TRPML1‐Mediated Ca2+ Signaling in Cerebral Artery Smooth Muscle

Abstract

Late endosomes and lysosomes (collectively referred to as endolysosomes) are small acidic organelles present in all cells that maintain a steep Ca2+ gradient compared with the cytosol. Therefore, activation of Ca2+ permeable ion channels within the endolysosomal membrane can transiently generate localized microdomains of elevated Ca2+ within the cytosol. The goal of the current study is to elucidate the importance of Ca2+ signals arising from acidic Ca2+ stores in cerebral artery smooth muscle cells (SMC). Members of the mucolipin transient receptor potential (TRPML) subfamily of cation channels are Ca2+ permeable and selectively localize to endolysosomes. Mutations in the human TRPML1 gene (MCOLN1) result in the heritable childhood neurodegenerative disease type IV mucolipidosis (ML‐IV). Quantitative RT‐PCR analysis showed the presence of TRPML1, TRPML2, and TRPML3 in mRNA extracted from cerebral pial arteries from C57Bl/6 mice, with TRPML1 being most abundant. Furthermore, freshly isolated cerebral pial artery SMC showed positive immunofluorescence labeling for TRPML1, which co‐localized with the endolysosomal marker LAMP‐1. Immunolabeling experiments also suggest colocalization of TRPML1 and NADPH oxidase isoform 2 (NOX2) in endolysosomes in pial artery SMC. Ca2+ signals arising from TRPML1‐mediated endolysosomal release were investigated using a TRPML1‐GCaMP3 fusion protein. This genetic construct is targeted to endolysosomes and selectively detects Ca2+ released into the cytosol from these organelles. In HEK293 cells expressing TRPML1‐GCaMP3, an increase in cytosolic fluorescence was detected in response to the selective TRPML1 activator ML‐SA1 (10 μM), signifying that Ca2+ release from acidic stores through TRPML1 can be detected using these methods. We also investigated the functional significance of TRPML1‐mediated intracellular Ca2+ signals. In cerebral SMC, large conductance Ca2+ activated K+ (BK) channels are activated by spatially and temporally limited release of Ca2+ from the sarcoplasmic reticulum via ryanodine receptors (RyR), which generate microdomains of high cytosolic [Ca2+] known as Ca2+ sparks. This pathway promotes smooth muscle cell hyperpolarization and vasodilation. The activity of RyR is elevated by localized increases in cytosolic [Ca2+] to cause Ca2+‐induced Ca2+ release. Therefore, we hypothesized that Ca2+ release from endolysosomes sensitizes RyR to increase Ca2+ spark and BK channel activity. Ca2+ sparks were recorded from cerebral SMC loaded with the fast Ca2+ indicator fluo‐4AM using high‐speed, high‐resolution confocal microscopy. We found that Ca2+ spark sites co‐localized with endolysosomes labeled with LysoTracker Red DND‐99. Furthermore, preliminary experiments show that Ca2+ spark frequency and BK channel activity are increased by ML‐SA1 (3 μM). These data suggest a novel pathway for cerebral blood flow regulation in which local Ca2+ release from endolysosomes through TRPML1 channels sensitizes RyR, stimulating Ca2+ spark and BK channel activity to promote vasodilation. It is possible that disrupted cerebral blood flow control resulting from loss of TRPML1 activity in SMC contributes to neurodegeneration associated with ML‐IV.Support or Funding InformationSupport: R01HL091905 (SE).