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 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.

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.

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.

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.

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.

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.

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.

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.