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.

Inward Ca2+ current through the polycystin-2-dependent channels of renal primary cilia.

In 15% of cases, autosomal dominant polycystic kidney disease arises from defects in polycystin-2 (PC2). PC2 is a member of the polycystin transient receptor potential subfamily of cation-conducting channels and is expressed in the endoplasmic reticulum and primary cilium of renal epithelial cells. PC2 opposes a procystogenic influence of the cilium, and it has been proposed that this beneficial effect is mediated in part by a flow of Ca2+ through PC2 channels into the primary cilium. However, previous efforts to determine the permeability of PC2 channels to Ca2+ have yielded widely varying results. Here, we report the mean macroscopic Ca2+ influx through native PC2 channels in the primary cilia of mIMCD-3 cells, which are derived from the murine inner medullary collecting duct. Under conditions designed to isolate inward Ca2+ currents, a small inward Ca2+ current was detected in cilia with active PC2 channels but not in cilia lacking those channels. The current was activated by the addition of 10 µM internal Ca2+, which is known to activate ciliary PC2 channels. It was blocked by 10 µM isosakuranetin, which blocks the same channels. On average, the current amplitude was -1.8 pA at -190 mV; its conductance from -50 to -200 mV averaged 20 pS. Thus, native PC2 channels of renal primary cilia are able to conduct a small but detectable Ca2+ influx under the conditions tested. The possible consequences of this influx are discussed.NEW & NOTEWORTHY In autosomal dominant polycystic kidney disease, it is proposed that Ca2+ entering the primary cilium through polycystin-2 (PC2) channels may limit the formation of cysts. Recent studies predict that any macroscopic Ca2+ influx through these channels should be small. We report that the native PC2 channels in primary cilia of cultured renal epithelial cells can allow a small macroscopic calcium influx. This may allow a significant accumulation of Ca2+ in the cilium in vivo.

The TRPP2-dependent channel of renal primary cilia also requires TRPM3.

Primary cilia of renal epithelial cells express several members of the transient receptor potential (TRP) class of cation-conducting channel, including TRPC1, TRPM3, TRPM4, TRPP2, and TRPV4. Some cases of autosomal dominant polycystic kidney disease (ADPKD) are caused by defects in TRPP2 (also called polycystin-2, PC2, or PKD2). A large-conductance, TRPP2-dependent channel in renal cilia has been well described, but it is not known whether this channel includes any other protein subunits. To study this question, we investigated the pharmacology of the TRPP2-dependent channel through electrical recordings from the cilia of mIMCD-3 cells, a murine cell line of renal epithelial origin. The pharmacology was found to match that of TRPM3 channels. The ciliary TRPP2-dependent channel is known to be activated by depolarization and by increasing cytoplasmic Ca2+. This activation was greatly enhanced by external pregnenolone sulfate, an agonist of TRPM3 channels. Pregnenolone sulfate did not change the single-channel current-voltage relation. The channels were effectively blocked by isosakuranetin, a specific inhibitor of TRPM3 channels. Both pregnenolone sulfate and isosakuranetin were effective at concentrations as low as 1 µM. Knocking out TRPM3 by CRISPR/Cas9 genome editing eliminated the ciliary channel. Thus the channel is both TRPM3-dependent and TRPP2-dependent, suggesting that it may include both types of subunit. Knocking out TRPM3 did not change the level of TRPP2 protein in the cilia, so it is unlikely that the absence of functional ciliary channels results from a failure of trafficking.

Primary cilia regulate the osmotic stress response of renal epithelial cells through TRPM3.

Primary cilia sense environmental conditions, including osmolality, but whether cilia participate in the osmotic response in renal epithelial cells is not known. The transient receptor potential (TRP) channels TRPV4 and TRPM3 are osmoresponsive. TRPV4 localizes to cilia in certain cell types, while renal subcellular localization of TRPM3 is not known. We hypothesized that primary cilia are required for maximal activation of the osmotic response of renal epithelial cells and that ciliary TRPM3 and TRPV4 mediate that response. Ciliated [murine epithelial cells from the renal inner medullary collecting duct (mIMCD-3) and 176-5] and nonciliated (176-5Δ) renal cells expressed Trpv4 and Trpm3 Ciliary expression of TRPM3 was observed in mIMCD-3 and 176-5 cells and in wild-type mouse kidney tissue. TRPV4 was identified in cilia and apical membrane of mIMCD-3 cells by electrophysiology and in the cell body by immunofluorescence. Hyperosmolal stress at 500 mOsm/kg (via NaCl addition) induced the osmotic response genes betaine/GABA transporter (Bgt1) and aldose reductase (Akr1b3) in all ciliated cell lines. This induction was attenuated in nonciliated cells. A TRPV4 agonist abrogated Bgt1 and Akr1b3 induction in ciliated and nonciliated cells. A TRPM3 agonist attenuated Bgt1 and Akr1b3 induction in ciliated cells only. TRPM3 knockout attenuated Akr1b3 induction. Viability under osmotic stress was greater in ciliated than nonciliated cells. Akr1b3 induction was also less in nonciliated than ciliated cells when mannitol was used to induce hyperosmolal stress. These findings suggest that primary cilia are required for the maximal osmotic response in renal epithelial cells and that TRPM3 is involved in this mechanism. TRPV4 appears to modulate the osmotic response independent of cilia.

The native TRPP2-dependent channel of murine renal primary cilia.

Autosomal dominant polycystic kidney disease (ADPKD) is the most common life-threatening monogenic renal disease. ADPKD results from mutations in either of two proteins: polycystin-1 (also known as PC1 or PKD1) or transient receptor potential cation channel, subfamily P, member 2 (TRPP2, also known as polycystin-2, PC2, or PKD2). Each of these proteins is expressed in the primary cilium that extends from many renal epithelial cells. Existing evidence suggests that the cilium can promote renal cystogenesis, while PC1 and TRPP2 counter this cystogenic effect. To better understand the function of TRPP2, we investigated its electrophysiological properties in the native ciliary membrane. We recorded directly from the cilia of mIMCD-3 cells, a murine cell line of renal epithelial origin. In one-third of cilia examined, a large-conductance channel was observed. The channel was not permeable to Cl¯ but conducted cations with permeability ratios PK:PCa:PNa of 1:0.55:0.14. The single-channel conductance ranged from 97 pS in typical physiological solutions to 189 pS in symmetrical 145 mM KCl. Open probability of the channel was very sensitive to membrane depolarization or increasing cytoplasmic free Ca2+ in the low micromolar range, with the open probability increasing in either case. Knocking out TRPP2 by CRISPR/Cas9 genome editing eliminated the channel current, establishing it as TRPP2 dependent. Possible mechanisms for activating the TRPP2-dependent channel in the renal primary cilium are discussed.

Calcium channels in primary cilia.

PURPOSE OF REVIEW: Primary cilia have become important organelles implicated in embryonic development, organogenesis, health, and diseases. Although many studies in cell biology have focused on changes in ciliary length or ciliogenesis, the most common readout for evaluating ciliary function is intracellular calcium. RECENT FINDINGS: Recent tools have allowed us to examine intracellular calcium in more precise locations, that is, the cilioplasm and cytoplasm. Advances in calcium imaging have also allowed us to identify which cilia respond to particular stimuli. Furthermore, direct electrophysiological measurement of ionic currents within a cilium has provided a wealth of information for understanding the sensory roles of primary cilia. SUMMARY: Calcium imaging and direct measurement of calcium currents demonstrate that primary cilia are sensory organelles that house several types of functional calcium channels. Although intracellular calcium now allows a functional readout for primary cilia, discussions on the relative contributions of the several channel types have just begun. Perhaps, all of these calcium channels are required and necessary to differentiate stimuli in different microenvironments.

A TRPM4-dependent current in murine renal primary cilia.

Defects in primary cilia lead to a variety of human diseases. One of these, polycystic kidney disease, can be caused by defects in a Ca²âº-gated ion channel (TRPP2) found on the cilium. Other ciliary functions also contribute to cystogenesis, and defects in apical Ca²âº homeostasis have been implicated. By recording directly from the native cilia of mIMCD-3 cells, a murine cell line of renal epithelial origin, we have identified a second Ca²âº-gated channel in the ciliary membrane: the transient receptor potential cation channel, subfamily M, member 4 (TRPM4). In excised primary cilia, TRPM4 was found to have a low sensitivity to Ca²âº, with an EC50 of 646 µM at +100 mV. It was inhibited by MgATP and by 9-phenanthrol. The channel was not permeable to Ca²âº or Cl⁻ and had a permeability ratio PK/PNa of 1.42. Reducing the expression of Trpm4 mRNA with short hairpin (sh) RNA reduced the TRPM4 current by 87% and shortened primary cilia by 43%. When phospholipase C was inhibited, the sensitivity to cytoplasmic Ca²âº greatly increased (EC50 = 26 µM at +100 mV), which is consistent with previous reports that phosphatidylinositol 4,5-bisphosphate (PIP2) modulates the channel. MgATP did not restore the channel to a preinactivation state, suggesting that the enzyme or substrate necessary for making PIP2 is not abundant in primary cilia of mIMCD-3 cells. The function of TRPM4 in renal primary cilia is not yet known, but it is likely to influence the apical Ca²âº dynamics of the cell, perhaps in tandem with TRPP2.

Electrical Signaling in Motile and Primary Cilia.

Cilia are highly conserved for their structure and also for their sensory functions. They serve as antennae for extracellular information. Whether the cilia are motile or not, they respond to environmental mechanical and chemical stimuli and signal to the cell body. The information from extracellular stimuli is commonly converted to electrical signals through the repertoire of ion-conducting channels in the ciliary membrane resulting in changes in concentrations of ions, especially Ca2+, in the cilia. These changes, in turn, affect motility and signaling pathways in the cilia and cell body to carry on the signal transduction. We review here the activities of ion channels in cilia from protists to vertebrates.

A selective PMCA inhibitor does not prolong the electroolfactogram in mouse.

BACKGROUND: 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. PRINCIPAL FINDINGS: 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. CONCLUSIONS: Under these experimental conditions, PMCA does not contribute detectably to the termination of the odor response.

A method for measuring electrical signals in a primary cilium.

BACKGROUND: Most cells in the body possess a single primary cilium. These cilia are key transducers of sensory stimuli, and defects in cilia have been linked to several diseases. Evidence suggests that some transduction of sensory stimuli by the primary cilium depends on ion-conducting channels. However, the tiny size of the cilium has been a critical barrier to understanding its electrical properties. We report a novel method that allows sensitive, repeatable electrical recordings from primary cilia. Adherent cells were grown on small, spherical beads that could be easily moved within the recording chamber. In this configuration, an entire cilium could be pulled into a recording microelectrode. RESULTS: In 47% of attempts, suction resulted in a seal with high input resistance. Single channels could be recorded while the cilium remained attached to the cell. When the pipette was raised into the air, the cell body was pulled off at the air-bath interface. The pipette retained the cilium and could then be immersed in various solutions that bathed the cytoplasmic face of the membrane. In excised cilia, ionic currents through ciliary channels were modulated by cytoplasmic Ca(2+) and transmembrane voltage. CONCLUSIONS: Ciliary recording is a direct way to learn the effects of second messengers and voltage changes on ciliary transduction channels.

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 µm 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. This may contribute to the efficient detection of weak stimuli.

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 electrochemical basis of odor transduction in vertebrate olfactory cilia.

Most vertebrate olfactory receptor neurons share a common G-protein-coupled pathway for transducing the binding of odorant into depolarization. The depolarization involves 2 currents: an influx of cations (including Ca2+) through cyclic nucleotide-gated channels and a secondary efflux of Cl- through Ca2+-gated Cl- channels. The relation between stimulus strength and receptor current shows positive cooperativity that is attributed to the channel properties. This cooperativity amplifies the responses to sufficiently strong stimuli but reduces sensitivity and dynamic range. The odor response is transient, and prolonged or repeated stimulation causes adaptation and desensitization. At least 10 mechanisms may contribute to termination of the response; several of these result from an increase in intraciliary Ca2+. It is not known to what extent regulation of ionic concentrations in the cilium depends on the dendrite and soma. Although many of the major mechanisms have been identified, odor transduction is not well understood at a quantitative level.

Identification of Cl(Ca) channel distributions in olfactory cilia.

Identification of detailed features of neuronal systems is an important challenge in the biosciences today. Transduction of an odor into an electrical signal occurs in the membranes of the cilia. The Cl(Ca) channels that reside in the ciliary membrane are activated by calcium, allow a depolarizing efflux of Cl(-) and are thought to amplify the electrical signal to the brain.In this paper, a mathematical model consisting of partial differential equations is developed to study two different experiments; one involving the interaction of the cyclic-nucleotide-gated (CNG) and Cl(Ca) channels and the other, the diffusion of Ca(2+) into cilia. This work builds on an earlier study (Mathematical modeling of the Cl(Ca) ion channels in frog olfactory cilia. Ph.D. Thesis, University of Cincinnati, Cincinnati, OH, 2006; Math. Comput. Modelling 2006; 43:945-956; Biophys. J. 2006; 91:179-188), which suggested that the CNG channels are clustered at about 0.28 of the length of a cilium from its open end. Closed-form solutions are derived after certain reductions in the model are made. These special solutions provide estimates of the channel distributions. Scientific computation is also used. This preliminary study suggests that the Cl(Ca) ion channels are also clustered at about one-third of the length of a cilium from its open end.

Mice lacking NKCC1 have normal olfactory sensitivity.

When olfactory receptor neurons respond to odors, a depolarizing Cl(-) efflux is a substantial part of the response. This requires that the resting neuron accumulate Cl(-) against an electrochemical gradient. In isolated olfactory receptor neurons, the Na(+)+K(+)+2Cl(-) cotransporter NKCC1 is essential for Cl(-) accumulation. However, in intact epithelium, a robust electrical olfactory response persists in mice lacking NKCC1. To determine whether NKCC1 is required for normal olfactory sensitivity, olfactory sensitivity was compared between knockout (KO) mice carrying a null mutation for NKCC1 and wild-type (WT) littermates. Using operant behavioral techniques, olfactory sensitivity was measured using a commercial liquid-dilution olfactometer. Detection thresholds for the simple odorants cineole, 1-heptanol, and 1-propanol were compared in KO and WT animals. Regardless of the stimulus conditions employed, no systematic differences in behavioral thresholds were evident between KO and WT animals. We conclude that NKCC1 is not required for normal olfactory sensitivity.

Mechanisms of neuronal chloride accumulation in intact mouse olfactory epithelium.

When olfactory receptor neurons respond to odours, a depolarizing Cl(-) efflux is a substantial part of the response. This requires that the resting neuron accumulate Cl(-) against an electrochemical gradient. In isolated olfactory receptor neurons, the Na(+)-K(+)-2Cl(-) cotransporter NKCC1 is essential for Cl(-) accumulation. However, in intact epithelium, a robust electrical olfactory response persists in mice lacking NKCC1. This response is largely due to a neuronal Cl(-) efflux. It thus appears that NKCC1 is an important part of a more complex system of Cl(-) accumulation. To identify the remaining transport proteins, we first screened by RT-PCR for 21 Cl(-) transporters in mouse nasal tissue containing olfactory mucosa. For most of the Cl(-) transporters, the presence of mRNA was demonstrated. We also investigated the effects of pharmacological block or genetic ablation of Cl(-) transporters on the olfactory field potential, the electroolfactogram (EOG). Mice lacking the common Cl(-)/HCO(3)(-) exchanger AE2 had normal EOGs. Block of NKCC cotransport with bumetanide reduced the EOG in epithelia from wild-type mice but had no effect in mice lacking NKCC1. Hydrochlorothiazide, a blocker of the Na(+)-Cl(-) cotransporter, had only a small effect. DIDS, a blocker of some KCC cotransporters and Cl(-)/HCO(3)(-) exchangers, reduced the EOG in epithelia from both wild-type and NKCC1 knockout mice. A combination of bumetanide and DIDS decreased the response more than either drug alone. However, no combination of drugs completely abolished the Cl(-) component of the response. These results support the involvement of both NKCC1 and one or more DIDS-sensitive transporters in Cl(-) accumulation in olfactory receptor neurons.

Clustering of cyclic-nucleotide-gated channels in olfactory cilia.

Olfactory cilia contain the known components of olfactory signal transduction, including a high density of cyclic-nucleotide-gated (CNG) channels. CNG channels play an important role in mediating odor detection. The channels are activated by cAMP, which is formed by a G-protein-coupled transduction cascade. Frog olfactory cilia are 25-200 microm in length, so the spatial distribution of CNG channels along the length should be important in determining the sensitivity of odor detection. We have recorded from excised cilia and modeled diffusion of cAMP into a cilium to determine the spatial distribution of the CNG channels along the ciliary length. The proximal segment, which in frog is the first 20% of the cilium, appears to express a small fraction of the CNG channels, whereas the distal segment contains the majority, mostly clustered in one region.

Neuronal chloride accumulation in olfactory epithelium of mice lacking NKCC1.

When stimulated with odorants, olfactory receptor neurons (ORNs) produce a depolarizing receptor current. In isolated ORNs, much of this current is caused by an efflux of Cl-. This implies that the neurons have one or more mechanisms for accumulating cytoplasmic Cl- at rest. Whether odors activate an efflux of Cl- in intact olfactory epithelium, where the ionic environment is poorly characterized, has not been previously determined. In mouse olfactory epithelium, we found that >80% of the summated electrical response to odors is blocked by niflumic acid or flufenamic acid, each of which inhibits Ca2+-activated Cl- channels in ORNs. This indicates that ORNs accumulate Cl- in situ. Recent evidence has shown that NKCC1, a Na+-K+-2Cl- cotransporter, contributes to Cl- accumulation in mammalian ORNs. However, we find that the epithelial response to odors is only reduced by 39% in mice carrying a null mutation in Nkcc1. As in the wild-type, most of the response is blocked by niflumic acid or flufenamic acid, indicating that the underlying current is carried by Cl-. We conclude that ORNs effectively accumulate Cl- in situ even in the absence of NKCC1. The Cl- -transport mechanism underlying this accumulation has not yet been identified.

Numerical Approximation of Solutions of a Nonlinear Inverse Problem Arising in Olfaction Experimentation.

Identification of detailed features of neuronal systems is an important challenge in the biosciences today. Cilia are long thin structures that extend from the olfactory receptor neurons into the nasal mucus. Transduction of an odor into an electrical signal occurs in the membranes of the cilia. The cyclic-nucleotide-gated (CNG) channels which reside in the ciliary membrane and are activated by adenosine 3',5'-cyclic monophosphate (cAMP) allow a depolarizing influx of Ca(2+) and Na(+) and are thought to initiate the electrical signal.In this paper, a mathematical model consisting of two nonlinear differential equations and a constrained Fredholm integral equation of the first kind is developed to model experiments involving the diffusion of cAMP into cilia and the resulting electrical activity. The unknowns in the problem are the concentration of cAMP, the membrane potential and, the quantity of most interest in this work, the distribution of CNG channels along the length of a cilium. A simple numerical method is derived that can be used to obtain estimates of the spatial distribution of CNG ion channels along the length of a cilium. Certain computations indicate that this mathematical problem is ill-conditioned.