Regenerative Calcium Currents in Renal Primary Cilia

Polycystic kidney disease (PKD) is a leading cause of end-stage renal disease. PKD arises from mutations in proteins, one a Ca2+-conducting channel, expressed in the primary cilia of renal epithelial cells. A common hypothesis is that Ca2+ entering through ciliary ion channels may reduce cystogenesis. The cilia have at least two Ca2+-conducting channels: polycystin-2 (PC2) and TRPV4 (transient receptor potential (TRP) cation channel, subfamily V, member 4), but how substantially they can increase intraciliary Ca2+ is unknown. By recording channel activities in isolated cilia, conditions are identified under which the channels can increase free Ca2+ within the cilium by at least 500-fold through regenerative (positive-feedback) signaling. Ca2+ that has entered through a channel can activate the channel internally, which increases the Ca2+ influx, and so on. Regenerative signaling is favored when the concentration of the Ca2+ buffer is reduced or when a slower buffer is used. Under such conditions, the Ca2+ that enters the cilium through a single PC2 channel is sufficient to almost fully activate that same channel. Regenerative signaling is not detectable with reduced external Ca2+. Reduced buffering also allows regenerative signaling through TRPV4 channels, but not through TRPM4 (TRP subfamily M, member 4) channels, which are activated by Ca2+ but do not conduct it. On a larger scale, Ca2+ that enters through TRPV4 channels can cause secondary activation of PC2 channels. I discuss the likelihood of regenerative ciliary Ca2+ signaling in vivo, a possible mechanism for its activation, and how it might relate to cystogenesis

A Selective PMCA Inhibitor Does Not Prolong the Electroolfactogram in Mouse

Within the cilia of vertebrate olfactory receptor neurons, Ca(2+) accumulates during odor transduction. Termination of the odor response requires removal of this Ca(2+), and prior evidence suggests that both Na(+)/Ca(2+) exchange and plasma membrane Ca(2+)-ATPase (PMCA) contribute to this removal.In intact mouse olfactory epithelium, we measured the time course of termination of the odor-induced field potential. Replacement of mucosal Na(+) with Li(+), which reduces the ability of Na(+)/Ca(2+) exchange to expel Ca(2+), prolonged the termination as expected. However, treating the epithelium with the specific PMCA inhibitor caloxin 1b1 caused no significant increase in the time course of response termination.Under these experimental conditions, PMCA does not contribute detectably to the termination of the odor response

Spatial Distribution of Calcium-Gated Chloride Channels in Olfactory Cilia

Background: In vertebrate olfactory receptor neurons, sensory cilia transduce odor stimuli into changes in neuronal membrane potential. The voltage changes are primarily caused by the sequential openings of two types of channel: a cyclic-nucleotide-gated (CNG) cationic channel and a calcium-gated chloride channel. In frog, the cilia are 25 to 200 mm in length, so the spatial distributions of the channels may be an important determinant of odor sensitivity. Principal Findings: To determine the spatial distribution of the chloride channels, we recorded from single cilia as calcium was allowed to diffuse down the length of the cilium and activate the channels. A computational model of this experiment allowed an estimate of the spatial distribution of the chloride channels. On average, the channels were concentrated in a narrow band centered at a distance of 29 % of the ciliary length, measured from the base of the cilium. This matches the location of the CNG channels determined previously. This non-uniform distribution of transduction proteins is consistent with similar findings in other cilia. Conclusions: On average, the two types of olfactory transduction channel are concentrated in the same region of the cilium

Limits of Calcium Clearance by Plasma Membrane Calcium ATPase in Olfactory Cilia

BACKGROUND: In any fine sensory organelle, a small influx of Ca(2+) can quickly elevate cytoplasmic Ca(2+). Mechanisms must exist to clear the ciliary Ca(2+) before it reaches toxic levels. One such organelle has been well studied: the vertebrate olfactory cilium. Recent studies have suggested that clearance from the olfactory cilium is mediated in part by plasma membrane Ca(2+)-ATPase (PMCA). PRINCIPAL FINDINGS: In the present study, electrophysiological assays were devised to monitor cytoplasmic free Ca(2+) in single frog olfactory cilia. Ca(2+) was allowed to enter isolated cilia, either through the detached end or through membrane channels. Intraciliary Ca(2+) was monitored via the activity of ciliary Ca(2+)-gated Cl(-) channels, which are sensitive to free Ca(2+) from about 2 to 10 microM. No significant effect of MgATP on intraciliary free Ca(2+) could be found. Carboxyeosin, which has been used to inhibit PMCA, was found to substantially increase a ciliary transduction current activated by cyclic AMP. This increase was ATP-independent. CONCLUSIONS: Alternative explanations are suggested for two previous experiments taken to support a role for PMCA in ciliary Ca(2+) clearance. It is concluded that PMCA in the cilium plays a very limited role in clearing the micromolar levels of intraciliary Ca(2+) produced during the odor response

The Na(+)/Ca(2+) exchanger NCKX4 governs termination and adaptation of the mammalian olfactory response

Sensory perception requires accurate encoding of stimulus information by sensory receptor cells. We identified NCKX4, a potassium-dependent Na(+)/Ca(2+) exchanger, as being necessary for rapid response termination and proper adaptation of vertebrate olfactory sensory neurons (OSNs). Nckx4(-/-) (also known as Slc24a4) mouse OSNs displayed substantially prolonged responses and stronger adaptation. Single-cell electrophysiological analyses revealed that the majority of Na(+)-dependent Ca(2+) exchange in OSNs relevant to sensory transduction is a result of NCKX4 and that Nckx4(-/-) mouse OSNs are deficient in encoding action potentials on repeated stimulation. Olfactory-specific Nckx4(-/-) mice had lower body weights and a reduced ability to locate an odorous source. These results establish the role of NCKX4 in shaping olfactory responses and suggest that rapid response termination and proper adaptation of peripheral sensory receptor cells tune the sensory system for optimal perception

A Systems Biology Approach Reveals the Role of a Novel Methyltransferase in Response to Chemical Stress and Lipid Homeostasis

Using small molecule probes to understand gene function is an attractive approach that allows functional characterization of genes that are dispensable in standard laboratory conditions and provides insight into the mode of action of these compounds. Using chemogenomic assays we previously identified yeast Crg1, an uncharacterized SAM-dependent methyltransferase, as a novel interactor of the protein phosphatase inhibitor cantharidin. In this study we used a combinatorial approach that exploits contemporary high-throughput techniques available in Saccharomyces cerevisiae combined with rigorous biological follow-up to characterize the interaction of Crg1 with cantharidin. Biochemical analysis of this enzyme followed by a systematic analysis of the interactome and lipidome of CRG1 mutants revealed that Crg1, a stress-responsive SAM-dependent methyltransferase, methylates cantharidin in vitro. Chemogenomic assays uncovered that lipid-related processes are essential for cantharidin resistance in cells sensitized by deletion of the CRG1 gene. Lipidome-wide analysis of mutants further showed that cantharidin induces alterations in glycerophospholipid and sphingolipid abundance in a Crg1-dependent manner. We propose that Crg1 is a small molecule methyltransferase important for maintaining lipid homeostasis in response to drug perturbation. This approach demonstrates the value of combining chemical genomics with other systems-based methods for characterizing proteins and elucidating previously unknown mechanisms of action of small molecule inhibitors

Effect of carboxyeosin (CE) on the ciliary transduction currents.

<p>(A) The current-voltage (I–V) relation of a cilium was measured with a 1-s voltage ramp in the following cytoplasmic baths: a control (gray) with no cAMP or CE; 2 µM cAMP (black); 2 µM cAMP and 50 µM CE (red); and 2 µM cAMP again (blue). Each bath also contained 0.1 µM free Ca²⁺, buffered with 2 mM BAPTA. Recordings were taken after 2 min in each bath, except that the last was taken after 4 min. (B) Summary of the effects of CE on the ciliary CNG current. Mean steady-state current was measured by averaging over a 2-s voltage step to −80 or +80 mV. Normalized current is the current measured in the presence of cAMP and CE divided by that measured in cAMP alone. The normalized currents shown are (left to right) 1.44±0.11, 2.35±0.32, 4.22±0.61, 2.72±0.37, 3.81±0.65, 1.17±0.03, 1.33±0.07, 1.54±0.06, 1.47±0.07, 1.82±0.18, 1.10±0.03, and 0.96±0.03. ***<i>P</i><0.001; **<i>P</i><0.02; *<i>P</i><0.05. In 3 cilia tested, the current measured in 2 µM cAMP at +80 mV was less than the saturating current (measured in 100 µM cAMP) by a factor of 0.62±0.07. (C) Summary of the effects of CE on the ciliary Ca²⁺-activated Cl⁻ current. Normalized current is the current measured in the presence of Ca²⁺ and CE divided by that measured in Ca²⁺ alone. The normalized currents shown are (left to right) 0.72±0.14, 0.96±0.22, 0.98±0.01, and 1.03±0.06. In 7 cilia tested, the current measured in 5 µM Ca²⁺ at +80 mV was less than the saturating current (measured in 300 µM Ca²⁺) by a factor of 0.52±0.09.</p

Cl⁻ current activated by longitudinal diffusion of Ca²⁺ from the bath is insensitive to ATP.

<p>(A) A cilium was held for 2 min in a control cytoplasmic bath containing 0.1 µM free Ca²⁺ and 2 mM MgITP. At <i>t</i> = 0 s, the cilium was transferred to a bath containing 300 µM free Ca²⁺ and 2 mM MgITP (recording shown in black). Membrane potential was held at −50 mV. The initial current (−20 pA) is a membrane leak current. The additional current that starts at <i>t</i> = 1.8 s is the Ca²⁺-activated Cl⁻ current. The experiment was repeated in control and test solutions in which MgITP was replaced by MgATP (recording shown in red). Ca²⁺ was buffered in all solutions with 2 mM BAPTA. (B) Summary of the effects of ATP on the onset time of the Cl⁻ current. Normalized onset time is the time measured in the presence of ATP divided by that measured in the control (ITP). The normalized onset times shown are 0.97±0.08 (7 µM free Ca²⁺), 1.13±0.06 (20 µM free Ca²⁺), and 0.97±0.06 (300 µM free Ca²⁺).</p

Cl⁻ current activated by membrane Ca²⁺ influx is insensitive to ATP.

<p>(A) A cilium was held at 0 mV for 2 min in a control cytoplasmic bath containing 0.1 µM free Ca²⁺, 100 µM cAMP, and 2 mM MgITP. Ca²⁺ was buffered with 2 mM BAPTA. At <i>t</i> = 0 s, the voltage was switched to −40 mV (recording shown in black). The initial current (−80 pA) is carried by cations, including Ca²⁺, primarily through CNG channels gated by cAMP. The additional current that starts at <i>t</i> = 2.9 s is the Ca²⁺-activated Cl⁻ current. The experiment was repeated in control and test solutions in which MgITP was replaced by MgATP (recording shown in red). (B) Summary of the effects of ATP on the onset time of the Cl⁻ current. Normalized onset time is as defined for <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0005266#pone-0005266-g001" target="_blank">Fig. 1</a>. The normalized onset times shown are 1.04±0.04 (left bar), 1.05±0.06 (middle bar), and 0.82±0.10 (right bar).</p