Permeation Properties of Two Pore Channel 2 (TPCN2)-Mediated Ca2+ Currents Recorded from Single Lysosomes

Abstract

Nicotinic acid adenine dinucleotide phosphate (NAADP) is a second messenger that releases Ca2+ from intracellular stores at low nanomolar concentrations. NAADP-evoked Ca2+-release has been demonstrated in invertebrates and numerous mammalian cell types including pancreatic acinar and beta-cells, cardiac and smooth muscle cells, T-lymphocytes, platelets and neurons. Several studies using NAADP binding assays and Ca2+ imaging experiments linked members of the two-pore channel family (TPCN1-3) with NAADP-induced Ca2+ release from lysosome-like acidic organelles. However, there has been no direct demonstration that TPCNs can act as NAADP-sensitive Ca2+ release channels. Recently, we developed a highly efficient method to record ionic currents in single isolated lysosomes. These recordings are performed using an automated patch clamp approach that involves the immobilization of isolated lysosomes on a solid matrix planar glass chip. Using this method we have provided direct evidence that TPCN2 is a highly selective Ca2+ channel. Furthermore, we identified an amino acid residue in the putative pore region that is crucial for conferring high Ca2+-selectivity. Here, we extend our biophysical characterization of TPCN2 and report a detailed analysis of the permeation properties of these channels. Overall, our study lays the groundwork for the understanding of TPCN2 function in lysosomal stores. 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.