Low-Resistance silver bromide electrodes for recording fast ion channel kinetics under voltage clamp conditions.

BACKGROUND

Two-electrode voltage clamp is a widely used technique for studying ionic currents. However, fast activation kinetics of ion channels are disguised by the capacitive transient during voltage clamp of Xenopus oocytes. The limiting factors of clamp performance include, but are not limited to, amplifier gain, membrane capacitance, and micropipette resistance. Previous work has focused on increasing amplifier gain (e.g.; high performing two-electrode amplifiers) or reducing the membrane capacitance (e.g.; the cut-open technique).

NEW METHOD

The use of an Ag-AgBr electrode with saturated KBr solution to reduce micropipette resistance.

RESULTS

The conductivity of 4 M KBr was 37 % higher compared to 3 M KCl and the micropipette resistance was reduced by 19 % when 4 M KBr was used, compared to the standard 3 M KCl solution. Micropipette resistances correlated positively with capacitive transient durations. Neither the current-voltage relationship of the voltage-gated sodium channel, Nav1.7, nor Xenopus oocyte stability were affected by bromide ions.

COMPARISON WITH EXISTING METHODS

The de facto standard for two-electrode voltage clamp is 3 M KCl and Ag-AgCl electrodes, which are associated an unnecessarily high micropipette resistance. Elsewise, cut-open voltage clamp techniques are technically demanding and require manipulation of the intracellular environment.

CONCLUSIONS

The use of an Ag-AgBr electrode with saturated KBr as micropipette solution reduces the capacitive transient in two-electrode voltage clamp recordings. Moreover, the exchange of chloride against bromide ions does not seem to affect oocyte physiology and ion channel kinetics.