Different CaV1.3 Channel Isoforms Control Distinct Components of the Synaptic Vesicle Cycle in Auditory Inner Hair Cells.

The mechanisms orchestrating transient and sustained exocytosis in auditory inner hair cells (IHCs) remain largely unknown. These exocytotic responses are believed to mobilize sequentially a readily releasable pool of vesicles (RRP) underneath the synaptic ribbons and a slowly releasable pool of vesicles (SRP) at farther distance from them. They are both governed by Cav1.3 channels and require otoferlin as Ca2+ sensor, but whether they use the same Cav1.3 isoforms is still unknown. Using whole-cell patch-clamp recordings in posthearing mice, we show that only a proportion (∼25%) of the total Ca2+ current in IHCs displaying fast inactivation and resistance to 20 µm nifedipine, a l-type Ca2+ channel blocker, is sufficient to trigger RRP but not SRP exocytosis. This Ca2+ current is likely conducted by short C-terminal isoforms of Cav1.3 channels, notably Cav1.342A and Cav1.343S, because their mRNA is highly expressed in wild-type IHCs but poorly expressed in Otof-/- IHCs, the latter having Ca2+ currents with considerably reduced inactivation. Nifedipine-resistant RRP exocytosis was poorly affected by 5 mm intracellular EGTA, suggesting that the Cav1.3 short isoforms are closely associated with the release site at the synaptic ribbons. Conversely, our results suggest that Cav1.3 long isoforms, which carry ∼75% of the total IHC Ca2+ current with slow inactivation and confer high sensitivity to nifedipine and to internal EGTA, are essentially involved in recruiting SRP vesicles. Intracellular Ca2+ imaging showed that Cav1.3 long isoforms support a deep intracellular diffusion of Ca2+SIGNIFICANCE STATEMENT Auditory inner hair cells (IHCs) encode sounds into nerve impulses through fast and indefatigable Ca2+-dependent exocytosis at their ribbon synapses. We show that this synaptic process involves long and short C-terminal isoforms of the Cav1.3 Ca2+ channel that differ in the kinetics of their Ca2+-dependent inactivation and their relative sensitivity to the l-type Ca2+ channel blocker nifedipine. The short C-terminal isoforms, having fast inactivation and low sensitivity to nifedipine, mainly control the fast fusion of the readily releasable pool (RRP); that is, they encode the phasic exocytotic component. The long isoforms, with slow inactivation and great sensitivity to nifedipine, mainly regulate the vesicular replenishment of the RRP; that is, the sustained or tonic exocytosis.

Generation of specific Ca(2+) signals from Ca(2+) stores and endocytosis by differential coupling to messengers.

It remains unclear how different intracellular stores could interact and be recruited by Ca(2+)-releasing messengers to generate agonist-specific Ca(2+) signatures. In addition, refilling of acidic stores such as lysosomes and secretory granules occurs through endocytosis, but this has never been investigated with regard to specific Ca(2+) signatures. In pancreatic acinar cells, acetylcholine (ACh), cholecystokinin (CCK), and the messengers cyclic ADP-ribose (cADPR), nicotinic acid adenine dinucleotide phosphate (NAADP), and inositol 1,4,5-trisphosphate (IP(3)) evoke repetitive local Ca(2+) spikes in the apical pole. Our work reveals that local Ca(2+) spikes evoked by different agonists all require interaction of acid Ca(2+) stores and the endoplasmic reticulum (ER), but in different proportions. CCK and ACh recruit Ca(2+) from lysosomes and from zymogen granules through different mechanisms; CCK uses NAADP and cADPR, respectively, and ACh uses Ca(2+) and IP(3), respectively. Here, we provide pharmacological evidence demonstrating that endocytosis is crucial for the generation of repetitive local Ca(2+) spikes evoked by the agonists and by NAADP and IP(3). We find that cADPR-evoked repetitive local Ca(2+) spikes are particularly dependent on the ER. We propose that multiple Ca(2+)-releasing messengers determine specific agonist-elicited Ca(2+) signatures by controlling the balance among different acidic Ca(2+) stores, endocytosis, and the ER.

The Ca2+-releasing messenger NAADP, a new player in the nervous system.

Many physiological processes are controlled by a great diversity of Ca2+ signals. Within cell, Ca2+ signals depend upon Ca2+ entry and/or Ca2+ release from internal Ca2+ stores. The control of Ca2+-store mobilization is ensured by a family of messengers comprising inositol 1,4,5 trisphosphate, cyclic ADP-ribose and nicotinic acid adenine dinucleotide phosphate (NAADP). From recent works, new concepts have emerged where activation of the cells by outside stimuli, acting at the plasma membrane, results in the synthesis of multiple Ca2+-releasing messengers which may interact and shape complex Ca2+ signals in the cytosol as well as in the nucleus. This contribution will cover the most recent advances on NAADP signalling with some emphasis on neurons.

X-ray scattering study of pike olfactory nerve: intensity of the axonal membrane, solution of the phase problem and electron density profile.

Synchrotron radiation X-ray scattering experiments were performed on unmyelinated pike olfactory nerves. The difference between the meridional and the equatorial traces of the 2-D spectra yielded the 1-D equatorial intensity of the macromolecular components oriented with respect to the nerve: axonal membranes, microtubules and other cytoskeletal filaments. These 1-D spectra display a diffuse band typical of bilayer membranes and, at small s, a few sharper bands reminiscent of microtubules. All the spectra merge at large s. The intensity of the axonal membrane was determined via a noise analysis of the nerve-dependent spectra, involving also the notion that the thickness of the membrane is finite. The shape of the intensity function indicated that the electron density profile is not centrosymmetric. The knowledge of intensity and thickness paved the way to the electron density profile via an ab initio solution of the phase problem. An iterative procedure was adopted: (i) choose the lattice D of a 1-D pseudo crystal, interpolate the intensity at the points sh = h/D, adopt an arbitrary set of initial phases and compute the profile; (ii) determine the phases corresponding to this profile truncated by the thickness D/2; (iii) repeat the operation with the updated phases until a stable result is obtained. This iterative procedure was carried out for different D-values, starting in each case from randomly generated phases: stable results were obtained in less than 10,000 iterations. Most importantly, for D in the vicinity of 200 A, the overwhelming majority of the profiles were congruent with each other. These profiles were strongly asymmetric and otherwise typical of biological membranes.

X-ray scattering study of pike olfactory nerve: elastic, thermodynamic and physiological properties of the axonal membrane.

The effects of several agents, sugars, isotonic KCl, and a variety of drugs, on the structure of the axonal membranes of unmyelinated pike olfactory nerve have been studied by synchrotron radiation X-ray scattering experiments. The main effects of the sugars are: (i) to increase the electron density of the extra-axonal space and thereby yield the absolute scale of the electron density profile; (ii) to osmotically stress the membrane and thus yield its elastic modulus of area compressibility, since the related strain, thickness dilation, is directly determined by the X-ray scattering experiments. Exposure to isotonic KCl, a depolarizing agent, induces membrane thickness to increase. The energy liberated in this process is a function of the amplitude of the dilation and of the elastic modulus of the membrane. This energy turns out to be close to the thermal energy liberated by the pike olfactory nerve during the initial phase of action potential that has previously been measured by others. Electrical depolarization thus seems to be accompanied by a thickness dilation of the axonal membrane. Another effect of isotonic KCl is to induce a large fraction of the membranes to pair by tight apposition of their extra-axonal faces. Local anaesthetics and some drugs have the effect of altering membrane thickness. All these observations are interpreted in terms of a modulation of the conformational disorder of the hydrocarbon chains of the lipid molecules.

Regulation of nuclear Ca2+ signaling by translocation of the Ca2+ messenger synthesizing enzyme ADP-ribosyl cyclase during neuronal depolarization.

In neurons, voltage-gated Ca(2+) channels and nuclear Ca(2+) signaling play important roles, such as in the regulation of gene expression. However, the link between electrical activity and biochemical cascade activation involved in the generation of the nuclear Ca(2+) signaling is poorly understood. Here we show that depolarization of Aplysia neurons induces the translocation of ADP-ribosyl cyclase, a Ca(2+) messenger synthesizing enzyme, from the cytosol into the nucleus. The translocation is dependent on Ca(2+) influx mainly through the voltage-dependent L-type Ca(2+) channels. We report also that specific nucleoplasmic Ca(2+) signals can be induced by three different calcium messengers, cyclic ADP-ribose, nicotinic acid adenine dinucleotide phosphate (NAADP), both produced by the ADP-ribosyl cyclase, and inositol 1,4,5-trisphosphate (IP(3)). Moreover, our pharmacological data show that NAADP acts on its own receptor, which cooperates with the IP(3) and the ryanodine receptors to generate nucleoplasmic Ca(2+) oscillations. We propose a new model where voltage-dependent L-type Ca(2+) channel-induced nuclear translocation of the cytosolic cyclase is a crucial step in the fine tuning of nuclear Ca(2+) signals in neurons.

Co-ordination of Ca(2+) signalling in mammalian cells by the new Ca(2+)-releasing messenger NAADP.

Ca(2+) signalling is one of the most important means in mammalian cells of relaying the action of hormones and neurotransmitters. The great diversity of agonist-induced Ca(2+) signatures, visualized by optical imaging techniques, can be explained by the production of intracellular messengers triggering Ca(2+) release from internal stores and/or by different coupling of Ca(2+) release to Ca(2+) entry. Several messengers, such as inositol trisphosphate and cyclic ADP-ribose, have been identified to date. More recent studies have reported the important role of a newly discovered Ca(2+) releasing messenger, nicotinic acid adenine dinucleotide phosphate (NAADP). These studies have shown important interactions of these messengers in the generation of specific Ca(2+) signals. NAADP acts at a very low concentration and seems to have a key role in sensitising cyclic ADP-ribose and inositol trisphosphate receptors. These points will be discussed in the present review.