Assessing Ion Channel Expression by Patch Clamp (Version 1.0)
Version
1.0
Notice
This page is the corresponding protocol tomestone page generated as part of the ATLAS-D2K shutdown in July 2025. Many links on this page may be broken.
Authors
Thomas Kleyman
Keywords
[‘hiPSC’, ‘kidney’, ‘mouse’, ‘nephron’, ‘organoid’, ‘patch clamp’, ‘electrophysiology’, ‘epithelial cells’]
Subjects
[‘Physiology’]
Release Date
2024-10-04
Abstract
Using cells derived from kidney organoids, we used whole cell and cell attached patch clamp techniques to examine the functional expression of three key K+ and Na+-selective ion channels that are found in distal nephron segments: the large-conductance Ca2+-activated K+ channel (BKCa), the renal outer medullary potassium channel (ROMK, Kir1.1) and the epithelial Na+ channel (ENaC). Electrophysiological analysis in combination with selective inhibitors for BKCa (iberiotoxin), ROMK (VU591) showed that these channels are expressed in different cell populations. Both cell populations also express other Ba2+-sensitive K+ channels. BKCa expression was confirmed at a single channel level, based on its high conductance and voltage-dependence of activation. We also found a population of cells expressing amiloride-sensitive ENaC currents.
Introduction
Equipment
Micropipette puller (Narishige ) Patch-clamp amplifier (Warner Instruments) Digidata 1440A (Molecular Devices) pClamp software (Molecular Devices)
Procedure
Isolation of cells from hiPSC-derived kidney organoids. To isolate cells and tubule segments, day 14 kidney organoids were transferred to a clean 15 ml falcon tube in 2 ml of Stage II medium. Kidney organoids were gently triturated with a fire-polished glass pipette and the cell suspension was centrifuged at 420 x g for 5 min. The pellet was resuspended in DMEM media supplemented with 10% fetal calf serum, 1% penicillin/streptomycin, and 1% minimal essential medium nonessential amino acids (Invitrogen). The centrifugation and resuspension steps were repeated three times. Finally, the pellet was resuspended in 1.5 ml of culture media, and the suspension was seeded on 8-mm diameter round glass cover slips coated with poly-L-Lysine. After an incubation of 2 h at 37°C with 5% CO2, 3 ml of warm culture media was added to each well and the tissue culture plate was returned to the incubator. Electrophysiological studies were performed within 2 to 10 h of plating.
Electrophysiological studies. Electrical activity of cells and tubules isolated from hiPSC-derived kidney organoids was evaluated using the patch-clamp technique. Glass coverslips containing isolated cells were transferred to a chamber mounted on the stage of a Nikon Ti inverted microscope equipped a Sedat Quad set (Chroma Technology), a Lambda XL light source (Sutter Instruments) and an ORCA-Flash 2.8 camera (Hamamatsu). Micropipettes were pulled from borosilicate glass capillary tubes with a PP-81 puller (Narishige). Fire-polished micropipettes with a tip resistance of 1.5-3 MOhms were used for voltage-clamp recordings. Experiments were performed at room temperature with a PC-505B patch-clamp amplifier (Warner Instruments). Signals were low-pass filtered at 1 kHz (four-pole Bessel filter) and digitized using a Digidata 1440A (Molecular Devices) at 5 kHz. Junction potential was cancelled when the patch-pipette was dipped in the bath solution close to the cell before sealing the cell membrane. Cell capacitance was obtained by reading the value for whole-cell input capacitance neutralization directly from the amplifier. Command protocols, data acquisition and analysis were controlled with pClamp 10 (Molecular Devices).
Cell attached recordings. For cell-attached patch studies, the recording chamber contained the following solution (in mM): 135 NaCl, 5 KCl, 1 MgCl2, 2.5 CaCl2, 10 glucose and 10 HEPES, pH 7.4. The pipette solution was composed of (in mM) 145 KCl, 1 MgCl2, 0.1 CaCl2, 1 EGTA, and 10 HEPES (pH 7.2). In cell-attached data, all potentials were expressed in terms of the voltage applied to the pipette (extracellular side of the patch). Cell-attached patches were voltage-clamped at a pipette potential of 0 mV. Currents were evoked by 1 sec, 10 mV depolarizing steps from -80 to +80 mV. The single channel slope conductance was obtained by fitting a straight line to the linear part of I-V curves.
Whole-cell K+ currents. K+ currents were recorded from isolated cells and tubules obtained from kidney organoids using the conventional whole-cell technique. Cell interior was accessed by mechanical rupture of the membrane attached to the patch pipette. Tubule segment were patched from the basolateral side. Bath solution was composed of (in mM) 135 NaCl, 5 KCl, 1 MgCl2, 2.5 CaCl2, 10 glucose and 10 HEPES, pH 7.4. The pipette solution was composed of (in mM) 145 KCl, 1 MgCl2, 1 EGTA, 10 HEPES (pH 7.2) and free Ca2+ was adjusted to 10 M using MaxChelator software (version 2.4). Whole-cell currents were evoked by 0.2 sec, 10 mV depolarizing steps from -100 to +100 mV. Whole-cell slope conductance was estimated from least mean square fitting to the linear portion of the I-V curve (+40 to +100 mV range for BKCa). To define the identity of the K+ channels mediating whole-cell currents the following inhibitors were added serially in this order: i) iberiotoxin (IbTX), ii) VU592 and iii) Ba2+. IbTX (Alomone), is a toxin purified from the Eastern Indian red scorpion Buthus tamulus that selectively binds to the BKCa alpha subunit and inhibits its function by decreasing channel open probability. IbTX was used at a final concentration of 100 nM to define the component of the K+ current-mediated by BKCa. VU591, a ROMK specific inhibitor, was added to the bath solution to obtain a final concentration of 10 uM. Ba2+ is non-selective K+ channel blocker and was added to the bath solution at final concentration of 5 mM. Steady-state conductance-voltage (G-V) relationships were obtained using the conventional whole-cell technique. The voltage of half-maximal activation (V50) was determined by fitting normalized G-V curves to a Boltzmann function: G/Gmax = 1 + exp[-(V-V50)QF/RT]-1. G is chord conductance at the command potential V assuming an EK of -85 mV, Gmax is the maximal conductance, Q is the equivalent gating charge (slope of the G-V relationship or “voltage dependence”). T was 20oC. F and R have their usual definition.
Whole-cell Na+ currents. Cell access was gained by adding amphotericin B to the pipette solution (perforated patch technique). Amphotericin B was added to the patch pipette to a final concentration of 120 ug/ml. The bath solution was composed of (in mM) 135 NaCl, 1 MgCl2, 2.5 CaCl2, 10 glucose and 10 HEPES, pH 7.4. The pipette solution was composed of (in mM) 135 NaCl, 1 MgCl2, 0.1 CaCl2, 1 EGTA, and 10 HEPES (pH 7.2). For current recordings, the membrane potential was initially held at -60 mV. Whole-cell currents were evoked by 0.2 sec, 10 mV depolarizing steps from -80 to +80 mV. The single channel slope conductance was obtained by fitting a straight line to the linear part of I-V curves. Amiloride, an ENaC blocker, was used at a final concentration of either 10 uM.
Acknowledgement
Nicolas Montalbetti, Shaohu Sheng, Aneta J. Przepiorski, Joseph C. Maggiore, Marcelo D. Carattino, Neil A. Hukriede, Thomas R. Kleyman
Consortium
(Re)Building a Kidney (RBK) Consortium
