Planar Patch Clamp Approach to Characterize Ionic Currents from Intact Lysosomes

Abstract

Since its launch in the early 1980s, the patch clamp method has been extensively used to study ion channels in the plasma membrane, but its application to the study of intracellular ion channels has been limited. Unlike the plasma membrane, intracellular membranes are usually not stable enough to withstand mechanical manipulation by glass electrodes during seal formation and rupturing of the membrane. To circumvent these problems, we developed a method involving the immobilization of isolated organelles on a solid matrix planar glass chip. This glass chip contains a microstructured hole that supports the formation of gigaseals and subsequent electrophysiological recordings despite the high fragility of intracellular membranes. Here, we report the experimental details of this method using lysosomes, which are the smallest cellular organelles, as a model system. We demonstrate that we can record endogenous ionic currents from wild-type lysosomes, as well as from lysosomes overexpressing ion channels, and expect that this method will provide electrophysiological access to a broad range of intracellular ion channels.

A small molecule restores function to TRPML1 mutant isoforms responsible for mucolipidosis type IV

“…Luminal solution was 60 Ca-MSA, 70 K-MSA, 10 HEPES (pH adjusted with MSA to 4.6). The membrane potential was held at þ 60 mV, and 500 ms voltage ramps from À 200 to þ 100 mV were applied every 5 s. Due to high luminal calcium used to ensure giga seals when performing whole-endolysosomal patch-clamp on the port-a-patch system 37,38 , the inward currents are less rectifying and reverse at more positive potentials, depending on current amplitude and protein expression level 9 . All recordings were obtained at room temperature and were analysed using PatchMaster (HEKA) and Origin 6.1 (OriginLab) software.…”

“…Whole-lysosome planar patch-clamp recordings and preparation of lysosomes (HEK293, Human Fibroblast) were performed as described previously 37,38 . In brief, late endosomes and lysosomes were enlarged by treating the cells with 1 mM vacuolin-1 overnight 12,37,38,49 . The following day, cells were homogenized in homogenization buffer (0.25 M sucrose, 10 mM Tris-Cl, pH 7.4) on ice using a potter homogenizer to obtain whole-cell lysates.…”

“…The supernatant was collected and the pellet washed in 150 mM KCl, 10 mM Tris-Cl, pH 7.4, followed by a final centrifugation step at 25,000 g for 15 min at 4°C. The planar patch-clamp technology combined with a pressure control system and microstructured glass chips containing an aperture of B1 mm diameter (resistances of 10-15 MO) (Port-a-Patch, Nanion Technologies) were applied as described 37,38 . Currents were recorded using an EPC-10 patch-clamp amplifier and PatchMaster acquisition software (HEKA).…”

“…We have previously reported that a plasma membrane variant (PM variant) of WT hTRPML1 (TRPML1DNC) can be activated by SF-22 (5-chloro-N-(2-piperidin-1-ylphenyl)thiophene-2-sulphonamide) in calcium imaging experiments 15 . Here, we applied the whole-lysosome planar patch-clamp technique 37,38 to investigate the small-molecule activation of WT hTRPML1 and selected mutant isoforms of TRPML1. We demonstrate that SF-22 elicits inwardly rectifying (from lysosomal lumen to cytosol) currents in lysosomes isolated from a HEK293 cell line stably expressing hTRPML1 (Fig.…”

High susceptibility to fatty liver disease in two-pore channel 2-deficient mice

“…There is evidence that TPC1 is mainly present in the proximal endosomal system, and TPC2 is predominantly expressed on late endosomes and lysosomes 16,18,19 . The activation mechanism of TPCs is complex and has been suggested to involve the second messenger nicotinic acid adenine dinucleotide phosphate (NAADP) and the endolysosomal membrane lipidphosphatidylinositol (3,5)bisphosphate (PI(3,5)P 2 ) 17,18,[20][21][22][23][24] . Independent of what the nature of the endogenous ligand of these channels might be, there is substantial evidence that on activation TPCs mediate the release of Ca 2 þ from lysosomal stores.…”