Development and Evaluation of Novel Solution Pairs to Enhance Seal Resistance in Automated Patch Clamp Electrophysiology and Ion Channel Experiments

Catherine Hodgson¹, Edward Stevens¹, Andrew James¹, Anissa Bara², Beatrice Badone², Damian Bell², Daniel Sauter², Gary Clark¹

¹Metrion Biosciences Ltd, Granta Centre, Granta Park, Cambridge, CB21 6AL, UK

²Sophion Bioscience A/S, Industriparken 39, 2750 Ballerup, Denmark

Overview of automated patch clamp electrophysiology and ion channels

Gigaohm seals, or ‘gigaseals’, are crucial for patch clamp electrophysiology, ensuring excellent electrical access to cells to enable high-quality recordings. These seals form through chemical bonds and electrostatic forces between the cell membrane and the glass pipette in manual patch clamp, or between the cell membrane and chip substrate in planar patch clamp. Planar patch clamp often employs ‘seal enhancers’ to increase seal resistances, with CaF₂ being the most commonly used. It is hypothesized that high extracellular Ca²⁺ and intracellular F^- concentrations lead to CaF₂ precipitate formation at the solution interface, promoting seal formation.

However, CaF₂ as a seal enhancer has limitations. F^- interacts with various internal components such as protein kinase A, adenylate cyclase, and lipid phosphatases, which can affect the biophysical properties of some ion channels. Additionally, F^- is not ideal when recording from Ca²⁺- activated ion channels due to the resulting unknown concentrations of free intracellular Ca²⁺.

In an effort to overcome these limitations, Sophion developed new solution pairs in 2017 that foster seal formation (Patent: WO2018100206A1). Building on this technology, Metrion and Sophion collaborated to further determine whether other insoluble salts can act as seal enhancers and how these solution pairs affect the biophysical properties and pharmacology of the investigated ion channels.

Materials and methods for gigaseal patch clamp electrophysiology experiments

Experiments were conducted using a Sophion Bioscience Qube 384 with QChip 384 (single hole) and QChip 384X (multihole) consumables. Temperature was maintained at 22 °C using the Qube temperature control module.

Analysis was conducted using Sophion Analyzer v9.0.42 and GraphPad Prism v10.2.2.

CHO-hNa_v1.5 and HEK293-hCa_v1.2 cell lines were provided by Metrion Biosciences.

All compounds were tested at: 0.001, 0.01, 0.1, 1, 10 and 100 µM.

Results from automated patch clamp electrophysiology and gigaseal studies

Table 1. Correlation between salt pair solubility product constants (K_sp) and gigaseal formation on Qube 384. Little correlation was found between the solubility product constants (K_sp) of Ca²⁺, Ba²⁺ and Sr²⁺ salts (A) and their ability to foster gigaseal formation (B). Despite the PO­₄^3- salts having very low K_sp values and SrCO₃ having a similar K_sp value to CaF­₂ and BaSO₄, these salts failed to produce gigaohm seals. Moderate seal resistances with PO₄^3- salts were transient and unstable. Median resistances calculated from 24 cells per salt pair.

Table 2. Resistances of seals formed using various concentrations of extracellular Ba²⁺ and intracellular SO₄^2-. Gigaseals only formed with ≥ 3 mM Ba²⁺ and in the presence of SO₄^2-. Median resistances calculated from 24 or 48 cells per condition.

Figure 1. Effects of CaF₂ and BaSO₄ on hNa_v1.5 channel biophysics. hNa_v1.5 V_0.5 inactivation with different cation and anion concentrations (mean ± S.D.; N ≥ 11). Increasing concentrations of intracellular F^- caused a depolarising shift in V_0.5 inactivation (A). In contrast, increasing concentrations of SO₄^2- had no effect on hNa_v1.5 V_0.5 inactivation (B). One-way ANOVAs conducted within each cation group followed by Tukey’s Honestly Significant Difference post-hoc tests: ** = p < .01; *** = p < .001; **** = p < .0001.

Figure 2. CaF₂ versus BaSO₄ – hNa_v1.5 pharmacology. A) Representative sweep plots (left) and current-time (I-t) plots (right) for hNa_v1.5 inhibition by amitriptyline. There was no difference in cumulative inhibition of hNa_v1.5 by increasing concentrations of amitriptyline between CaF₂ and BaSO₄. B) Screening of a range of inhibitory compounds showed no difference in hNa_v1.5 pharmacology between CaF₂ and BaSO₄. Concentration-response curves for amitriptyline against hNa_v1.5 using CaF₂ or BaSO₄ as the seal enhancer (mean ± S.D.; N = 12 wells per concentration for CaF₂, N = 8 wells per concentration for BaSO₄).

Figure 3. CaF₂ versus BaSO₄ – hCa_v1.2 kinetics. hCa_v1.2 exhibits Ca²⁺-dependent inactivation when CaF₂ is used as the seal enhancer (A). BaSO₄ as the seal enhancer (using Ba²⁺ as a surrogate carrier ion) (B) confers loss of the Ca²⁺-dependent inactivation of hCa_v1.2 observed with CaF₂. Example sweep plots derived from Sophion Analyzer v9.0.42.

Figure 4. CaF₂ versus BaSO₄ – hCa_v1.2 pharmacology. Concentration-response curves for two common inhibitors against hCa_v1.2, nifedipine (A) and verapamil (B) (CaF₂: N = 2‑5 wells per concentration; BaSO₄: N = 6-12 wells per concentration). Compound potencies (IC₅₀ values) did not differ between CaF₂ and BaSO₄. Data displayed as mean ± S.D.

Findings of automated patch clamp electrophysiology experiments

• BaSO₄ was identified as an equivalent seal enhancer to CaF₂
• BaSO₄ and CaF₂ characterized across two ion channels: hNav1.5 and hCav1.2.
• Intracellular F^- caused depolarising shifts in the voltage dependence of inactivation of hNav1.5 where no such effects were observed with SO₄^2- in the intracellular solution.
• No difference in pharmacological effects of inhibitory compounds against hNav1.5 or hCav1.2 was observed between the two solution pairs, CaF₂ and BaSO₄.
• BaSO₄ is well-suited as a seal enhancer for recording from non-K⁺-conducting Ca²⁺-activated channels, such as TMEM16A. In particular as BaSO₄ allows more accurate estimation of free intracellular Ca2+ concentration.