Calcium Homeostasis Modulator 1-Like Currents in ... - Oxford Journals

Chemical Senses, 2017, Vol 42, 343–359 doi:10.1093/chemse/bjx013 Original Article Advance Access publication March 10, 2017

Original Article

Calcium Homeostasis Modulator 1-Like Currents in Rat Fungiform Taste Cells Expressing Amiloride-Sensitive Sodium Currents Albertino Bigiani Dipartimento di Scienze Biomediche, Metaboliche e Neuroscienze, Università di Modena e Reggio Emilia, Via G. Campi 287, 41125 Modena, Italy Correspondence to be sent to: Albertino Bigiani, Dipartimento di Scienze Biomediche, Metaboliche e Neuroscienze, Università di Modena e Reggio Emilia, Via G. Campi 287, 41125 Modena, Italy. e-mail: [email protected] Editorial Decision 13 February 2017.

Abstract Salt reception by taste cells is still the less understood transduction process occurring in taste buds, the peripheral sensory organs for the detection of food chemicals. Although there is evidence suggesting that the epithelial sodium channel (ENaC) works as sodium receptor, yet it is not clear how salt-detecting cells signal the relevant information to nerve endings. Taste cells responding to sweet, bitter, and umami substances release ATP as neurotransmitter through a nonvesicular mechanism. Three different channel proteins have been proposed as conduit for ATP secretion: pannexin channels, connexin hemichannels, and calcium homeostasis modulator 1 (CALHM1) channels. In heterologous expression systems, these channels mediate outwardly rectifying membrane currents with distinct biophysical and pharmacological properties. I  therefore tested whether also salt-detecting taste cells were endowed with these currents. To this aim, I  applied the patch-clamp techniques to single cells in isolated taste buds from rat fungiform papillae. Saltdetecting cells were functionally identified by exploiting the effect of amiloride, which induces a current response by shutting down ENaCs. I  looked for the presence of outwardly rectifying currents by using appropriate voltage-clamp protocols and specific pharmacological tools. I found that indeed salt-detecting cells possessed these currents with properties consistent with the presence, at least in part, of CALHM1 channels. Unexpectedly, CALHM1-like currents in taste cells were potentiated by known blockers of pannexin, suggesting a possible inhibitory action of this protein on CALMH1. These findings indicate that communication between salt-detecting cells and nerve endings might involve ATP release by CALMH1 channels. Key words: CALHM1, connexin, ENaC, pannexin, sodium taste

Introduction Taste reception relies on the activity of specific receptors, enzymes, and ion channels expressed in taste cells, the peripheral detectors of food chemicals (Liman et  al. 2014). Detailed studies on mice have established that a specific subset of taste cells, called Type II cells, is endowed with the molecular machinery to detect sweet, bitter, and umami substances (Clapp et al. 2006; DeFazio et al. 2006) © The Author 2017. Published by Oxford University Press. All rights reserved. For permissions, please e-mail: [email protected]

and transmits the relevant sensory information to nerve endings by nonvesicular ATP release (Finger et  al. 2005; Huang et  al. 2007; Romanov et  al. 2007). Three different channel proteins have been proposed as the main conduit for ATP secretion: pannexin channels, connexin hemichannels, and recently calcium homeostasis modulator 1 (CALHM1) channels. All these proteins share functional and structural similarities (Siebert et al. 2013), but their specific role in

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344 taste cell functioning is not yet completely defined. Although earlier experiments suggested that pannexin 1 (Panx1) channels could be responsible for taste-evoked ATP release (Huang et  al. 2007; Murata et al. 2010), recent studies with Panx1 knockout (KO) mice do not support this view (Romanov et al. 2012; Tordoff et al. 2015; Vandenbeuch, Anderson, et al. 2015). The possible role of connexin hemichannels in ATP release (Romanov et  al. 2007, 2008) is still unclear (Medler 2015). Currently, there is compelling evidence for the involvement of CALMH1 channel as a key element in Type II cells for cell-to-cell communication (Taruno, Vingtdeux, et al. 2013). Sensory transduction for sour stimuli is performed by another taste cell subset, Type III cells, which releases serotonin and likely other transmitters using conventional calcium-dependent vesicle exocytosis to communicate sensory information to nerve endings (Huang et al. 2008; Kataoka et al. 2008; Chang et al. 2010; Vandenbeuch et al. 2010; Larson et al. 2015; Ye et al. 2016). In both Type II and Type III cells, action potential firing seems to be linked to chemical detection (Yoshida, Miyauchi, et al. 2009; Murata et al. 2010), although there is also evidence for ATP secretion not related to firing activity (Huang and Roper 2010). In addition to the direct communication with taste nerve fibers, lateral cross-talk occurs between Type II and Type III, and this interaction among adjacent cells inside the taste buds may play a role in processing information before relaying sensory input to the brain (Roper 2013; Chaudhari 2014). The chain of molecular events involved in the transduction of salty stimuli is less understood. In mice, the amiloride-sensitive, epithelial sodium channel (ENaC) has been demonstrated to work as sodium receptor (Chandrashekar et  al. 2010). ENaC seems to be segregated in Type I  cells (Vandenbeuch et  al. 2008), a separate subset of taste cells thought to serve a glial-like role within taste buds (Bigiani and Prandi 2011). However, it is not clear how these cells transmit sensory information to nerve endings and/or adjacent taste cells, since they do not express voltage-gated Na+ (and Ca2+) channels (Vandenbeuch et al. 2008) required for firing activity. Indeed, salt-detecting cells generate action potentials during stimulation with NaCl (Avenet and Lindemann 1991; Ohtubo et al. 2001; Yoshida, Horio, et al. 2009) and likely release ATP as suggested by studies with mice lacking the purinergic receptors on nerve fibers (Finger et  al. 2005; Eddy et  al. 2009; Vandenbeuch, Larson, et  al. 2015). Recently, behavioral and nerve recording experiments with CALHM1 KO mice have shown the involvement of this channel in salt taste, at least for some NaCl concentrations (Tordoff et al. 2014). Interestingly, Tordoff and collaborators “observed no effect of amiloride on the size of chorda tympani responses to NaCl in CALHM1 KO mice, which suggests that ENaCs and CALHM1 are part of the same transduction pathway.” Thus, there are indications that taste cells responding to salty stimuli might communicate saltiness to nerve endings via ATP release possibly through CALHM1 channels. These findings, however, are in contrast with the observation that CALHM1 channels are expressed only by Type II taste cells (Moyer et al. 2009; Taruno, Vingtdeux, et al. 2013), which are endowed with membrane receptors for sweet, bitter, and umami compounds, but seemingly not with ENaCs (Vandenbeuch et al. 2008). In this study, I have performed an electrophysiological analysis of salt-sensitive taste cells in rat fungiform papillae to test whether these cells possessed membrane currents consistent with the occurrence of ATP-releasing channels (pannexins, connexins, CAHLM1). In exogenous expression systems, such as Xenopus oocytes or mammalian cell lines, these channels generate voltage-dependent, outwardly rectifying currents that can be distinguished on the basis of their biophysical properties and pharmacological profiles

(e.g., Eskandari et  al. 2002; Bruzzone et  al. 2005; Qiu and Dahl 2009; Ma et al. 2009, 2012, 2016; Patel et al. 2014; Romanov et al. 2012; Dahl et  al. 2013; Fasciani et  al. 2013; Siebert et  al. 2013). Thus, I  looked for evidence of these currents in rat taste cells by using the patch-clamp technique in whole-cell, voltage-clamp configuration, and by evaluating the effect of specific channel inhibitors. Salt-detecting cells were identified by exploiting the known effect of amiloride, which is a selective blocker of ENaCs at submicromolar concentrations (Lindemann 1996). In standard physiological saline, constitutively open ENaCs mediate a stationary inward current (amiloride-sensitive sodium current [ASSC]): by shutting off these channels, amiloride causes a typical reduction in ASSC, called “response to amiloride,” which can be easily recorded with the patch-clamp recording technique (Bigiani 2016). The occurrence of this response was used as an electrophysiological fingerprint for the presence of ENaCs in rat taste cells.

Materials and methods Ethical approval Experiments were performed in compliance with the Italian law on animal care no.  116/1992 and in accordance with the European Community Council Directive (EEC/609/86). All efforts were made to reduce both animal suffering and the number of animals used. This study was approved by the institutional review board “Organismo Preposto al Benessere degli Animali.”

Animals Sprague-Dawley rats (male and female, 50–70  days of age, 200– 350  g of weight) were used in this study. Animals were housed 2 per cage on a 12-h light/dark cycle in climate-controlled conditions with ad libitum access to water and food. I used the Sprague-Dawley rat as animal model because ENaC-mediated component of sodium taste response recorded from chorda tympani nerve is much larger than in other rodents, including mice (Halpern 1998). Chorda tympani fibers innervate fungiform taste buds: thus, a larger nerve response sensitive to amiloride likely reflects either a larger number of sodium-detecting taste cells, or a larger response to sodium in single taste cells, or both, in fungiform taste buds. To isolate taste tissue, animals were deeply anesthetized by inhalation of isoflurane (Merial Italia), and then euthanized by cervical dislocation.

Isolation of rat fungiform taste buds Electrophysiological experiments were performed on single taste cells in isolated taste buds. My procedure to isolate taste buds from fungiform papillae closely followed published protocols (e.g., Doolin and Gilbertson 1996; Kossel et  al. 1997; Liu et  al. 2005). During taste bud isolation, amiloride (10 µM) was added to all solutions to avoid enzymatic degradation of ENaCs (e.g., Doolin and Gilbertson 1996). Taste buds were plated on the bottom of a chamber consisting of a standard glass slide onto which a silicon ring 1–2 mm thick and 15 mm ID was pressed. The glass slide was precoated with CellTak (~3 µg/cm2; Becton Dickinson) to improve adherence of isolated taste buds to the bottom of the chamber. The chamber was placed on the stage of an upright Olympus microscope (model BHWI), and taste buds were viewed with Nomarski optics using a water immersion objective. During the experiments, isolated taste buds were continuously perfused with Tyrode’s solution (containing [in mM] 140 NaCl, 5 KCl, 2 CaCl2, 1 MgCl2, 10 glucose, 10 sodium pyruvate, and 10 HEPES; pH 7.4) by means of a gravity-driven system.

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Patch-clamp recording

Chemicals

Whole cell patch-clamp recordings were made from cells in isolated taste buds as described previously (e.g., Doolin and Gilbertson 1996; Kossel et al. 1997; Liu et al. 2005). Signals were filtered at 5 kHz with the patch-clamp amplifier (Axopatch 1-D; Molecular Devices) and sampling rate was adjusted according to the type of membrane current. Data were acquired and analyzed with the pCLAMP9 software (Molecular Devices). Recording pipettes were made from soda lime glass capillaries (Globe Scientific) on a 2-stage vertical puller (PP-830; Narishige). Typical pipette resistances were 2–3 MΩ when filled with a standard pipette solution containing (in mM) 120 KCl, 1 CaCl2, 2 MgCl2, 10 HEPES, 11 EGTA, and 2 ATPNa2, pH 7.2 adjusted with KOH. The liquid junction potential of ~4 mV measured between pipette solution and Tyrode (bath) solution was neglected. Patch pipette was positioned onto taste cells by means of a water hydraulic micromanipulator (MHW-3; Narishige). Patched taste cells were identified on the basis of their voltage-gated ion currents as described previously (Bigiani and Cuoghi 2007). The access resistance of the patch pipette tip was estimated by dividing the amplitude of a 20-mV voltage step by the peak of the cell capacitive transient. Typical values were about 5–15 MΩ and were not compensated. Access resistance is the main determinant of series resistance (Rseries) that can affect the behavior of voltage-gated currents (Marty and Neher 1995; Sontheimer and Ransom 2002). However, the voltage error due to uncompensated Rseries was negligible for the outward current values recorded in my experiments. Rseries was monitored throughout the experiments by checking the kinetic behavior of voltage-gated sodium currents, which are very sensitive even to small changes of Rseries (Marty and Neher 1995). The presence of functional ENaCs in taste cells was monitored by studying the effect of bath-applied amiloride, a selective blocker of ENaC at submicromolar concentrations, on the whole-cell current recorded at a given holding potential (Bigiani 2016). To assure specificity, I used as a probe an amiloride concentration of 1 µM, which is above the inhibition constant for ENaCs in these cells and below the dose range affecting other membrane transporters (Lindemann 1996; Halpern 1998). I  included in the analysis taste cells with response to amiloride ≥50 pA. Cloned ATP-releasing channels expressed in heterologous systems produce voltage-dependent outward currents that activate at positive membrane potentials (e.g., Ma et al. 2012; Romanov et al. 2012; Fasciani et al. 2013). Since rat taste cells are endowed with voltage-dependent outward K+ currents (IK) (Chen et al. 1996; Liu et al. 2005; Bigiani and Cuoghi 2007), I typically performed electrophysiological recordings by holding the cell membrane at −40 mV to exploit inactivation of K+ currents or, if necessary, in the presence of 10 mM tetraethylammonium chloride (TEA) in the extracellular solution to exploit sensitivity to TEA block by K+ currents (e.g., Liu et  al. 2005). I  usually preferred not to use CsCl in the patch pipette to block IK because the amplitude of these currents was used as criterion to distinguish different subsets of “mature” taste cells (for details, see Bigiani and Cuoghi 2007). Nonetheless, to make sure the above procedure abolished any contribution of potassium currents to the records, I  also performed some experiments by using CsCl instead of KCl in the patch pipette. I found no difference between the 2 recording conditions (Figure 2F) and data were pooled together. To reduce the background current due to open ENaCs, 1  µM amiloride was added to all drug solutions. The linear leakage current remaining in the presence of amiloride was subtracted mathematically from the whole current in order to evaluate outward current amplitude at +80 mV, unless otherwise stated.

Pharmacological profile of outwardly rectifying currents was determined by evaluating the effect of known blockers of ATP-releasing channels (e.g., Dahl et al. 2013; Patel et al. 2014; Ma et al. 2016). Specifically I  tested benzoylbenzoyl-ATP (BzATP, 100  µM), carbenoxolone (CBX, 10–500  µM), Gd3+ (100  µM), octanol (1  mM), and probenecid (1 mM). BzATP, CBX, and Gd3+ were directly dissolved in Tyrode’s solution. Also octanol could be dissolved directly in Tyrode’s only by stirring due to its partial solubility in water (~500 mg/l, corresponding to ~3.8 mM concentration). Probenecid was predissolved in 200  mM NaOH solution at a concentration of 200  mM (stock solution) and then diluted in Tyrode’s solution to obtain a final concentration of 1 mM. Amiloride was dissolved in dimethyl sulfoxide at a concentration of 0.5 M, and then diluted to its final concentration of 1 µM. Drugs were delivered through the gravity-driven flow perfusion system, which assured a solution change in less than 1 min (see Figure 2A). To quantify the drug effect, I evaluated the percentage change in the amplitude of the current elicited at +80 mV from a holding potential of −40 mV and measured at the end of the voltage pulse. Dose–response curve for the effect of CBX was obtained by measuring the amplitude of the outward currents at the end of a 3-s pulse to +80 mV (I+80 mV) from a holding potential of −40 mV in the presence of increasing concentration of CBX into the bath solution. The data were fitted to the 4-parameter logistic equation: I +80 mV = IBottom +

I Top − IBottom

1 + 10(

(LogEC50 − X)⋅ nH )

where IBottom is the lowest amplitude of the current in the absence of CBX, ITop is the largest amplitude of the current in the presence of CBX, EC50 is the concentration of agonist that gives a response half way between IBottom and ITop, X is the variable CBX concentration, and nH is the Hill slope describing the steepness of the curve. Noninteger nH may indicate cooperativity and allosteric interactions underlying dose–response relationship (Prinz 2010).

Statistics Analysis and plotting were performed using Prism 6.0 software (Graph Pad Software). Results are presented as means ± SE of the means (SEM) of the specified number of cells (n). Paired Student’s t-test was used for statistical significance in the analysis of I–V plots. Data distribution among patched cells was represented in the form of box and whiskers plots. Boxes show the middle half of the data (the 25th and 75th percentiles) and the horizontal line marks the median, whereas the “whiskers” extending from the top and the bottom of the boxes show the main body of the data (the 10th and 90th percentiles). Outliers or extreme values are plotted individually with circles.

Results Taste cells are electrophysiologically heterogeneous (Lindemann 1996; Bigiani and Prandi 2011) and undergo continuous turnover (Barlow 2015). The expression and the amplitude of specific voltagegated membrane currents depend on the development stage (immature vs. mature) and on the functional role played inside taste buds (glial-like vs. sensory). The presence of large voltage-gated sodium currents (INa) is considered to be an electrophysiological hallmark for mature sensory cells (Doolin and Gilbertson 1996; Mackay-Sim et  al. 1996; Medler et  al. 2003; Vandenbeuch et  al. 2008; Kimura et  al. 2014). Thus, for this study, I  selected rat fungiform taste cells with sizeable INa (maximum peak value >1 nA). As described

346 previously (Bigiani and Cuoghi 2007; Bigiani 2015), a subset of these cells exhibits the so-called “response to amiloride,” that is, a reduction in the stationary membrane current recorded at negative membrane potential during application of amiloride (Bigiani 2016). This response is due to the presence of ASSC mediated by functional ENaCs.

Outwardly rectifying currents in salt-detecting taste cells In response to long-lasting (seconds) depolarizing voltage steps, cloned ATP-releasing channels mediate voltage-dependent currents that show outward rectification at positive membrane potentials in regular physiological saline (e.g., Gómez-Hernández et al. 2003; Bruzzone et al. 2005; Ma et al. 2012). Thus, in preliminary experiments, I looked for the presence of outwardly rectifying currents in salt-sensitive taste cells. As described previously (Bigiani and Cuoghi 2007),

Chemical Senses, 2017, Vol. 42, No. 4 these cells are characterized by expressing, in addition to large INa, TEA-sensitive voltage-gated potassium currents (IK) and ASSCs. Figure  1A shows their typical electrophysiological profile. INa and IK were evoked with a standard voltage-clamp protocol consisting in the application of 40-ms voltage steps from a holding potential of −80 mV (Figure 1A, left; see also Bigiani and Cuoghi 2007). In the same cell, bath-application of amiloride (1 µM) induced a clear current response (IAm: Figure 1A, right), indicating the presence of functional ENaCs. By using long-lasting (3 s) depolarizing voltage steps, it was possible to elicit outward currents (hereafter referred to as Ilate) with biophysical properties that differed from IK. Unlike potassium currents, which inactivated with a time constant of about 1–2 s (see also Liu et al. 2005), Ilate did not show any amplitude reduction during 3-s voltage steps (Figure  1B, left). In addition, Ilate showed tail currents at −80 mV (Figure  1B, left, arrow at the end of current records), although their magnitude and time course were quite variable among cells and were also affected by the experimental

Figure 1.  Electrophysiological profile of a salt-detecting taste cell. Whole cell, voltage-clamp recordings from the same cell. (A) Left: Voltage-gated ion currents elicited by applying 40-ms depolarizing voltage steps (in 10-mV increments) to +100 mV from a holding potential of −80 mV. INa: voltage-gated sodium currents; IK: voltage-gated potassium currents. Right: Response to amiloride (IAm) obtained by bath-applying 1 µM amiloride (Am, box over the record) to the cell membrane held at −80 mV. Im: membrane current; IS: stationary inward current. (B) Left: Voltage-gated ion currents elicited by applying long-lasting (3 s) voltage steps. Cell membrane was depolarized to +100 mV in 20-mV increments from a holding potential of −80 mV. With strong depolarizations (larger than +40 mV in this case), slowly developing outward currents appeared in the records (black traces). For their kinetics, these currents were clearly distinguishable from INa and IK (gray traces). Note the typical inactivation of IK (e.g., Liu et al, 2005). Background currents through ENaCs were not blocked in these recordings. Arrow at the end of current indicates tail currents. Right: Current–voltage relationship for the outward currents (Iout) obtained by measuring current amplitude at the end of 3-s voltage steps (black bar on the current traces on the left). Vm: membrane potential.

Chemical Senses, 2017, Vol. 42, No. 4 conditions, as described below. Current–voltage (I–V) relationship showed that in this cell, Ilate was evident only at very positive values of membrane potential (Figure  1B, right). Figure  2A–C shows another salt-sensitive taste cell (Figure 2A) in which application of TEA (to block IK) and amiloride (to block background currents due to open ENaCs) had no evident effect on Ilate (Figure 2B). I–V plot in the presence of TEA and amiloride clearly indicated that threshold for Ilate activation was about 0 mV (Figure 2C), that is, more positive than IK activation threshold (about −30 mV; see also Liu et al. 2005; Bigiani and Cuoghi 2007). Holding potential had no effect on activation threshold and amplitude of Ilate (Figure 2D,E), suggesting that this current did not inactivate even over prolonged time periods. Consistent with this finding, Ilate did not show any time-dependent reduction even during the application of 10-s voltage step (data not shown). Thus, Ilate resembled heterologous connexin or CALHM1 currents, which do not inactivate (e.g., Gómez-Hernández et  al. 2003; Bruzzone et al. 2005; Ma et al. 2012). Cloned pannexins, on the contrary, produce slowly inactivating currents in the same time range (Bruzzone et al. 2005). To compare Ilate among cells, I used adequate experimental conditions to reduce as much as possible the contribution of other ion channels to the outward currents. Specifically, recordings were performed in the presence of 1 µM amiloride to inhibit ASSCs, and by setting the holding potential to −40 mV to inactivate IK (see Materials and methods). Under these controlled conditions, I  found that all taste cells endowed with ASSCs displayed also Ilate. A similar result was also achieved by using Cs+ instead of K+ in the patch pipette solution to block IK. Figure 2F shows the I–V plots for Ilate obtained with different pipette solutions: the curves did not differ significantly (paired t-test, P =  0.3195). Both the response to 1  µM amiloride (Figure 2G) and the amplitude of Ilate evaluated at the end of a voltage step to +80 mV from a holding potential of −40 mV (Figure 2H) were highly variable among patched cells. However, there was no correlation between these 2 electrophysiological parameters (r2 =  0.05776 for linear regression). Neither 10  mM TEA nor 1  µM amiloride exerted a significant effect on isolated Ilate (see pharmacological profile shown in Figure 9). One interesting feature of Ilate was the noise in the current records, which tended to increase with depolarization (see, e.g., current traces in Figure  2D). This noise disappeared upon membrane repolarization (Figure 2D), suggesting that it was likely due to channel activity (e.g., Castro et al. 1999).

Pharmacology of outwardly rectifying currents Pharmacological profiling has been proved useful to distinguish different cloned ATP-releasing channels given the absence of specific inhibitors for some of them (e.g., Dahl et  al. 2013; Siebert et  al. 2013; Patel et al. 2014; Ma et al. 2016). Therefore, I tested whether commonly used blockers for these channels had any effect on Ilate in salt-sensitive taste cells. Gadolinium (Gd3+; 100  µM) completely inhibits heterologous CALHM1 currents (Ma et al. 2012; Siebert et al. 2013) but has no effect on Panx1 currents at 300 µM (Ma et al. 2009). However, Gd3+ blocks also some connexin hemichannels, although not totally (e.g., Cx46 and Cx50: Eskandari et al. 2002). In rat taste cells, 100 µM Gd3+ inhibited Ilate (Figure 3A). The blocking effect was scarcely reversible even after prolonged washout (several minutes; Figure 3A), as demonstrated already in other preparations (e.g., Mlinar and Enyeart 1993; Tokimasa and North 1996). Gd3+ shifted the I–V relationship towards more positive membrane potentials (Figure 3B). Thus, these

347 findings were consistent with the presence of either CALHM1 channels or Gd3+-sensitive connexin hemichannels, although the partial inhibition by Gd3+ suggested the involvement of other channels as well. It is worth noting that in CALHM1-expressing CHO cells, Gd3+ strongly suppresses, although not completely, the outwardly rectifying currents (Dreses-Werringloer et  al. 2008). Nevertheless, it is important to underscore that Gd3+ is a rather nonselective channel blocker (Adding et al. 2001), and therefore its inhibitory effect on Ilate could be also interpreted as the consequence of the blockade of ion channels other than CALHM1 or connexin hemichannels. To establish whether connexin hemichannels could give a contribution to Ilate, I tested the effect of the connexin blocking agent, octanol (Patel et  al. 2014), which does not affect CALHM1 channels (Ma et al. 2012). Figure 4A shows a salt-sensitive cell in which application of 1 mM octanol had no evident effect on Ilate, although 100 µM Gd3+ applied after washout strongly inhibited the currents. On average, the I–V relationship was scarcely affected by octanol (Figure 4B; paired t-test, P = 0.086), ruling out a possible involvement of connexin hemichannels. Unlike Ilate, octanol affected INa (Horishita and Harris 2008) in the same cell shown in Figure 4A confirming its solubilization into the taste cell membrane (Figure 4C). As a whole, these results were consistent with the view that Ilate was in part mediated by CALHM1 channels, whereas connexin hemichannels seemed not to be involved. To test the contribution of pannexin channels to Ilate, I  used CBX, a potent inhibitor of Panx1 at low concentration (Bruzzone et al. 2005; Ma et al. 2009; Romanov et al. 2012), which is ineffective on CALHM1 channels (Ma et al. 2012). I found that CBX did not inhibit Ilate, but unexpectedly potentiated the currents (Figure  5A). This effect was surprising because cloned CALHM1 channels expressed in Xenopus oocytes are insensitive to 200 µM CBX (Ma et al. 2012). The effect of CBX (50/100 µM) was observed in all taste cells tested (n = 25) and was so remarkable that in some of them CBX seemed able even to unmask Ilate (Figure  5B). CBX also affected the activation threshold of Ilate, as shown by I–V plot (Figure  5C). CBX effect was completely reversible upon washout (Figure 5A). Dose–response curves indicated that Ilate was very sensitive to CBX. Figure 6A shows the effect of increasing concentrations of CBX. Note that CBX slowed down significantly current deactivation, as indicated by the appearance of long-lasting tail currents (Figure 6A, arrows). I–V plots showed that activation threshold of Ilate was shifted towards more negative voltage values as CBX concentration was increased (Figure 6B). The current amplitude evaluated at +80 mV plotted against CBX concentration could be fitted nicely to a logistic equation (Figure 6C): on average, CBX exerted its effect with an EC50 of 39.7 µM and a Hill coefficient (nH) of 1.6 (n = 6). Although cloned pannexin channels are very sensitive to blockade by CBX (Bruzzone et al. 2005; Romanov et al. 2012), this drug at the concentration range used in my experiments blocks also connexin hemichannels (Bruzzone et al. 2005). Therefore, I evaluated the effect of probenecid, a specific inhibitor of Panx1 that does not affect connexin (Silverman et  al. 2008) or CALHM1 channels (Siebert et  al. 2013; Taruno, Vingtdeux, et  al. 2013). Probenecid (1  mM) enhanced Ilate as CBX did (Figure 7A). Like CBX, probenecid shifted I–V curve towards more negative values (Figure 7B). The effect of probenecid was completely reversible, and subsequent applications of the drug produced similar effect (Figure 7C). Interestingly, the different potency of probenecid and CBX on Ilate (Figure 7C) matched nicely the pharmacological properties of cloned Panx1 channels expressed in Xenopus oocytes, which are more sensitive to CBX than

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Figure 2.  Outwardly rectifying currents in salt-detecting taste cells. (A) Response to amiloride (IAm) obtained by bath-applying 1 µM amiloride (Am, box over the record) to the cell membrane held at −80 mV. IS: stationary inward current. (B) voltage-gated ion currents elicited by applying long-lasting (3 s) voltage steps and recorded in regular Tyrode’s solution (Control) and during perfusion with 10 mM TEA and 1 µM amiloride (TEA/AM). Cell membrane was depolarized to +80 mV in 20-mV increments from a holding potential of −80 mV. Note the outward currents remaining after blocking background amiloride-sensitive currents and voltage-gated potassium currents (right traces). INa: voltage-gated sodium current; dashed line: zero-current level. Recordings from the same cell shown in (A). (C) Current–voltage relationships for the outward currents (Iout) shown in (B) and obtained by measuring current amplitude at the end of 3-s voltage steps in Tyrode’s solution and during application of TEA and amiloride. Vm: membrane potential. (D) Effect of holding potential (Vh) on the membrane ion currents elicited by applying long-lasting (3 s) voltage steps and recorded in Tyrode’s solution containing 10 mM TEA and 1 µM amiloride. Cell membrane was depolarized to +80 mV in 20-mV increments from the indicated Vh. The outward current remaining after blocking voltage-gated potassium currents and background amiloridesensitive currents is termed Ilate. (E) Current–voltage relationships for the outward currents shown in (D) and obtained by measuring current amplitude at the end of 3-s voltage steps. Vm: membrane potential. (F) Current–voltage (I–V) relationships for outward currents (Ilate) obtained by using either KCl (filled circles) or CsCl (open circles) in the patch pipettes. Cell membrane was depolarized to +80 mV in 20-mV increments from a holding potential (Vh) of −40 mV (see Materials and methods). Bath solution contained 1 µM amiloride to block ENaC background currents. Points represent means ± SEM (n = 15 cells with both pipette solutions). I–V plots were not significantly different (paired t-test, P = 0.3195). (G) Distribution of the amplitude (IAm) of the response to 1 µM amiloride in salt-detecting taste cells analyzed in this study (n = 45) and represented in the form of box and whiskers plot. Dashed line: amplitude threshold used to include taste cells in the analysis. (H) Distribution of the amplitude of outward currents (Ilate) measured at the end of a 3-s voltage step to +80 mV from a holding potential of −40 mV and in the presence of 1 µM amiloride (see Materials and methods) in 32 salt-detecting taste cells. Data were represented in the form of box and whiskers plot.

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Figure  3.  Effect of Gd3+ on Ilate in salt-detecting taste cells. (A) Outwardly rectifying currents (Ilate) elicited by applying long-lasting (3  s) voltage steps. Cell membrane was depolarized from −40 mV to +80 mV in 20-mV increments from a holding potential of −40 mV to inactivate IK and in the presence of 1  µM amiloride to reduce background amiloride-sensitive current (see Materials and methods). In Tyrode’s solution (Control), outward currents were present in the records. Bath-application of Gd3+ (100 µM) reduced significantly these currents. Note that the effect was slightly reversible after 7-min washout (Wash). Arrow: tail currents. (B) Current–voltage relationships for Ilate obtained by measuring current amplitude at the end of 3-s voltage steps in Tyrode’s solution (Control, filled circles) and during application of 100 µM Gd3+ (Gd3+, open circles). Points represent means ± SEM (n = 11 cells). I–V plots differed significantly (paired t-test, P = 0.0005). Vm: membrane potential.

probenecid (IC50 ~ 2–5  µM and ~150  µM, respectively; Bruzzone et al. 2005; Silverman et al. 2008). The enhancement effect of both CBX and probenecid on Ilate obviously raised questions about the mechanism underlying their action. Recent findings on other tissues suggest that pannexins can interact with membrane proteins, including ion channels and receptors (Isakson and Thompson 2014; Wicki-Stordeur and Swayne 2014; Dahl et al. 2016). I therefore put forward the hypothesis that the effect of CBX or probenecid on Ilate could be explained in terms of an inhibitory action by pannexins on the ion channels mediating such currents: by blocking pannexins, inhibition on Ilate channels would be removed and they could open up easily upon depolarization. If this was true, then the obvious question was what molecule could remove this inhibition in physiological conditions, that is, when taste cells work. A good candidate was ATP for this nucleotide inhibits Panx1 (Ma et al. 2009; Qiu and Dahl 2009) and is released inside taste buds during transduction (Kinnamon and Finger 2013; Chaudhari 2014). To test this possibility, I used the ATP analogue, benzoylbenzoyl-ATP (BzATP), which exerts a stronger inhibitory effect on cloned Panx1 as compared to ATP (IC50 ~ 100  µM and ~1 mM, respectively: Qiu and Dahl 2009). BzATP (100 µM) indeed potentiated Ilate as expected. Figure 8 shows an example of the effect of BzATP. This taste cell was endowed with quite large ASSCs, as indicated by the response to amiloride (Figure 8A), and part of the leakage current present in the current records shown in Figure  8B were due to open ENaCs not completely blocked by the dose of amiloride used. It worth noting that in Xenopus oocytes, BzATP blocks Panx1 currents but is without effect on connexin currents (Qiu and Dahl 2009). Like CBX and probenecid, BzATP shifted the I–V plot towards more negative value (Figure 8C). The pharmacological profile of Ilate in salt-detecting taste cells is summarized in Figure 9, where it is compared with the corresponding profiles for cloned ATP-releasing channels. Drug effect was quantified as the percentage change in the amplitude of Ilate elicited at +80 mV from a holding potential of −40 mV (Figure  9, inset). Positive values indicated an increase in current amplitude, whereas negative values a decrease. Data in Figure 9 clearly indicate that Ilate was potentiated (↑) by drugs known to block (↓) pannexin (CBX, probenecid, BzATP).

Although the data were suggestive of an involvement of pannexin in mediating the effect of CBX on Ilate, yet sensitivity to CBX (IC50 ~ 40 µM; Figure 6C) did not match the one observed for CBX block of pannexin expressed in heterologous system (IC50 ~ 2–5 µM; Bruzzone et  al. 2005). A  possible explanation for this discrepancy could be provided by the value of Hill coefficient (nH). For cloned Panx1, nH is about 1, and this led the authors to conclude that “the channel closure is caused by a simple 1: 1 interaction between CBX and Px1 hemichannels, without cooperativity effect” (Bruzzone et al. 2005). The dose–response relationship for the effect of CBX on Ilate in rat fungiform taste cells yielded a nH of about 1.6, which suggested multiple ligand binding and cooperativity (Prinz 2010). In other words, it is possible that a single CAHLM1 channel interacted with more than one pannexin molecule. This protein–protein interaction might affect some properties of Panx1 itself, including the binding properties for CBX (lower sensitivity). To establish whether CBX really unmasked a CAHLM1-mediated current rather than affecting other unknown channels, I tested the effect of Gd3+ in the presence of CBX. Figure 10A shows an example of such an experiment and clearly demonstrates that the CBX-induced outward current was very sensitive to Gd3+. As indicated by the I–V plot, application of Gd3+ completely abolished the current unmasked by CBX (Figure 10B).

Activation and deactivation of outwardly rectifying currents in the presence of CBX In Xenopus oocytes, cloned CALHM1 and connexins generate slowly (seconds) activating outward currents that exhibit large tail currents upon membrane repolarization after the application of depolarizing voltage steps (Bruzzone et al. 2005; Ma et al. 2012). On the contrary, recombinant taste pannexins expressed in several cell lines produce outward currents that activate and deactivate quickly (ms), without exhibiting tail currents (Romanov et al. 2012). In my experiments, I found a quite large variability of Ilate in terms of activation and deactivation kinetics. This variability was not surprising given the electrophysiological heterogeneity that characterizes taste cells and could be due to several factors, including the contribution of channels other than the ATP-releasing ones to the outward currents. Indeed, the partial inhibitory effect

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Figure 4.  Effect of octanol on Ilate in salt-detecting taste cells. (A) Outwardly rectifying currents elicited in a fungiform taste cell by applying long-lasting (3 s) voltage steps. Cell membrane was depolarized from −40 mV to +80 mV in 20-mV increments from a holding potential of −40 mV and in the presence of 1 µM amiloride to reduce background amiloride-sensitive current (see Materials and methods). In Tyrode’s solution (Control), outward currents (Ilate) were present in the records. Bath-application of 1 mM octanol (Octanol) did not affect significantly Ilate. After washout (Wash), 100 µM Gd3+ strongly reduced Ilate (Gd3+). Pipette solution contained Cs+ instead of K+. (B) Current–voltage (I–V) relationships for Ilate obtained by measuring current amplitude at the end of 3-s voltage steps in Tyrode’s solution (Control, filled circles) and during application of 1 mM octanol (Octanol, open circles). Points represent means ± SEM (n = 7 cells). I–V plots did not differ significantly (paired t-test, P = 0.086). Vm: membrane potential. (C) Voltage-gated Na+ currents (INa) recorded from the same taste cell shown in (A) and elicited by applying 40-ms depolarizing voltage steps (in 10-mV increments) to −10 mV from a holding potential of −80 mV. Currents were recorded in Tyrode’s solution (Control, just after the “Control traces” in panel A) and during perfusion with 1 mM octanol (Octanol, just after the “Octanol traces” in panel A). Note that this treatment strongly depressed INa, indicating the presence of octanol in the taste cell membrane. During washing-out, INa recovered in part (Wash, just after the “Wash traces” in panel A).

of Gd3+ (see above) suggested this possible scenario. I  therefore exploited the enhancing effect of CBX on Ilate to get some insights on the activation and deactivation processes due to presumptive ATP-releasing channels in salt-detecting taste cells. I  isolated the CBX-induced currents by subtracting algebraically records obtained in Tyrode’s solution from those obtained in the presence of 50  µM CBX (Figure  11). I  chose this concentration because larger doses seemed to slow down considerably deactivation (e.g., see Figure 6A). In these experiments, holding potential was set to −40 mV to inactivate IK and both the current trace at +60 mV and the corresponding tail current at −40 mV were fitted to a standard exponential function. I found that the activation time constant (τa) at +60 mV was 1.66 ± 0.39 s, and the deactivation time constant (τd) at −40 mV was 0.15  ±  0.04  s (means ± SEM; n  =  9). These values were roughly in good agreement with those obtained for heterologous CALHM1 currents (τa ~ 3 s at +60 mV; τd ~ 0.2 s at −80 mV; Ma et al. 2012) if we consider that for Xenopus oocytes the voltage-clamp protocol consisted in applying 5-s depolarizing steps from a holding potential of −80 mV (Ma et  al. 2012). On the contrary, kinetics of CBX-induced currents was not quite compatible with connexin hemichannels. In transfected human HeLa cells, connexin45 hemichannel currents activate by following the

sum of 2 exponentials (τa1  =  0.42  s; τa2  =  4.3  s), whereas deactivate exponentially with a single time constant that at −40 mV is of 1.5  s (Bader and Weingart 2004). Similar kinetic behavior of hemichannel activation has been described also for other connexins expressed in Xenopus oocytes (Cx46: τa1 = 0.89 s, τa2 = 5.9 s; Cx43•46: τa1 = 0.69 s, τa2 = 4.3 s; Hoang et al. 2010).

Outwardly rectifying currents in “isolated” fungiform taste cells In this study, I patch-recorded taste cells in taste buds isolated from rat fungiform papillae (see Materials and methods). Although this preparation is widely used for single taste cell electrophysiology, caution is required in interpreting the data. Because cells communicate within taste buds either via electrical connections (Bigiani and Roper 1995; Yoshii 2005) and chemical interactions (Kinnamon and Finger 2013; Roper 2013; Chaudhari 2014), recordings could reflect contributions from other cells in the taste bud. To test this possibility, I extracted a few patched cells from the taste bud by pulling them apart with the patch pipette. I  found that also in these conditions cells showed the outwardly rectifying currents: an example of this finding is provided in Figure 12. Thus, Ilate was only due to the activity of the patched cells.

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Figure 5.  Effect of carbenoxolone (CBX) on Ilate in salt-detecting taste cells. (A) Outwardly rectifying currents elicited in a fungiform taste cell by applying longlasting (3 s) voltage steps. Cell membrane was depolarized from −80 mV to +80 mV in 20-mV increments from a holding potential of −40 mV to inactivate IK and in the presence of 1 µM amiloride to reduce background amiloride-sensitive current. In Tyrode’s solution (Control), outward currents were present in the records. Bath-application of carbenoxolone (CBX 50 µM) enhanced significantly these currents. The effect was completely reversible upon washout (Wash). Note the large tail currents (arrow) in the presence of CBX. (B) Sample recordings from another taste cell in Tyrode’s solution (Control) and during bath-application of 100 µM CBX. Cell membrane was held at −40 mV to inactivate IK and step depolarized in 20-mV increments to +80 mV. Amiloride (1 µM) was added to all solutions to reduce background currents through ENaCs. In this cell, Ilate exhibited small amplitude in Tyrode’s solution, whereas it was strongly potentiated during application of CBX. Also, current record seemed to possess 2 components in Tyrode’s solution (Control): a rapid, initial current (*) followed by a slowly activating one (**). (C) Current–voltage (I–V) relationships for Ilate were obtained by measuring current amplitude at the end of 3-s voltage steps in Tyrode’s solution (Control, filled circles) and during application of 100 µM CBX (CBX, open circles). Points represent mean values ± SEM (n = 4 cells). I–V plots differed significantly (paired t-test, P = 0.0002). Vm: membrane potential.

Discussion The present report shows that salt-detecting taste cells of rat fungiform papillae possess outwardly rectifying currents (Ilate) with features that are consistent with those of heterologous currents mediated by cloned ATP-releasing channels. In particular, electrophysiological findings suggest that Ilate can include, at least in part, a CALHM1-like component. Since CALHM1 channels are thought to mediate ATP release in taste cells responding to sweet, bitter, and umami substances (Taruno, Matsumoto, et  al. 2013; Taruno, Vingtdeux, et al. 2013), data suggest that also salt-detecting cells could use the same mechanism (nonvesicular secretion of ATP) for transferring sensory information to nerve endings. In addition, the pharmacological profile of Ilate was suggestive of a possible role for pannexin as regulator of CALHM1 channels in rat taste cells.

Outwardly rectifying currents in rat taste cells When expressed in Xenopus oocytes or mammalian cell lines, cloned pannexin, connexin, and CALHM1 proteins mediate outwardly

rectifying currents with distinct biophysical and pharmacological properties (e.g., Eskandari et  al. 2002; Gómez-Hernández et  al. 2003; Bruzzone et  al. 2005; Qiu and Dahl 2009; Ma et  al. 2009, 2012; Romanov et al. 2012; Siebert et al. 2013). Here, I used these properties as guidelines for identifying the ion channels underlying outwardly rectifying currents (Ilate) recorded in salt-detecting taste cells. As nicely pointed out by Romanov and collaborators (2008), it is important to underscore that the behavior of the currents recorded in native taste cells might be quite different from those studied in heterologous systems, since these cells express several channels as well as multiple isoforms (Huang et al. 2007; Romanov et al. 2007; Moyer et al. 2009) that can form heterooligomers or can interact as channel complex. Indeed, I found that the biophysical properties and the pharmacological profile of Ilate in rat taste cells were quite unique. The more remarkable finding was the enhancement of these currents by CBX, a known blocker of both pannexin channels and connexin hemichannels expressed in Xenopus oocytes (e.g., Bruzzone et al. 2005). Moreover, the current unmasked by the action of CBX showed activation and deactivation kinetics similar to those of heterologous CALHM1 currents (Ma et al. 2012). On one hand, these

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Figure 6.  Sensitivity of Ilate to carbenoxolone (CBX) in salt-detecting taste cells. (A) Outwardly rectifying currents elicited in a fungiform taste cell by applying long-lasting (3 s) voltage steps. Cell membrane was held at −40 mV to inactivate IK and step depolarized in 20-mV increments to +80 mV. Amiloride (1 µM) was added to all solutions to reduce background currents through ENaCs. Bath-application of increasing concentrations of CBX (10 µM: CBX 10; 50 µM: CBX 50; 100 µM: CBX 100; 500 µM: CBX 500) induced an increase in the amplitude of outward currents as compared to currents recorded in Tyrode’s solution (Control). Note the large tail currents (arrows) in the presence of CBX. The potentiating effect by CBX was completely reversible upon washing-out (Wash). (B) Current– voltage relationships for current records shown in (A) and obtained by measuring current amplitude at the end of 3-s voltage steps. Vm: membrane potential. (C) Dose–response curves for the effect of CBX on Ilate. Amplitude of the current elicited by a voltage step to +80 mV was plotted against the logarithm of CBX concentration. Points represent means ± SEM (n = 6 cells). Sigmoidal curve is the best fit of the data obtained with the logistic equation, which yielded the following parameters: EC50 = 39.7 µM; nH = 1.6.

results clearly ruled out a possible involvement of either pannexin channels or connexin hemichannels in generating Ilate. On the other hand, the unexpected effect questioned whether rat taste cells really expressed bona fide CALHM1 channels. Current through CLHM1 channels, the CALMH1 homolog found in Caenorhabditis elegans, is enhanced by CBX (Tanis et  al. 2013). Rat CALHM1 protein shares sequence identity of 90 and 97% with the corresponding human and mouse proteins, respectively. The identity falls to 17% when comparing CALHM1 gene with C. elegans CLHM1 gene. This strong sequence identity suggests that rat CALHM1 channels should share biophysical and pharmacological properties with the human/ mouse counterparts. Yet, human CALHM1 channel is not affected by CBX (Ma et al. 2012). In addition, C. elegans CLHM1 currents are also strongly potentiated by octanol, whereas in rat taste cells this alcohol had a small effect on Ilate in agreement with findings on human cloned channels (Ma et al. 2012). A possible explanation for this strange behavior of outwardly rectifying currents in rat taste cells was that in these native cells, CALHM1 channel functioning and properties were affected by

interaction with other endogenous membrane proteins, a condition that perhaps does not occur when the channel is heterologously expressed in Xenopus oocytes. A good candidate was represented by pannexin 1 (Panx1), which is abundant in taste tissue (Huang et al. 2007) and which is involved in protein–protein interaction in other cell types (Wicki-Stordeur and Swayne 2014). Indeed, the potentiating effect of drugs known to block Panx1 (probenecid, BzATP; Figure 9) was consistent with the idea that in salt-detecting taste cells CALHM1 channels were under the inhibitory control of Panx1. This could explain, for example, the large variability in current amplitude (Figure 2H) observed among taste cells. Indeed, in some cells, CBX (a pannexin blocker) even unmasked the outward currents, suggesting a strong inhibitory action (Figure 5B). Also tail currents, which characterize cloned CALHM1 channels (Ma et  al. 2012), become more evident in the presence of CBX (Figures 5 and 6A). It is tempting to speculate that CALHM1 channels, when interacting with pannexin, might show pharmacological properties that differ from cloned channels, such as the lower sensitivity to Gd3+ (Figure  3B). Interestingly, current unmasked by CBX was completely blocked by

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Figure  7.  Effect of probenecid on Ilate in salt-detecting taste cells. (A) Outwardly rectifying currents elicited by applying long-lasting (3  s) voltage steps. Cell membrane was depolarized to +80 mV in 20-mV increments from a holding potential of −40. Amiloride (1 µM) was added to all solutions to reduce background currents through ENaCs. In Tyrode’s solution (Control), outward currents were present in the records. Bath-application of 1  mM probenecid (Probenecid) enhanced significantly these currents. The effect was completely reversible upon washout (Wash). Pipette solution contained Cs+ instead of K+. (B) Current– voltage (I–V) relationships for Ilate were obtained by measuring current amplitude at the end of 3-s voltage steps in Tyrode’s solution (Control, filled circles) and during application of 1 mM probenecid (Probenecid, open circles). Points represent mean values ± SEM (n = 7 cells). I–V plots differed significantly (paired t-test, P = 0.0066). Vm: membrane potential. (C) Left: Outwardly rectifying currents recorded from another taste cell. Voltage-clamp protocol as in (A). In Tyrode’s solution (Control), small-amplitude outward currents were present in the records. Bath-application of 1 mM probenecid (Probenecid 1) induced an increase in Ilate, which reversed upon washout (Wash 1). Subsequent application of 100  µM CBX (CBX) caused a more pronounced enhancement of the currents, which could be completely reversed during a subsequent washout (Wash 2). A second application of 1 mM probenecid (Probenecid 2) mimicked the effect of its first application. Pipette solution contained K+. Right: Current–voltage relationships for current records shown on the left and obtained by measuring current amplitude at the end of 3-s voltage steps. Vm: membrane potential. C: control; P1: first application of probenecid; W1: first washout; CBX: application of carbenoxolone; W2: second washout; P2: second application of probenecid.

Gd3+ (Figure 10B). Furthermore, the effect of BzATP suggested that in physiological conditions, ATP could be the ligand able to remove the inhibition of pannexin on CALHM1 channels. Cloned taste Panx1 channels expressed in mammalian cell lines are unable to release ATP (Romanov et al. 2012), yet taste tissue expresses high level of this protein (Huang et al. 2007). Thus the present findings suggest a possible role for pannexin as ion channel regulator in rat taste cells. Further studies are clearly required to get detailed information on the biophysical properties of CALHM1 channels in taste cell membrane and on their possible interaction with pannexins. At this regards, it worth mentioning that the possibility that pannexin 1 and/or connexins expressed in taste cells act in complex with CALHM1 has been proposed recently (Taruno, Vingtdeux, et al. 2013).

Although the pharmacological experiments suggested a possible involvement of pannexin as regulatory molecule, yet there was no evidence of pannexin-mediated currents in rat taste cells. This was surprising, since cloned taste Panx1 expressed in mammalian cell lines produces outwardly rectifying currents blocked by 10–50 µM CBX (Romanov et  al. 2012). I  do not know how to explain this discrepancy. It is possible that current through pannexin channels was only a small portion of Ilate (see also Romanov et al. 2007) and could not be detected under my recording conditions. Activation of pannexin currents is quick (milliseconds: Bruzzone et al. 2005; Romanov et al. 2012) as compared to CALHM1 currents (seconds: Ma et al. 2012; Taruno, Vingtdeux, et al. 2013). Thus, in response to depolarizing voltage steps that activate both currents, one might

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Figure 8.  Effect of benzoylbenzoyl-ATP (BzATP) on Ilate in salt-detecting taste cells. (A) Response to amiloride (IAm) obtained by bath-applying 1 µM amiloride (Am, box over the record) to the cell membrane held at about −80 mV. Im: membrane current; IS: stationary inward current. (B) Outwardly rectifying currents elicited by applying long-lasting (3 s) voltage steps to the cell shown in (A). Cell membrane was depolarized to +80 mV in 20-mV increments from a holding potential of −40 mV to inactivate IK and in the presence of 1 µM amiloride to block most of the background amiloride-sensitive current, which was particularly large in this cell (A). In Tyrode’s solution (Control), outward currents were present in the records. Bath-application of 100 µM BzATP (BzATP) enhanced significantly these currents. The effect was reversible (Wash). (C) Current–voltage (I–V) relationships for Ilate were obtained by measuring current amplitude at the end of 3-s voltage steps in Tyrode’s solution (Control, filled circles) and during application of 100 µM benzoylbenzoyl-ATP (BzATP, open circles). Points represent mean values ± SEM (n = 4 cells). I–V plots differed significantly (paired t-test, P = 0.0002). Vm: membrane potential.

Figure 9.  Pharmacological profile of Ilate in salt-detecting taste cells. Cell membrane was held at −40 mV and step depolarized to +80 mV for 3 s. Current was measured at the end of the voltage step after 5-min application of indicated drug. The percent variation was referred to the current amplitude immediately before drug application (inset showing the effect of BzATP as an example). The effect of amiloride was evaluated by substracting the leakage current from outward currents recorded before and during drug application. For all other drugs, test solution contained always 1 µM amiloride in addition to the indicated chemical. Data are shown in the form of box and whiskers plots. Dotted line indicated the zero level for variation. Number of tested cells in parentheses. Amiloride (1 µM), TEA (10 mM), CBX (50 µM), Probenecid (1 mM), Gd3+ (100 µM), octanol (1 mM), BzATP (100 µM). For comparison, the pharmacological profile of cloned ATPreleasing channels is summarized on the right. CALHM1: calcium homeostasis modulator 1 channels; Panx 1: pannexin 1 channels; Cx: connexin hemichannels. ↓: inhibition; ↑: enhancement. Data from: Eskandari et al. (2002); Bruzzone et al. (2005); Ma et al. (2009, 2012); Qiu and Dahl (2009); Romanov et al. (2012); Silverman et al. (2008).

expect to see 2 current components in the records: an initial rapid outward current followed by a slowly activating current. Indeed, in some taste cells, it was possible to observe this situation (see, e.g., Figure 5B, control traces). In any case, without any specific inhibitor for CALHM1 channels (Ma et al. 2016) it is difficult to isolate endogenous pannexin currents in rat taste cells. Alternatively, but this is only speculative, pannexin might not function as a channel, instead just as a “sensor” for the extracellular ATP to allow proper

functioning of CAHLM1 channels. It is worth mentioning that a regulatory role for Panx1 was proposed by Romanov et al. (2012), who hypothesized that “taste cells might utilize a Panx1-dependent regulatory circuit to control activity of ATP-permeable channels of non-Panx1 nature.” Panx1 KO mice release ATP and respond to taste stimuli, including NaCl, like wild-type littermates (Tordoff et  al. 2015; Vandenbeuch, Anderson, et  al. 2015). According to my findings,

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Figure 10.  Sensitivity of carbenoxolone-induced outward current to Gd3+. (A) Outwardly rectifying currents (Ilate) elicited by applying long-lasting (3 s) voltage steps in a salt-sensitive taste cell. Cell membrane was depolarized to +80 mV in 20-mV increments from a holding potential of −40. Amiloride (1 µM) was added to all solutions to reduce background currents through ENaCs. In Tyrode’s solution (Control), outward currents were present in the records. Bath-application of 50 µM carbenoxolone (CBX) enhanced significantly these currents. The addition of 100 µM Gd3+ to the CBX solution (CBX + Gd3+) strongly reduced the outward currents. (B) Current–voltage (I–V) relationships for Ilate were obtained by measuring current amplitude at the end of 3-s voltage steps in Tyrode’s solution (Control, filled circles), during application of 50  µM carbenoxolone (CBX, open circles), and subsequent application of 50  µM carbenoxolone together with 100 µM Gd3+ (CBX + Gd3+, open squares). Points represent mean values ± SEM (n = 7 cells). CBX and CBX+Gd3+ I–V plots did differ significantly (paired t-test, P = 0.0005). Vm: membrane potential.

Figure 11.  Activation and deactivation of Ilate unmasked by carbenoxolone. Outwardly rectifying currents (Ilate) were elicited in a taste cell by applying long-lasting (3 s) voltage steps. Cell membrane was depolarized to +80 mV in 20-mV increments from a holding potential of −40 mV. In this experiment, solutions did not contain amiloride. In Tyrode’s solution (a: Control) outward currents were present in the records superimposed to the background currents. Bath-application of 50 µM carbenoxolone (b: CBX 50 µM), enhanced the outward currents. The algebraic subtraction of control currents from those obtained during perfusion with CBX yielded the currents “unmasked” by this drug (b − a). Note that background currents disappeared. Time constant of activation and deactivation could now be computed from the current records. Dashed line: zero-current level.

this would imply that the regulatory mechanism affecting CALHM1 channels are perfectly functioning even in the absence of Panx1. I do not know how to reconcile these observations with the available data. The large variability of Ilate amplitude among rat taste cells (Figure 2H) suggested that the inhibitory tone on CAHLM1 channels changed significantly from cell to cell. Thus, it is conceivable that some cells could be able to respond to salty stimuli even with Panx1 knocked-out. Rat fungiform taste cells express a Ca2+-dependent Cl− conductance (Wladkowski et  al. 1998) that produces outward currents when cells are patch-recorded with ionic conditions similar to those used in this study. Then an obvious question is whether Ilate could include also some chloride currents mediated by channels other than CALHM1. My findings cannot exclude this possibility, and indeed the partial blocking effect of Gd3+ (Figure  3) suggested that other

channels could be involved as well. Indeed, Gd3+ does not block the current through recombinant Ca2+-activated Cl− channels in HEK293 cells (Bulley et al. 2012). Still, a major component of Ilate had some unique features consistent with the presence of ATP-releasing channels, likely CALHM1 channels. Earlier electrophysiological studies led to identification of outwardly rectifying currents in taste cells from mouse circumvallate papillae (Romanov et al. 2007, 2008; Taruno, Vingtdeux, et al. 2013). However, the pharmacological analysis of these currents produced 2 different interpretations in terms of underlying ATP-releasing channels. I have summarized the main findings of these studies in Table 1, so a direct comparison with the present results from rat taste cells can be made. It is worth noting that studies on mice were performed on Type II cells responding to sweet, bitter, and umami substances, whereas here I investigated rat taste cells assumed to be salt-sensitive

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Figure 12.  Carbenoxolone-sensitive outward currents from an “isolated” fungiform taste cell. Whole cell, voltage-clamp recordings from the same cell. Pipette solution contained Cs+ instead of K+. (A) Response to amiloride (IAm) obtained by bath-applying 1 µM amiloride (Am, box over the record) to the cell membrane held at about −80 mV. Im: membrane current; IS: stationary inward current. (B) Voltage-gated Na+ currents (INa) elicited by applying 40-ms depolarizing voltage steps (in 10-mV increments) to 60 mV from a holding potential of −80 mV. (C) Outwardly rectifying currents elicited by applying long-lasting (3 s) voltage steps and recorded in Tyrode’s solution (Control) and during bath-application of 50 µM carbenoxolone (CBX). Cell membrane was held at −40 mV and step depolarized in 20-mV increments to +80 mV. Amiloride (1 µM) was added to all solutions to reduce background currents through ENaCs.

Table 1.  Biophysical and pharmacological properties of outwardly rectifying currents in mammalian taste cells

Biophysics

Current activation threshold (mV) Current inactivation (depolarizing voltage step duration) Tail currents Pharmacology Gadolinium (Gd3+) Heptanol Octanol Carbenoxolone (CBX) Probenecid Benzoylbenzoyl-ATP (BzATP) Various blockers of anion channels 43 GAP26 Proposed ATP-releasing channels underlying outwardly rectifying currents

Mouse

Rat

Circumvallate papillae, Type II cells, sweet, bitter, and umami

Fungiform papillae, unknown cell type, salty

Romanov et al. (2007, 2008)

Taruno, Vingtdeux, et al. 2013

Present work

≈0 No (100 ms)

≈0 No (1 s)

≈0 No (3 s)

Yes = 

Yes ↓ = 

Yes ↓

↓ =  =  =  ↓ Connexin hemichannels

CALHM1 channels

=  ↑ ↑ ↑

Pannexin–CALHM1 complex

↓: inhibiting effect; ↑: potentiating effect; =: no effect.

on the basis of the occurrence of functional ENaCs (response to amiloride). Work by Kolesnikov’s laboratory (Romanov et  al. 2007, 2008) provided evidence supporting the hypothesis that connexin hemichannels were responsible for the outwardly rectifying currents in mouse taste cells. The strongest evidence was the effect of octanol (3  mM) and of the connexin mimetic peptide 43GAP26 (500  µM), which reduced the currents. It is important to point out, however, that the GAP26 peptide partially inhibits also Panx1 channels (Patel et al. 2014). On the contrary, CBX had no evident effect on the I–V relationship for outward currents. However, CBX was tested at 10 µM (Romanov et al. 2007) and 20 µM (Romanov et al. 2008), which according to the present data from rat taste cells represent the lower concentration limit for eliciting a clear-cut effect (Figure  6). Interestingly, 20  µM CBX affected current deactivation (tail currents) in mouse taste cells (figure  1A in Romanov et  al. 2008) as found in rat taste cells (see, e.g., Figure 5A). Also, Gd3+ did not block

completely the outward current in rat taste cells (see Figure 3), suggesting the possible occurrence of connexin hemichannels, which were apparently not affected by this cation in mouse taste cells (Romanov et al. 2007). The role for CALHM1 channels in mediating the outwardly rectifying currents in mouse taste cells has been proposed recently in a thorough, multidisciplinary study by Taruno, Vingtdeux, et al. (2013). Although the pharmacological analysis was not extensive (Table 1), nevertheless the significant decrease of outward current amplitude in taste cells from CALHM1 KO mice strongly indicated that these channels were responsible for the outward currents. It is important to note that knocking out CALHM1 did not abolish completely the outward current, suggesting that other channels were possibly involved. Outwardly rectifying current in rat taste cells share some similarities with the CALHM1-mediated currents described by Taruno, Vingtdeux, et al. (2013), including sensitivity to Gd3+ but not to connexin blocking agents (heptanol, octanol). However, probenecid (a

Chemical Senses, 2017, Vol. 42, No. 4 pannexin inhibitor) potentiated currents in rat taste cells whereas it was without effect on mouse CALHM1 currents. Unfortunately, CBX was not tested by Taruno, Vingtdeux, et al. (2013). It is difficult to provide a reasonable explanation for the conflicting results from separate laboratories (Table  1). A  possible factor affecting data interpretation might be the overlapping sensitivity to many pharmacological blockers shown by ATP-release channels (Dahl et al. 2013; Patel et al. 2014; Ma et al. 2016), and perhaps also the recording conditions. For example, voltage-clamp protocol to elicit outward currents involved depolarizing steps of different durations: 100 ms (Romanov et al. 2007, 2008), 1 s (Taruno, Vingtdeux, et al. 2013), 3 s (this report). Slowly developing currents were clearly excluded when using short periods of observations.

Transduction pathway in salt-detecting taste cells The present findings provide some clues on the mechanism used by salt-detecting cells to transmit sensory information to nerve endings. An increase in saliva sodium content causes an inward current through apical ENaCs that depolarizes taste cells and triggers action potential firing (Avenet and Lindemann 1991; Ohtubo et al. 2001; Yoshida, Horio, et al. 2009). In turn impulse firing, by depolarizing the membrane to about +40 mV (peak of action potentials: Chen et al. 1996) would open up CAHLM1 channels allowing ATP secretion, as it is the case for taste cells sensitive to sweet, bitter, and umami substances (Taruno, Matsumoto, et  al. 2013; Taruno, Vingtdeux, et al. 2013). This mechanism is consistent with the observation that amiloride does not affect chorda tympani nerve response to salt in mice lacking CALHM1 channels (Tordoff et  al. 2014). Nerve fibers in the chorda tympani collect taste information raised in the fungiform taste buds, that is, those used in this work. In 2-bottle choice tests, CALHM1 KO mice did not prefer NaCl over water for a wide range of salt concentrations (Taruno, Vingtdeux, et al. 2013; Tordoff et al. 2014), further suggesting the involvement of CALHM1 channels in relaying information about salt content to nerve endings. Action potential firing in salt-detecting cells is required in order to reach the membrane depolarization able to open up CALHM1 channels (Taruno, Matsumoto, et al. 2013). An intriguing question then is how the speed of electrical signaling (action potential discharge) can be reconciled with the slow activation kinetics of CALHM1 currents (Figure 11). Presumably, regulation of the channel activity by pannexin (see above) could be a key factor for boosting up CALHM1 currents. An initial release of ATP, by inhibiting pannexin, would remove the blocking action on CALHM1 channels, which can now operate more efficiently during action potential firing. In short, CALHM1 channel would be responsible for ATP release but negative feedback on Panx1 is necessary to boost voltage-sensitivity of CALHM1 channel in order to transform firing pattern into adequate neurotransmitter release. It is interesting to note that salt-detecting taste cells keep on generating spikes during the entire period of salt stimulation (e.g., Avenet and Lindemann 1991; Yoshida, Horio, et al. 2009). Also this electrophysiological behavior may be important for CAHLM1 channel activation, which is strongly time-dependent (see, e.g., control traces in Figure 5). ATP is the main neurotransmitter necessary to stimulate nerve endings (Finger et  al. 2005) but it also plays an intriguing role as autocrine/paracrine messenger within mouse vallate taste buds (Huang et  al. 2009, 2011). In particular, compelling evidence suggests that this nucleotide enhances its own release via purinergic receptors located on taste cell membranes (Huang et  al. 2011). It is not known whether this feedback mechanism operates also in salt-detecting cells in rat fungiform taste buds. Nonetheless, my data are consistent with the view that cell-to-cell communication in

357 taste buds requires a boosting step, either mediated by activation of purinergic receptors (Huang et al. 2009, 2011) or by inhibition of pannexin proteins. Given the presence of regulatory mechanisms for ATP release, it is tempting to speculate that detection threshold for taste stimuli depends not only on the operation of apical taste receptors (such as the sodium receptor, ENaC), but also by the activity level of the ATP-releasing channel in the basolateral membrane. Salt-detecting taste cells, by expressing large INa and CALHM1like currents, resemble mouse Type II cells sensitive to sweet, bitter, and umami compounds (Moyer et al. 2009; Taruno, Vingtdeux, et al. 2013). On the other hand, previous observations suggest that functional ENaC occur in presumptive glial-like cells (Type I cells), which do not possess INa (Vandenbeuch et al. 2008). How to explain then these conflicting results? Of course, one possibility is that the chain of molecular events underlying taste detection and signaling for sodium ions may differ, in part or completely, between mice and rats, as it is the case for other transduction pathways (e.g., Lin et al. 2002; Ugawa et al. 2003; Richter et al. 2004; Huang et al. 2006; Ma et al. 2007). Yet, one aspect of the biology of taste cells that needs to be taken into account is their continuous turnover. Immature cells may display membrane properties that can be erroneously assigned to a given subtype of mature cells (e.g., Mackay-Sim et al. 1996; see also Bigiani 2016). Indeed, functional ENaCs are found also in developing cells (e.g., Doolin and Gilbertson 1996; Kossel et al. 1997). In their work, Vandenbeuch and collaborators (2008) identified gliallike (Type I) cells by exclusion, namely they considered cells lacking specific marker for Type II or Type III cells as Type I cells. However, in doing so they might have included in the analysis developing cells. In my study, I used a functional marker for cell maturity, namely the occurrence of large INa (e.g., Doolin and Gilbertson 1996; Kimura et al. 2014), and therefore I could exclude other types of cells. Despite the simplicity of the taste stimulus (Na+, a monovalent cation), sodium reception is a rather complex sensory process involving both amiloride-sensitive (i.e., mediated by ENaC) and amiloride-insensitive components (Halpern 1998; McCaughey and Scott 1998; Roper 2015). The contribution of these different pathways may change greatly among species (Halpern 1998). Thus, my findings need to be read in this framework and support the view that the ENaC-mediated component of sodium reception, or at least part of it, may depend on the activity of 2 proteins co-expressed in the same taste cell: the sodium receptor ENaC and the ATP-releasing CAHLM1 channel. Clearly, other transduction mechanisms may involve different molecular elements, as it is the case for the amiloride-insensitive sodium response (Lewandowski et al. 2016).

Limitations and future directions The goal of this work was to establish whether salt-sensitive taste cells exhibited membrane currents with properties compatible with those of ATP-releasing channels (pannexins, connexins, CALHM1) expressed in heterologous systems. To this aim, I used rat fungiform taste buds as an experimental model because salt-sensitive cells could be easily identified by monitoring the response to amiloride. However, it is important to underscore that all the available background information on ATP-release mechanism in taste cells is based on mice (for review, see Kinnamon and Finger 2013; Roper 2013; Chaudhari 2014). Besides a master thesis showing the occurrence of connexin immunoreactivity in rat circumvallate taste buds (Bond 2012), it is not known if rat taste cells express CALHM1 and/or panx1, although the corresponding genes occur in rat genome. Therefore, the electrophysiological data presented in this paper will need to be supported by molecular evidence regarding the expression of channel mRNA

358 and/or protein before making any definitive conclusion on the cellular events occurring in salt-detecting cells of the rat fungiform papillae.

Funding This work was supported in part by Università di Modena e Reggio Emilia (FAR 2015).

Acknowledgements I am grateful to Mr Fausto Vaccari and Mr Claudio Frigeri for their excellent technical assistance, to Dr Roberto Tirindelli for evaluating the sequence identity between CALHM1 and CLHM1 proteins, and to Dr Rita Bardoni for helpful discussion on pannexin biophysics and pharmacology.

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