A novel gain of function mutation in TPC2 reiterates pH-pigmentation interplay: Emerging role of ionic homeostasis as a master pigmentation regulator

Abstract

Pigmentation is a complex physiological phenomenon that protects from UV induced damage. Perturbations in pigmentation pathways lead to pigmentary disorders such as vitiligo, albinism and Darier...s disease. Emerging literature implicates a critical role of ionic homeostasis and pH in regulating pigmentation. In a recent study, Wang et al. identified a novel gain of function mutation in a non-selective cation channel “Two Pore Channel 2” (TPC2) that is responsible for albinism in a human patient. The authors demonstrate that this mutation leads to constitutive activation of TPC2 thereby modulating cellular calcium dynamics and inducing changes in the lysosomal pH. Further, authors generated a knock in mice with homologous TPC2 mutation and corroborated a causative role for this mutation in albinism. It is an exciting study that reports a novel TPC2 mutation, which is responsible for albinism in an autosomal dominant inheritance fashion. Since TPC2 is localized on melanosomes as well, going forward it would be interesting to investigate the role of this mutation on melanosomal calcium dynamics and alterations in melanosomal pH.

Melanogenesis is a physiological phenomenon that provides skin pigmentation and protects from UV induced DNA damage. Melanin synthesis occurs in melanocytes present in the basal layer of skin epidermis. Melanocytes contain melanosomes, lysosome derived organelles, which serve as the site for melanin synthesis and deposition. The cascade of signaling events leading to melanin generation and deposition is an intricate circuit involving a variety of factors like hormones, transcription factors, ion channels, melanin bio-synthetic enzymes and structural proteins. All components of this cascade must function together flawlessly for induction of melanogenesis. Any perturbations in the melanogenic cascade manifest as pigmentation disorders such as albinism, vitiligo etc.

One of the key cellular mechanisms governing melanogenesis is inter and intra-organelle homeostasis of ions such as Ca²⁺, H⁺, Na⁺ and Cl⁻[1–4]. Ion channels working on the plasma membrane or sub cellular organelles such as melanosomes and lysosomes critically regulate melanin synthesis. The functioning of the ion transport system is dependent on the electrochemical gradient across organelle membrane. Lysosomes and lysosome derived organelles like melanosomes play a significant role in maintaining cellular ionic homeostasis by functioning as intracellular stores of ions like Ca²⁺, H⁺ etc. Importantly, melanosomal pH is a crucial determinant of melanogenesis. A neutral pH environment in the melanosome is required for efficient activity of tyrosinase, rate limiting enzyme of the melanogenic pathway. The precise mechanisms regulating pH within the melanosomes remain poorly characterized. Earlier studies have shown that Ca²⁺ and Cl⁻ ions can modulate melanosomal pH thereby regulating pigmentation [3,5]. The flux of Na⁺, Ca²⁺and Cl⁻ ions plays a vital role in regulating the activity of melanosomal V-ATPase, which mediates H⁺ ion influx and controls melanosomal pH [1,3,5]. Please refer to Fig. 1 for diagrammatic illustration of lysosome-melanosome ionic homeostasis and its impact on melanin synthesis.

Fig. 1.

Ionic homeostasis within lysosome and related organelle melanosome regulates melanin synthesis. In lysosomes and related organelle melanosomes, TPC2 mediated Ca²⁺/Na⁺ efflux, along with CLC7/OCA2 mediated H⁺ and Cl⁻ flux, regulates the activity of V-ATPase to maintain luminal ionic homeostasis and pH. Modulation of V-ATPase activity governs functioning of tyrosinase (Tyr), the rate limiting enzyme of melanogenesis pathway. A recent study reported a novel variant of human TPC2 (R210C), which is constitutively active on the plasma membrane and lysosomes with enhanced sensitivity and affinity to the endogenous TPC2 agonist, PI(3,5)P₂. The elevated TPC2 activity leads to increase in Ca²⁺release and hyper-acidification of the lysosomal lumen inducing hypopigmentation. However, TPC2 (R210C) mediated melanosome specific changes in Ca²⁺ and pH dynamics remain unexplored.

As melanosomes are lysosome derived organelles it is likely that a variety of lysosomal ion channels might localize to melanosomes. Indeed, a number of melanosomal ion channels such as TPC2 and CLC7 were initially characterized on lysosomes [1,5,6]. These ion channels play an important role in regulating ionic homeostasis in melanosomes, consequently regulating melanogenesis. One such identified melanosomal channel is TPC2. TPC2 mediated Ca²⁺ and Na⁺ mobilization plays a pivotal role in regulating melanosomal pH, melanosomal size and melanogenesis [1,5]. Moreover, TPC2 gain of function mutations in European population i.e. M484L and G734E have been associated with hypopigmentation phenotype. This suggests that TCP2 is a negative regulator of melanogenesis [7]. TPC family members were initially identified as NAADP sensitive Ca²⁺ release channels regulating pH of acidic organelles [8]. Later on, TPCs were reported to function as Na⁺ selective channels activated by PI(3,5)P2 (phosphatidylinositol 3, 5-bisphosphate) [9]. Recent literature suggests that TPC2 can function as both Ca²⁺ and Na⁺ channel depending on the agonist.

Recently, Wang et al. identified a novel TPC2 gain of function mutation, R210C from a Chinese patient with albinism [10]. Clinically, this mutation manifested as a hypopigmentation phenotype of the skin and hair but had no effect on retinal pigmentation. Interestingly, analysis of cryo-EM structure of TPC2 revealed that this mutation is localized to PI (3,5)P₂ binding region of the protein. Importantly, patch-clamp studies with plasma membrane localizing TPC2 overexpression construct harboring R210C mutation showed significantly higher affinity and sensitivity for PI(3,5)P₂ than that of WT TPC2. Interestingly, the authors found that this enhanced level of affinity sensitized the mutant TPC2 to basal levels of PI(3,5)P₂ thus prompting constitutive channel activation. Further, pharmacological inhibition of the PIKfyve phosphoinositide kinase, which generates PI(3,5)P₂, led to reduction in basal R210C activity thereby highlighting the contribution of resting PI(3,5)P₂ to constitutive R210C activation. Moreover, electrophysiological studies on whole endo-lysosome demonstrated that TPC2 R210C was constitutively active on endo-lysosomal membranes as well. Another interesting observation is that R210C showed significant outward currents, indicating K⁺ export from the endo-lysosomal compartment and/or a functional coupling of TPC2 R210C to K⁺/non-selective cation channel. Future studies aimed at deciphering this electrophysiological phenomenon would shed light on role of TPC2 R210C in modulating endo-lysosomal ionic homeostasis and its implication on pigmentation.

Notably, Wang et al. generated knock-in mice with the homologous R194C mutation in TPC2 and established a causative role for TPC2 R210C in albinism. The mice having heterozygous and homozygous TPC2 mutations showed a graded reduction in pigmentation in the fur and tails along with reduced melanosome number in the retinal pigment epithelia. Mouse Embryonic Fibroblasts (MEFs) from homozygous R194C mice demonstrated significant Ca²⁺ release from lysosomes even with low doses of TPC2 agonist whereas as wild type cells did not show Ca²⁺ efflux. This observation further supported the fact that gain of function TPC2 mutation could be activated by low doses of agonist. Next, Wang et al. looked at lysosomal acidification in mutant MEFs and found significantly enhanced lysosomal acidification as well as increase in lysosome size. The reduction in pH and enlargement of lysosomes and by extension lysosome derived organelles, melanosomes points to an inhibition of melanogenesis through acidification. Although, the melanosomal membrane can be considered an extension of the lysosomes, utilization of melanosome specific pH sensors might provide a more clear understanding regarding the role of TPC2 mutations in regulating melanogenesis. Overall, Wang et al. have clearly demonstrated the role of TPC2 in Ca²⁺ mediated regulation of pH in the endo-lysosomal compartment. Conventional wisdom suggests that reduced pH of endo-lysosomes due to gain of function TPC2 mutation might correspond to melanosomal acidification and hypopigmentation. However, the precise molecular mechanism that bridges TPC2 R210C mutation to hypopigmentation remains largely uncharacterized. Further, direct association between TCP2 hyperactivation and reduction in the melanogenic activity, particularly that of key melanogenic enzymes like tyrosinase needs to be established. Moreover, TCP2 mediated acidification might induce structural changes in melanosomes (as evident by enlarged lysosomes), which might inhibit melanogenesis independent of ionic homeostasis. Most importantly, the specific role of TPC2 R210C mutation on melanosomal Ca²⁺ and pH levels still remains unexplored. Future studies with genetically encoded Ca²⁺ and pH sensors localized specifically to the melanosome lumen would provide such critical information.

Data availability

Data will be made available on request.