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Use of a Planar Patch Electrode to Measure NBCe1‐A Function in Mammalian Cells

Standard whole cell‐patch clamping is labor‐intensive whereas automated planar patch clamp devices offer the possibility of higher throughput. Planar patch clamp electrodes are also attractive because their geometry facilitates obtaining electrical and optical recordings simultaneously. Previous studies have utilized the Port‐a‐Patch system (Nanion Technologies GmbH), to study ion channel activity however there are no studies which have demonstrated the utility of this approach with electrogenic cotransporters, which in general have a lower conductance. In this study, we utilized a modified Port‐a‐Patch system to measure the functional activity of the electrogenic sodium bicarbonate cotransporter, NBCe1‐A. HEK‐293T cells permanently expressing NBCe1‐A were generated. Cell monolayers were detached using 0.025% Trypsin/0.22 mM EDTA in PBS and were re‐suspended at a concentration of 500,000 cells/ml in 50% DMEM/PBS. The cells were kept at room temperature until measurements were made. Approximately 2,500 cells were placed on a planar electrode chip from which a single cell was automatically selected. Planar electrode chips with variable resistances were assessed. Using chips with a resistance of 2 – 3.5 MOhm, we routinely obtained GOhm seals using this approach. Whole cell currents were elicited by voltage ramps from −100 mV to +30 mV from a holding potential of −100 mV. Cell membrane capacitance for the HEK‐293T cells was 7.14 ± 0.67 pF (n = 21) and access resistance was 8.6 ± 0.5 MOhm (n =21). The DIDS‐inhibitable Na+‐and HCO3−‐dependent NBCe1‐A‐mediated average whole cell current was 29.3 ± 3.2 pA (n =21) at +30 mV. These results represent the first reported use of a planar patch‐clamp device to study ionic currents mediated by a heterologously expressed electrogenic cotransporter in mammalian cells.