C / Q000737:chemistry*; D047428:Protein Kinase Inhibitors / Q000737:chemistry; D012371:Robotics; D013755:Tetradecanoylphorbol Acetate / Q000737:chemistry*; D014162:Transfection
D000072237:ERG1 Potassium Channel; C000606913:KCNH2 protein, human; D010455:Peptides; D057786:Peptidomimetics; D020033:Protein Isoforms; D047428:Protein Kinase Inhibitors; D011493:Protein Kinase C; D013755:Tetradecanoylphorbol Acetate
PKC; PatchXpress; hERG; intracellular peptide addition; membrane impermeable compounds; protein-protein interaction