Abstract
Two-pore channels (TPCNs) have been proposed to form lysosomal Ca(2+) release channels that are activated by nicotinic acid adenine dinucleotide phosphate. Here, we employ a glass chip-based method to record for the first time nicotinic acid adenine dinucleotide phosphate -dependent currents through a two-pore channel (TPCN2) from intact lysosomes. We show that TPCN2 is a highly selective Ca(2+) channel that is regulated by intralysosomal pH. Using site-directed mutagenesis, we identify an amino acid residue in the putative pore region that is crucial for conferring high Ca(2+) selectivity. Our glass chip-based method will provide electrophysiological access not only to lysosomal TPCN channels but also to a broad range of other intracellular ion channels.
Highlights
Two-pore channels (TPCNs) have been proposed to form lysosomal Ca2؉ release channels that are activated by nicotinic acid adenine dinucleotide phosphate
We employ a glass chip-based method to record for the first time nicotinic acid adenine dinucleotide phosphate -dependent currents through a two-pore channel (TPCN2) from intact lysosomes
We show that TPCN2 is a highly selective Ca2؉ channel that is regulated by intralysosomal pH
Summary
Two-pore channels (TPCNs) have been proposed to form lysosomal Ca2؉ release channels that are activated by nicotinic acid adenine dinucleotide phosphate. We employ a glass chip-based method to record for the first time nicotinic acid adenine dinucleotide phosphate -dependent currents through a two-pore channel (TPCN2) from intact lysosomes. 3 The abbreviations used are: NAADP, nicotinic acid adenine dinucleotide phosphate; NADP, nicotinamide adenine dinucleotide phosphate; TPCN, two-pore channel; TRP, transient receptor potential; GFP, green fluorescent protein; WLR, whole lysosome recordings; F, farad; m, murine; h, human.
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