Current Methods to Unravel the Functional Properties of Lysosomal Ion Channels and Transporters

Abstract

A distinct set of channels and transporters regulates the ion fluxes across the lysosomal membrane. Malfunctioning of these transport proteins and the resulting ionic imbalance is involved in various human diseases, such as lysosomal storage disorders, cancer, as well as metabolic and neurodegenerative diseases. As a consequence, these proteins have stimulated strong interest for their suitability as possible drug targets. A detailed functional characterization of many lysosomal channels and transporters is lacking, mainly due to technical difficulties in applying the standard patch-clamp technique to these small intracellular compartments. In this review, we focus on current methods used to unravel the functional properties of lysosomal ion channels and transporters, stressing their advantages and disadvantages and evaluating their fields of applicability.

Keywords: ion channels; lysosomes; patch-clamp; plant vacuole; transporters.

Figure 1

Schematic representation of a planar lipid bilayer system. Left: Solvent-free planar lipid membranes (PLM) (also called folded bilayers) are typically formed across a hole (~50–200 μm) present in a thin Teflon septum dividing two distinct compartments drilled in a Teflon chamber. The lipids are dissolved in n-alkane solvents (typically n-hexane) and spread at the air–water interface of the ionic solutions present in both compartments. The lipid bilayer is formed by the spontaneous apposition of the hydrocarbon chains of the monolayers when the liquid levels are simultaneously raised above the aperture as described by [56]. Alternatively, planar bilayers can be formed by dissolving the lipids in n-decane that can be painted or brushed onto a larger (~1 mm) hole to form so-called painted membranes or black lipid membranes (BLM) [57]. Unfortunately, there is some concern about the possibility that some n-decane molecules remain in the bilayer affecting the properties of the transport proteins. Improved techniques have recently been developed to obtain more stable and high-performance lipid bilayers to study channels/transporters under conditions as close to natural conditions as possible (for a review, see [58]). Right: Proteins can be directly added to the monolayer(s); however, this approach frequently destabilizes the formation of the folded bilayers; as an alternative they can be added to the bath solution of the cis (as well as the trans) compartment(s) pending the stochastic interaction with the bilayer and the subsequent incorporation. Furthermore, proteins can be incorporated into amphiphilic spherical systems (see Figure 2), such as liposomes or membrane vesicles (here shown not to scale) operating as protein carriers to the bilayer. The electrical properties of the protein enriched bilayer can be investigated by means of Ag/AgCl electrodes that connect the chamber compartments to the headstage of a patch-clamp amplifier. The ion transport properties of the channels/transporters result in variations of the very tiny current flowing through the hydrophobic lipid bilayer almost impermeable to the ionic fluxes. The currents are typically recorded under voltage-clamp conditions controlled by a PC: Single channel transitions or macroscopic currents are recorded depending on the number of channels/peptides incorporated into the lipid bilayer phase.

Figure 2

Schematic representation of liposomes containing channels/transporters. Liposomes are synthetic lipid vesicles where a lipid milieu separates the internal aqueous medium from the external ionic environment. After the dispersion of lipids in water solutions liposomes of different sizes (from 20–30 nm to several microns) and lamellarity can be obtained by changing the preparation method. Large and small unilamellar vesicles can be obtained from multilamellar vesicles by different methods such as ultrasonic treatment, or several extrusion cycles either through a small orifice under high pressure or through a polycarbonate membrane [59] (for a complete review, see Ref. [60]). Protein enriched liposomes (proteoliposomes) containing ion channels/transporters can be reconstructed from an incredible number of lipids that incorporate purified transport proteins from a variety of plasma or organellar membranes [60]. In turn, the reconstitution of cell/organellar planar membranes is achieved by the fusion of proteoliposomes to preformed PLM or BLM. Furthermore, native vesicles can be obtained by standard methods of cell fractionation, homogenization and centrifugation of native plasma as well as organellar membranes, including lysosomes [55,61]. Liposome and vesicle fusion with artificial bilayers can be facilitated by different procedures and tricks such as the presence of organic n-alkane solvents in the bilayer, the stirring of the bath solutions, the addition to the bath of millimolar concentrations of divalent cations, the existence of an osmotic pressure between the solutions present in the two compartments, the presence of nystatin and ergosterol in the proteoliposomes or even by centrifugal forces.

Figure 3

Schematic representation of the system for electrophysiological measurements on solid-supported membranes. SSM comprises a glass slide (light blue) covered by a very thin gold layer (in yellow) that binds thiol groups (in grey) of an alcanethiol reagent. A lipid monolayer completes the system. Proteoliposomes or native membrane vesicles as well as membrane fragments are adsorbed to the lipid monolayer. A change in the charge across the proteoliposome due to the activation of ion channels or transporters induces a correspondent change in the charge across the SSM, which is supplied by the amplifier (A). Therefore, as indicated by the box, a capacitive electrical coupling is established. The transient currents due to this coupling provide information on the properties of the channels/transporters (in green and orange) present in the adsorbed means (for a detailed and exhaustive description, see [68].

Figure 4

Cartoon of the patch-clamp recording configuration on enlarged lysosomes and current convention. The patch clamp technique is applied in the whole-lysosome configuration after lysosome enlargement by treatment with vacuolin-1. Positive currents correspond to the movement of cations from the cytosolic to the luminal side of the lysosome or to the opposite movement of anions.

Figure 5

Biosynthetic and endocytic traffic routes to vacuoles/lysosomes. The color code of each arrow corresponds to that of the vesicle type involved. Traffic pathways are as follows. Blue, COPII mediated: BFA-sensitive ER to GC; anterograde intra GC cisternae; BFA insensitive ER to MVBs. Gray, COPI mediated: BFA sensitive GC to ER; retrograde intra GC cisternae. Wine, clathrin mediated: AP1-, AP3-, AP4-, GGA-mediated TGN to MVBs (plant cells) or TGN to EEs (animal cells); AP3-mediated GC to VAC (plant cells); AP2-mediated PM to TGN (plant cells) or EEs (animal cells). Notice that the TGN in plant cells also plays the role of animal EEs. Green, secretory vesicles: GC to TGN; TGN to PM. The TGN to MVBs/LEs indirect pathway could involve secretory vesicles from TGN to PM, followed by a possible AP2-mediated clathrin-coated vesicle internalization from PM to MVBs/LEs. Pink, retromer vesicles: MVBs/LE to TGN. Yellow: AP5-mediated traffic involved in maintaining lysosome integrity. Its exact role has to be elucidated, both in animals and plants. Violet: MVBs/LEs to VAC/LYS. Notice that this pathway seems to be a direct fusion event that does not require specific vesicles or coats. Abbreviations: ER, endoplasmic reticulum; GC, Golgi complex; TGN, trans-Golgi network; EEs, early endosomes; MVBs/LEs, multivesicular bodies/late endosomes; VAC/LYS, vacuole/lysosome; PM, plasma membrane; AP, adaptor protein complex; GGA, GGA adaptor-related proteins; BFA, Brefeldin A.

Figure 6

Flow chart of the experimental procedures showing how Arabidopsis vacuoles can be a heterologous expression system for animal lysosomal ion channels and transporters. Transformation can be performed on protoplasts from Arabidopsis wild-type plants or mutants lacking a specific endogenous channels or transporters.

Figure 7

“Yeast Vacuoles”: The whole vacuole patch clamp recording of a giant yeast. (A–C) Haploid yeast (S. cerevisiae) was enlarged to giant yeast, and exposed vacuole with patch pipette. Bar = 5 µm. (D) Representative profile of whole vacuole currents elicited by a series of voltage steps ranging from −60 to 80 mV in 20 mV steps of the ∆yvc1 strain (left) and the NtTPK1-expressing cells (right).