Cut-open Technique Research Articles

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

BackgroundTwo-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 methodThe use of an Ag-AgBr electrode with saturated KBr solution to reduce micropipette resistance. ResultsThe 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 methodsThe 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. ConclusionsThe 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.

The <em>Xenopus</em> Oocyte Cut-open Vaseline Gap Voltage-clamp Technique With Fluorometry

The cut-open oocyte Vaseline gap (COVG) voltage clamp technique allows for analysis of electrophysiological and kinetic properties of heterologous ion channels in oocytes. Recordings from the cut-open setup are particularly useful for resolving low magnitude gating currents, rapid ionic current activation, and deactivation. The main benefits over the two-electrode voltage clamp (TEVC) technique include increased clamp speed, improved signal-to-noise ratio, and the ability to modulate the intracellular and extracellular milieu. Here, we employ the human cardiac sodium channel (hNaV1.5), expressed in Xenopus oocytes, to demonstrate the cut-open setup and protocol as well as modifications that are required to add voltage clamp fluorometry capability. The properties of fast activating ion channels, such as hNaV1.5, cannot be fully resolved near room temperature using TEVC, in which the entirety of the oocyte membrane is clamped, making voltage control difficult. However, in the cut-open technique, isolation of only a small portion of the cell membrane allows for the rapid clamping required to accurately record fast kinetics while preventing channel run-down associated with patch clamp techniques. In conjunction with the COVG technique, ion channel kinetics and electrophysiological properties can be further assayed by using voltage clamp fluorometry, where protein motion is tracked via cysteine conjugation of extracellularly applied fluorophores, insertion of genetically encoded fluorescent proteins, or the incorporation of unnatural amino acids into the region of interest(1). This additional data yields kinetic information about voltage-dependent conformational rearrangements of the protein via changes in the microenvironment surrounding the fluorescent molecule.