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BK channel agonist represents a potential therapeutic approach for lysosomal storage diseases

View Article: PubMed Central - PubMed

ABSTRACT

Efficient lysosomal Ca2+ release plays an essential role in lysosomal trafficking. We have recently shown that lysosomal big conductance Ca2+-activated potassium (BK) channel forms a physical and functional coupling with the lysosomal Ca2+ release channel Transient Receptor Potential Mucolipin-1 (TRPML1). BK and TRPML1 forms a positive feedback loop to facilitate lysosomal Ca2+ release and subsequent lysosome membrane trafficking. However, it is unclear whether the positive feedback mechanism is common for other lysosomal storage diseases (LSDs) and whether BK channel agonists rescue abnormal lysosomal storage in LSDs. In this study, we assessed the effect of BK agonist, NS1619 and NS11021 in a number of LSDs including NPC1, mild cases of mucolipidosis type IV (ML4) (TRPML1-F408∆), Niemann-Pick type A (NPA) and Fabry disease. We found that TRPML1-mediated Ca2+ release was compromised in these LSDs. BK activation corrected the impaired Ca2+ release in these LSDs and successfully rescued the abnormal lysosomal storage of these diseases by promoting TRPML1-mediated lysosomal exocytosis. Our study suggests that BK channel activation stimulates the TRPML1-BK positive reinforcing loop to correct abnormal lysosomal storage in LSDs. Drugs targeting BK channel represent a potential therapeutic approach for LSDs.

No MeSH data available.


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Activation of BK by NS1619 facilitates ML1-mediated Ca2+ release.(A,B) NS1619 (15 μM) treatment increased ML-SA1 (10 μM)-mediated GECO-TRPML1 response, which was inhibited by co-applying Paxilline (PAX, 3 μM) in HEK293T cells. (C) NS1619 (15 μM) treatment did not alter GECO-TRPML1 response to GPN (200 μM), suggesting lysosomal Ca2+ content was not affected. (D,E) NPC1 human fibroblasts exhibited impaired GECO-TRPML1 response to ML-SA1 (10 μM). NS1619 (15 μM) treatment increased GECO-TRPML1 response to ML-SA1, and this was inhibited by co-applying Paxilline (PAX, 3 μM). (F) GECO-TRPML1 responses to GPN (200 μM) was not altered by NS1619 (15 μM) in NPC1 cells, suggesting that NS1619 does not change lysosomal Ca2+ content. (G) GECO-TRPML1 responses to Ionomycin (1 μM) were not altered, indicating a similar level of GECO-TRPML1 expression.
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f3: Activation of BK by NS1619 facilitates ML1-mediated Ca2+ release.(A,B) NS1619 (15 μM) treatment increased ML-SA1 (10 μM)-mediated GECO-TRPML1 response, which was inhibited by co-applying Paxilline (PAX, 3 μM) in HEK293T cells. (C) NS1619 (15 μM) treatment did not alter GECO-TRPML1 response to GPN (200 μM), suggesting lysosomal Ca2+ content was not affected. (D,E) NPC1 human fibroblasts exhibited impaired GECO-TRPML1 response to ML-SA1 (10 μM). NS1619 (15 μM) treatment increased GECO-TRPML1 response to ML-SA1, and this was inhibited by co-applying Paxilline (PAX, 3 μM). (F) GECO-TRPML1 responses to GPN (200 μM) was not altered by NS1619 (15 μM) in NPC1 cells, suggesting that NS1619 does not change lysosomal Ca2+ content. (G) GECO-TRPML1 responses to Ionomycin (1 μM) were not altered, indicating a similar level of GECO-TRPML1 expression.

Mentions: We have shown that BK activation facilitates TRPML1 function by maintaining a beneficial lysosomal membrane potential for TRPML17. To test whether the beneficial effect of NS1619 in NPC1 is dependent on TRPML1 activation, we tested whether NS1619 increases TRPML1-mediated Ca2+ release using GECO-TRPML1, a single-wavelength genetically encoded Ca2+ indicator727. In cells bathed in a low Ca2+ (free Ca2+ < 10 nM) solution, an increase in GECO-TRPML1 signal which is monitored by measuring GECO fluorescence (F470) represents TRPML1 activation. GECO-TRPML1 was expressed in HEK293T cells, and GECO-TRPML1 fluorescence in response to ML-SA1 was compared between NS1619 and control vehicle. As shown in Fig. 3A,B, NS1619 (15 μM) significantly increased GECO-TRPML1 fluorescence signal in response to ML-SA1, as compared to the control. This was suppressed by Paxilline (PAX), the selective BK channel blocker7. Notably, NS1619 did not affect lysosomal Ca2+ content, because glycyl-phenylalanine 2-naphthylamide (GPN, 200 μM), a substrate of the lysosomal exopeptidase cathepsin C that induces lysosome osmolysis to deplete lysosomal Ca2+ pools4, induced a comparable GECO-ML1 response between the control and NS1619 treatment (Fig. 3C). These data suggest that NS1619 facilitates lysosomal Ca2+ release through TRPML1.


BK channel agonist represents a potential therapeutic approach for lysosomal storage diseases
Activation of BK by NS1619 facilitates ML1-mediated Ca2+ release.(A,B) NS1619 (15 μM) treatment increased ML-SA1 (10 μM)-mediated GECO-TRPML1 response, which was inhibited by co-applying Paxilline (PAX, 3 μM) in HEK293T cells. (C) NS1619 (15 μM) treatment did not alter GECO-TRPML1 response to GPN (200 μM), suggesting lysosomal Ca2+ content was not affected. (D,E) NPC1 human fibroblasts exhibited impaired GECO-TRPML1 response to ML-SA1 (10 μM). NS1619 (15 μM) treatment increased GECO-TRPML1 response to ML-SA1, and this was inhibited by co-applying Paxilline (PAX, 3 μM). (F) GECO-TRPML1 responses to GPN (200 μM) was not altered by NS1619 (15 μM) in NPC1 cells, suggesting that NS1619 does not change lysosomal Ca2+ content. (G) GECO-TRPML1 responses to Ionomycin (1 μM) were not altered, indicating a similar level of GECO-TRPML1 expression.
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f3: Activation of BK by NS1619 facilitates ML1-mediated Ca2+ release.(A,B) NS1619 (15 μM) treatment increased ML-SA1 (10 μM)-mediated GECO-TRPML1 response, which was inhibited by co-applying Paxilline (PAX, 3 μM) in HEK293T cells. (C) NS1619 (15 μM) treatment did not alter GECO-TRPML1 response to GPN (200 μM), suggesting lysosomal Ca2+ content was not affected. (D,E) NPC1 human fibroblasts exhibited impaired GECO-TRPML1 response to ML-SA1 (10 μM). NS1619 (15 μM) treatment increased GECO-TRPML1 response to ML-SA1, and this was inhibited by co-applying Paxilline (PAX, 3 μM). (F) GECO-TRPML1 responses to GPN (200 μM) was not altered by NS1619 (15 μM) in NPC1 cells, suggesting that NS1619 does not change lysosomal Ca2+ content. (G) GECO-TRPML1 responses to Ionomycin (1 μM) were not altered, indicating a similar level of GECO-TRPML1 expression.
Mentions: We have shown that BK activation facilitates TRPML1 function by maintaining a beneficial lysosomal membrane potential for TRPML17. To test whether the beneficial effect of NS1619 in NPC1 is dependent on TRPML1 activation, we tested whether NS1619 increases TRPML1-mediated Ca2+ release using GECO-TRPML1, a single-wavelength genetically encoded Ca2+ indicator727. In cells bathed in a low Ca2+ (free Ca2+ < 10 nM) solution, an increase in GECO-TRPML1 signal which is monitored by measuring GECO fluorescence (F470) represents TRPML1 activation. GECO-TRPML1 was expressed in HEK293T cells, and GECO-TRPML1 fluorescence in response to ML-SA1 was compared between NS1619 and control vehicle. As shown in Fig. 3A,B, NS1619 (15 μM) significantly increased GECO-TRPML1 fluorescence signal in response to ML-SA1, as compared to the control. This was suppressed by Paxilline (PAX), the selective BK channel blocker7. Notably, NS1619 did not affect lysosomal Ca2+ content, because glycyl-phenylalanine 2-naphthylamide (GPN, 200 μM), a substrate of the lysosomal exopeptidase cathepsin C that induces lysosome osmolysis to deplete lysosomal Ca2+ pools4, induced a comparable GECO-ML1 response between the control and NS1619 treatment (Fig. 3C). These data suggest that NS1619 facilitates lysosomal Ca2+ release through TRPML1.

View Article: PubMed Central - PubMed

ABSTRACT

Efficient lysosomal Ca2+ release plays an essential role in lysosomal trafficking. We have recently shown that lysosomal big conductance Ca2+-activated potassium (BK) channel forms a physical and functional coupling with the lysosomal Ca2+ release channel Transient Receptor Potential Mucolipin-1 (TRPML1). BK and TRPML1 forms a positive feedback loop to facilitate lysosomal Ca2+ release and subsequent lysosome membrane trafficking. However, it is unclear whether the positive feedback mechanism is common for other lysosomal storage diseases (LSDs) and whether BK channel agonists rescue abnormal lysosomal storage in LSDs. In this study, we assessed the effect of BK agonist, NS1619 and NS11021 in a number of LSDs including NPC1, mild cases of mucolipidosis type IV (ML4) (TRPML1-F408&#8710;), Niemann-Pick type A (NPA) and Fabry disease. We found that TRPML1-mediated Ca2+ release was compromised in these LSDs. BK activation corrected the impaired Ca2+ release in these LSDs and successfully rescued the abnormal lysosomal storage of these diseases by promoting TRPML1-mediated lysosomal exocytosis. Our study suggests that BK channel activation stimulates the TRPML1-BK positive reinforcing loop to correct abnormal lysosomal storage in LSDs. Drugs targeting BK channel represent a potential therapeutic approach for LSDs.

No MeSH data available.


Related in: MedlinePlus