Functional study of Smartox’s Huwentoxin IV and Ergtoxin
Introduction
The following case study gives details of patch-clamp recordings using the Nanion Port-a-Patch® system with Nav1.5 and hERG expressed in HEK293 cells provided by Creacell (France).
The experiment demonstrated the high levels of bioactivity of Smartox toxins Huwentoxin IV and Ergtoxin, further validating their use as tools in ion channel research.
Methods
Cells
HEK293 cells expressing Nav1.5 or hERG channel (Creacell, France).
Patch Clamp solutions
External solution: 140 mM NaCl, 4 mM KCl, 1 mM MgCl2, 2 mM CaCl2, 5 mM D-Glucose monohydrate, 10 mM HEPES/NaOH pH 7.4. Internal solution: 50 mM CsCl, 10 mM NaCl, 60 mM CsF, 20 mM EGTA, 10 mM HEPES/CsOH, pH 7.2. For hERG experiments, Internal solution: 50 mM KCl, 10 mM NaCl, 60 mM KF, 20 mM EGTA, 10 mM HEPES/KOH, pH 7.2.
Electrophysiology
Whole cell Nav1.5 currents were observed using voltage step protocol: -80 mV to 60 mV for 20 ms increasing in 10 mV increments from a holding potential of -80 mV (sweep interval 1 s). Whole cell hERG current were observed using a voltage step protocol: -80 mV to +40 mV step for 500 ms and to -120 mV to +80 mV for 500 ms increasing in 20 mV increments from a holding potential of -80 mV (sweep interval 10 s).
Results
Figure 1 shows whole-cell recording on Nav1.5 cell line and the effect of the application of 1 µM and 2 µM Huwentoxin IV from Smartox. The current is blocked by the addition of 1 µM and 2 µM Huwentoxin IV.
Figure 1: Effect of Huwentoxin IV on Nav1.5 currents. The figure shows the current response to a single step voltage protocol from -80 mV to 20 mV and subsequent block by external addition of Huwentoxin IV, 1 μM and 2 μM (blue).
Figure 2 shows a recording from a HEK293 cell expressing hERG channel and the inhibition by 1 µM ergtoxin from Smartox.
Figure 2: Example of hERG channel. Panel A shows an example recording of an I-V pulse step protocol. Panel B shows the the IV relationship curve in control and in presence of 1 μM Smartox's ergtoxin. Panel C shows the effect of 1 μM of ergtoxin application (blue) compared to the control (black) with a single step protocol at -80 mV.
Summary
The inhibition of ion channels using toxins from Smartox was done by a simple manual exchange of external solution. Different concentration of toxins were used for the different channels. The HEK293 cells were nicely sealed and really stable over time (hERG recording lasted more than 100 minutes) enough to do a full pharmacology. We only measured few concentration of toxins to block both channels Nav 1.5 and hERG. The relative ease with which cells could be captured to the aperture with good seals indicates that these cells are ideally suited for use on the Nanion Port-a-Patch®.
Study location: Smartox, Biopolis La Tronche, France
Date of study: November 26nd 2010
Author: Mohamed Kreir,
This e-mail address is being protected from spambots. You need JavaScript enabled to view it