Complex regulation of voltage-dependent activation and inactivation properties of retinal voltage-gated Cav1.4 L-type Ca2+ channels by Ca2+-binding protein 4 (CaBP4).

Cav1.4 L-type Ca(2+) channels are crucial for synaptic transmission in retinal photoreceptors and bipolar neurons. Recent studies suggest that the activity of this channel is regulated by the Ca(2+)-binding protein 4 (CaBP4). In the present study, we explored this issue by examining functional effects of CaBP4 on heterologously expressed Cav1.4. We show that CaBP4 dramatically increases Cav1.4 channel availability. This effect crucially depends on the presence of the C-terminal ICDI (inhibitor of Ca(2+)-dependent inactivation) domain of Cav1.4 and is absent in a Cav1.4 mutant lacking the ICDI. Using FRET experiments, we demonstrate that CaBP4 interacts with the IQ motif of Cav1.4 and that it interferes with the binding of the ICDI domain. Based on these findings, we suggest that CaBP4 increases Cav1.4 channel availability by relieving the inhibitory effects of the ICDI domain on voltage-dependent Cav1.4 channel gating. We also functionally characterized two CaBP4 mutants that are associated with a congenital variant of human night blindness and other closely related nonstationary retinal diseases. Although both mutants interact with Cav1.4 channels, the functional effects of CaBP4 mutants are only partially preserved, leading to a reduction of Cav1.4 channel availability and loss of function. In conclusion, our study sheds new light on the functional interaction between CaBP4 and Cav1.4. Moreover, it provides insights into the mechanism by which CaBP4 mutants lead to loss of Cav1.4 function and to retinal disease.

Characterization of two-pore channel 2 (TPCN2)-mediated Ca2+ currents in isolated lysosomes.

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.

Calmodulin is a functional regulator of Cav1.4 L-type Ca2+ channels.

Cav1.4 channels are unique among the high voltage-activated Ca2+ channel family because they completely lack Ca2+-dependent inactivation and display very slow voltage-dependent inactivation. Both properties are of crucial importance in ribbon synapses of retinal photoreceptors and bipolar cells, where sustained Ca2+ influx through Cav1.4 channels is required to couple slow graded changes of the membrane potential with tonic glutamate release. Loss of Cav1.4 function causes severe impairment of retinal circuitry function and has been linked to night blindness in humans and mice. Recently, an inhibitory domain (ICDI: inhibitor of Ca2+-dependent inactivation) in the C-terminal tail of Cav1.4 has been discovered that eliminates Ca2+-dependent inactivation by binding to upstream regulatory motifs within the proximal C terminus. The mechanism underlying the action of ICDI is unclear. It was proposed that ICDI competitively displaces the Ca2+ sensor calmodulin. Alternatively, the ICDI domain and calmodulin may bind to different portions of the C terminus and act independently of each other. In the present study, we used fluorescence resonance energy transfer experiments with genetically engineered cyan fluorescent protein variants to address this issue. Our data indicate that calmodulin is preassociated with the C terminus of Cav1.4 but may be tethered in a different steric orientation as compared with other Ca2+ channels. We also find that calmodulin is important for Cav1.4 function because it increases current density and slows down voltage-dependent inactivation. Our data show that the ICDI domain selectively abolishes Ca2+-dependent inactivation, whereas it does not interfere with other calmodulin effects.

The two-pore channel TPCN2 mediates NAADP-dependent Ca(2+)-release from lysosomal stores.

Second messenger-induced Ca(2+)-release from intracellular stores plays a key role in a multitude of physiological processes. In addition to 1,4,5-inositol trisphosphate (IP(3)), Ca(2+), and cyclic ADP ribose (cADPR) that trigger Ca(2+)-release from the endoplasmatic reticulum (ER), nicotinic acid adenine dinucleotide phosphate (NAADP) has been identified as a cellular metabolite that mediates Ca(2+)-release from lysosomal stores. While NAADP-induced Ca(2+)-release has been found in many tissues and cell types, the molecular identity of the channel(s) conferring this release remained elusive so far. Here, we show that TPCN2, a novel member of the two-pore cation channel family, displays the basic properties of native NAADP-dependent Ca(2+)-release channels. TPCN2 transcripts are widely expressed in the body and encode a lysosomal protein forming homomers. TPCN2 mediates intracellular Ca(2+)-release after activation with low-nanomolar concentrations of NAADP while it is desensitized by micromolar concentrations of this second messenger and is insensitive to the NAADP analog nicotinamide adenine dinucleotide phosphate (NADP). Furthermore, TPCN2-mediated Ca(2+)-release is almost completely abolished when the capacity of lysosomes for storing Ca(2+) is pharmacologically blocked. By contrast, TPCN2-specific Ca(2+)-release is unaffected by emptying ER-based Ca(2+) stores. In conclusion, these findings indicate that TPCN2 is a major component of the long-sought lysosomal NAADP-dependent Ca(2+)-release channel.

Planar patch clamp approach to characterize ionic currents from intact lysosomes.

Since its launch in the early 1980s, the patch clamp method has been extensively used to study ion channels in the plasma membrane, but its application to the study of intracellular ion channels has been limited. Unlike the plasma membrane, intracellular membranes are usually not stable enough to withstand mechanical manipulation by glass electrodes during seal formation and rupturing of the membrane. To circumvent these problems, we developed a method involving the immobilization of isolated organelles on a solid matrix planar glass chip. This glass chip contains a microstructured hole that supports the formation of gigaseals and subsequent electrophysiological recordings despite the high fragility of intracellular membranes. Here, we report the experimental details of this method using lysosomes, which are the smallest cellular organelles, as a model system. We demonstrate that we can record endogenous ionic currents from wild-type lysosomes, as well as from lysosomes overexpressing ion channels, and expect that this method will provide electrophysiological access to a broad range of intracellular ion channels.