Ion and Molecule Transport in Lysosomes

Bruno Gasnier,Michael X. Zhu

• Publisher: CRC Press
• Year: 2020
• ISBN:
  9781351361132
• Edition: 1
• Topic: Medicina
• Subtopic: Anatomia

Lysosomes are key subcellular organelles that regulate the cell function. Many of the essential activities of the cell are dependent on lysosomes. Dysfunction is linked to multiple diseases - storage disorders, neurodegeneration, immunological diseases and cancer. This book discusses concepts and methods used to study lysosome ion and small molecule transport. The contents will not only attract accomplished investigators in need of a broad review and synthesis of this important subject but will also appeal to young investigators and trainees needing to acquire comprehensive knowledge and technical skills working with lysosomal ion channels and small molecule transporters.

Key selling features:

• Summarizes the endocellular role that lysosomes play with respect to cellular waste disposal
• Reviews essential cellular functions of lysosomes
• Explores how lysosome dysfunction is the cause of many metabolic disorders
• Examines how lysomes are involved in storage diseases
• Describes various technologies and methods used in lysosome research

1 Endosomal and Lysosomal Electrophysiology

Xiaoli Zhang, Mingxue Gu, Meiqin Hu, Yexin Yang, and Haoxing Xu

CONTENTS

1.1 Introduction

1.2 Lysosomal Ionic Composition

1.3 Lysosomal Ion Channels

1.3.1 Lysosomal Ca²⁺ Channels

1.3.2 Lysosomal Na⁺ Channels

1.3.3 Lysosomal K⁺ Channels

1.4 Methods to Study Lysosomal Channels

1.4.1 Endolysosomal Electrophysiology

1.4.1.1 Chemical Enlargement of Endolysosomes

1.4.1.2 Genetic Enlargement of Endolysosomes

1.4.1.3 Lysosome Isolation

1.4.1.4 Limitations of Current Lysosomal Patch-Clamp Methods

1.4.2 Optical Imaging of Lysosomal Physiology

1.4.2.1 Chemical Ion Indicators

1.4.2.2 Genetically Encoded Ion Indicators

1.5 Protocol of Endolysosomal Patch Clamp

1.5.1 Recording Solutions

1.5.2 Equipment

1.5.3 Whole-Endolysosomal Recording Procedures

Step 1: Transfection

Step 2: Enlargement of LELs

Step 3: Preparation of Glass Electrodes

Step 4: Whole-Endolysosomal Patch Clamp

Step 5. Recordings from Excised Endolysosome Membranes

1.6 Electrophysiological Studies of Lysosome Function

1.7 Perspectives and Future Directions

Acknowledgement

References

1.1 Introduction

Lysosomes, acidic organelles containing more than 60 types of hydrolytic enzymes in the lumen, were traditionally viewed as the “digestion centre” of the cell “passively” involved in the digestion and recycling of cellular macromolecules (Kolter and Sandhoff, 2005; Perera and Zoncu, 2016). A more “active” signal transduction role of lysosomes has recently been discovered, and lysosomes are now known to regulate biomaterial recycling, membrane trafficking, catabolite export, nutrient sensing, and energy homeostasis (Perera and Zoncu, 2016; Xu and Ren, 2015). The mechanistic target of rapamycin (mTOR), the primary nutrient sensor in the cell, is found to be localized on the lysosomal membrane to regulate various lysosomal functions (Zoncu et al., 2011). The ionic composition of the lysosome lumen plays an essential role in both “digestive” and “signalling” functions of the lysosome (Xiong and Zhu, 2016; Xu and Ren, 2015). For instance, the 1,000–5,000 fold concentration gradients for H⁺ and Ca²⁺ in the lumen vs. cytosol are required for both degradation and nutrient-dependent signal transduction of lysosomes (Xu and Ren, 2015). Lysosomal ion homeostasis is established and maintained by lysosomal ion channels and transporters that mediate ionic flux across the lysosomal membranes in response to various cellular cues that are derived from either the cytoplasm or the lysosome lumen (Perera and Zoncu, 2016; Xu and Ren, 2015). Dysregulation of lysosomal ion flux leads to a lysosome storage phenotype with the characteristic accumulation of enlarged vacuoles, cellular wastes, and lipofuscin in the cell (Ferreira and Gahl, 2017; Kolter and Sandhoff, 2005; Xu and Ren, 2015).

Unlike their plasma membrane counterparts, ion channels and transporters localized on the intracellular membranes have been inaccessible for conventional electrophysiology, e.g., the patch-clamp method, in studying their ionic selectivity and gating/modulation mechanisms (Xu et al., 2015). The primary challenge is that the size of lysosomes, typically 100–500 nm in diameter, is sub-optimal for patch clamping (Xu et al., 2015). Early studies of lysosomal channels relied on liposome reconstitution or cell surface re-routing that allows examination of intracellular channels using the whole-cell patch-clamp technique (Arai et al., 1993; Sawada et al., 2008). However, the non-native membrane environment (e.g., the lack of lysosome-specific phospholipids and interacting proteins) has limited the physiological studies of lysosomal membranes and channels (Xu et al., 2015). In contrast, the development of the whole-endolysosome patch-clamp technique on artificially enlarged endolysosomes has allowed direct studies of lysosomal channels in a more “native” environment under more “physiological” conditions (Dong et al., 2008; Schieder et al., 2010; Xiong and Zhu, 2016; Xu and Ren, 2015). Hence, a new avenue has been opened for lysosomal channel research, which provides a promising platform for the discovery of new lysosomal channels and their cellular modulators (Dong et al., 2008; Schieder et al., 2010; Xiong and Zhu, 2016; Xu and Ren, 2015). In this chapter, we describe detailed protocols of endosomal and lysosomal electrophysiology and discuss several examples of lysosomal channels that have been characterized using this method.

1.2 Lysosomal Ionic Composition

The lysosome lumen contains H⁺, Na⁺, K⁺, Ca²⁺, and Cl⁻ (Mindell, 2012; Morgan et al., 2011; Xu and Ren, 2015). With the exceptions of H⁺ and Ca²⁺, luminal concentrations of other ions have not been accurately determined (Xu and Ren, 2015). Indeed, it remains controversial whether lysosomes are Na⁺ or K⁺ enriched compartments (Steinberg et al., 2010; Wang et al., 2012). Given that lysosomes constantly undergo membrane fusion or fission with other intracellular membrane compartments, the ionic compositions are likely heterogeneous for individual lysosomes. For example, peripheral lysosomes are less acidic than the perinuclear lysosomes (Johnson et al., 2016), suggesting that lysosomal positioning may affect the luminal ionic composition. Likewise, the primary and terminal lysosomes are likely Na⁺-dominant, whereas secondary lysosomes (e.g., the newly formed autolysosomes) may have a much lower luminal Na⁺ concentration due to the prior fusion with the cytosol-generated K⁺-dominant autophagosomes (Xu and Ren, 2015). Furthermore, due to the small volume of individual lysosomes, ion flux mediated by transient openings of organellar channels may be sufficient to cause drastic changes in the luminal ionic composition (Xu et al., 2015; Xu and Ren, 2015).

H⁺: A hallmark feature of the lysosome is its acidic pH (pH 4.6) in the lumen, which is required for the activity of most lysosome hydrolases (Kolter and Sandhoff, 2005; Mindell, 2012). During endosome maturation, the V-ATPase is responsible for decreasing luminal pH from 6.5 in early endosomes to 4.6 in late endosomes and lysosomes (LELs) (Huotari and Helenius, 2011). Disruption of lysosomal pH gradient using V-ATPase inhibitors (e.g., bafilomycin-A1) or protonophores results in accumulation of the endocytic and autophagic cargos (Kawai et al., 2007; Padman et al., 2013). In addition, lysosomal pH or V-ATPase regulates other lysosomal functions, including autophagosome-lysosome fusion (Kawai et al., 2007; Mauvezin and Neufeld, 2015) and nutrient sensing (Zoncu et al., 2011).

Na⁺/K⁺: The lysosome lumen was thought to be high in K⁺, but low in Na⁺, suggesting that like endoplasmic reticulum (ER), there are no significant concentration gradients of Na⁺ or K⁺ across lysosomal membranes (Morgan et al., 2011; Steinberg et al., 2010; Xu and Ren, 2015). This view has been challenged by several recent lysosomal physiological studies. First, whole-endolysosome recordings have revealed the presence of multiple Na⁺-selective and K⁺-selective channels in the lysosome (Cang et al., 2015; Cao et al., 2015b; Wang et al., 2017; Wang et al., 2012). Second, isolated lysosomes, like the extracellular space, may contain high concentrations of Na⁺ (i.e., >100 mM) (Wang et al., 2012). The ion transporters that are required to establish the Na⁺ gradient are not known. Importantly, activation of Na⁺ and K⁺-selective channels may rapidly change lysosome membrane potential (∆ψ, defined as ∆ψ = ψ_cytosol−ψ_lumen), which may be required for various lysosomal functions, such as catabolite export (Cang et al., 2013) and Ca²⁺ import (Wang et al., 2017).

Ca²⁺: [Ca²⁺]_lumen (~0.5 mM) is about 5,000 times higher than [Ca²⁺]_cyto (100 nM). Hence, like ER, lysosomes are recognized as important intracellular Ca²⁺ stores (Yang et al., 2018). The uptake/import mechanisms that maintain such high Ca²⁺ gradient across lysosomal membranes are not clear (Yang et al., 2018). Many lysosomal functions, including lysosomal membrane trafficking and lysosome biogenesis, are reportedly regulated by lysosomal Ca²⁺ through various downstream Ca²⁺ effectors, such as synaptotagmin VII, ALG-2, calcineurin, and calmodulin (Chu et al., 2015; Li et al., 2016a; Li et al., 2016b; Medina et al., 2015). Ca²⁺-permeable channels in the lysosome mediate lysosomal Ca²⁺ release in response to changes in nutrient availability, redox status, and lipid abundance (Cao et al., 2015a; Dong et al., 2010; Shen et al., 2012; Xiong and Zhu, 2016; Xu and Ren, 2015; Zhang et al., 2016).

Cl⁻: Lysosomes also store high concentrations of [Cl⁻]_lumen (Chakraborty et al., 2017), which are connected with the luminal pH, presumably through the Cl⁻ and H⁺-dependent transporter CLC7 (Graves et al., 2008; Jentsch, 2007; Neagoe et al., 2010).

1.3 Lysosomal Ion Channels

Lysosomal ion channels (LICs) include those that reside primarily on LELs, e.g., TRPML1–TRPML3, TPC1–TPC2, and TMEM175, the so-called “committed” lysosomal channels, as well as plasma membrane channels that are also localized in the lysosomes, i.e., the large conductance Ca²⁺- and voltage-activated K⁺ (BK) channels and P2X4 purinergic receptors/channels, referred to as “non-committed” lysosomal channels (Figure 1.1) (Cang et al., 20...

Table of contents

• Cover
• Half-Title
• Series
• Title
• Copyright
• Contents
• Preface
• Editors
• Contributors
• Chapter 1 Endosomal and Lysosomal Electrophysiology
• Chapter 2 Chloride Transport across the Lysosomal Membrane
• Chapter 3 Endolysosomal Patch Clamping: Approaches to Measure Vesicular Ion Channel Activities
• Chapter 4 TRPML Subfamily of Endolysosomal Channels: Concepts and Methods
• Chapter 5 Investigating the Role of Two-Pore Channel 2 (TPC2) in Zebrafish Neuromuscular Development
• Chapter 6 Functional Study of Lysosomal Nutrient Transporters
• Chapter 7 Lysosomal Vitamin B12 Trafficking
• Chapter 8 Detection of Lysosomal Membrane Permeabilization
• Chapter 9 Rapid Isolation of Lysosomes from Cultured Cells Using a Twin Strep Tag
• Chapter 10 A Transcriptomic Analysis and shRNA Screen for Intracellular Ion Channels and Transporters Regulating Pigmentation
• Index