TRP Channels as Interior Designers: Remodeling the Endolysosomal Compartment in Natural Killer Cells

Abstract

Cytotoxic lymphocytes, including natural killer (NK) cells and T cells are distinguished by their ability to eliminate target cells through release of secretory lysosomes. Conventional lysosomes and secretory lysosomes are part of the pleomorphic endolysosomal system and characterized by its highly dynamic nature. Several calcium-permeable TRP calcium channels play an essential role in endolysosomal calcium signaling to ensure proper function of these organelles. In NK cells, the expression of self MHC-specific inhibitory receptors dynamically tunes their secretory potential in a non-transcriptional, calcium-dependent manner. New insights suggest that TRPML1-mediated lysosomal calcium fluxes are tightly interconnected to NK cell functionality through modulation of granzyme B and perforin content of the secretory lysosome. Lysosomal TRP channels show a subset-specific expression pattern during NK differentiation, which is paralleled with gradually increased loading of effector molecules in secretory lysosomes. Methodological advances, including organellar patch-clamping, specific pharmacological modulators, and genetically-encoded calcium indicators open up new possibilities to investigate how TRP channels influence communication between intracellular organelles in immune cells. This review discusses our current understanding of lysosome biogenesis in NK cells with an emphasis on the TRP mucolipin family and the implications for NK cell functionality and cancer immunotherapy.

Keywords: NK cell; TRP cation channel; calcium signaling; cytotoxic lymphocytes; mucolipin; secretory lysosomes.

Figure 1

The lysosomal compartment is an important signaling hub and integrates a diverse range of signals. Secretory lysosomes are dual-functional organelles consisting of a lysosomal limiting membrane and a proteoglycan electron-dense core as safe storage unit for effector molecules like granzymes and perforin. Many different signals form the cell surface, or from the inside, converge at the limiting lysosomal membrane and can be detected by specialized metabolic-, energetic-, stress-, pH-, and lipid-moiety-sensor proteins. For NK cells, one of the central metabolic sensors is called mTOR complex 1, which can detect amino acids (AA) and growth factor signals. AMP-activated kinase (AMPK) reacts to stress signals, such as reactive-oxygen species (ROS) and can trigger autophagy induction to recover nutrients. A remarkable class of signal integrators, is the transient receptor potential (TRP) channel family, most importantly TRPML1, localized on the lysosomal membrane. TRP channels can integrate signals of diverse nature, translated into calcium signals. TRPML1 calcium signals control lysosomal trafficking membrane dynamics and TFEB-dependent activation of the CLEAR gene network. A network of genes associated with lysosomal biogenesis and autophagy, and commonly regulated by transcription factors of the MiT/TFE family. Lysosomal calcium signals and lipid membrane composition, as well as integral lysosomal surface proteins are essential for the recruitment of e.g., motor proteins, the small Rab27a GTPase, Munc 13-4, and SNARE proteins as mediators of plasma membrane fusion. Altogether, these are critical components for orchestrating exocytosis of secretory lysosomes in NK cells. iKIR, inhibitory killer immunoglobulin-like receptors.

Figure 2

The dense-core secretory lysosomes are the terminal storage unit for acidic hydrolases and effector molecules in NK cells. The lysosome as such is a highly dynamic compartment and can engage in several fusion events in order to deliver its degradative capacity for endocytosis, autophagy or killing of target cells by NK cells. Termination of signal transduction is an important task for the orchestration of NK cell effector functions. Early endosomal cargo can be propagated by gradual-maturation of an early endosome to a late endosome, which finally can fuse with a lysosome for degradation by acidic hydrolases. As a result, an endolysosomal hybrid organelle is formed. Lysosomes can be reformed through tubulation from the latter compartment and leads to formation of a proto-lysosome, which undergoes further maturation and content condensation. In analogy to that, lysosomes can also engage in autophagy. In immune cells, autophagy fulfills the important task of eradicating worn-out mitochondria. Damaged organelles or proteins can be entrapped in autophagosomes, ultimately fusing with lysosomes. Upon termination of autophagy, again, through tubulation and membrane extrusions, consumed proto-lysosomes are regenerated and mature to lysosomes. During target cell killing, NK cells go through step-wise receptor cross-linking, granule polarization and eventually lysosome-related organelle fusion with the plasma membrane at the immunological synapse. During this process, the effector molecules and the electron-dense core can be ejected and the lysosomal membrane will be recovered through endocytosis. MVB, multi-vesicular body; MTOC, microtubule-organizing center.

Figure 3

TRP channels as master regulators in the endolysosomal compartment. The TRP channel conductance is affected by a broad range of stimuli and integrates information about pH and signals from within the lipid bilayer, as well as the surrounding cytosol and vesicular lumen. This is illustrated for TRPML1, which is described to be activated by phosphatidylinositol-(3,5)-bisphosphate, PI(3,5)P₂, and inhibited by phosphatidylinositol-(4,5)-bisphosphate. PI(3,5)P₂ and PI(4,5)P₂ play an delicate role in controlling lysosomal membrane dynamics, hence their synthesis needs to be highly coordinated in time and space. Disparity in the concentration of TRP ligands, in the context of lysosomal storage disorders, impairs proper lysosomal lipid transfer, trafficking and calcium signaling capacity. Niemann-Pieck disease type A/B is characterized by lysosomal accumulation of sphingomyelins (SM). In this disease, SM has been suggested to be the causing agent for the grave pathogenic conditions due to its presumable antagonistic function on TRPML1. On the other site, Niemann Pieck type C is denoted by a defect in cholesterol transporters and might also affect SM levels in the lysosome.