Bernd Lindemann

Receptors and transduction in taste.

Taste is the sensory system devoted primarily to a quality check of food to be ingested. Although aided by smell and visual inspection, the final recognition and selection relies on chemoreceptive events in the mouth. Emotional states of acute pleasure or displeasure guide the selection and contribute much to our quality of life. Membrane proteins that serve as receptors for the transduction of taste have for a long time remained elusive. But screening the mass of genome sequence data that have recently become available has provided a new means to identify key receptors for bitter and sweet taste. Molecular biology has also identified receptors for salty, sour and umami taste.

Hyperpolarization-activated channels HCN1 and HCN4 mediate responses to sour stimuli

Sour taste is initiated by protons acting at receptor proteins or channels. In vertebrates, transduction of this taste quality involves several parallel pathways. Here we examine the effects of sour stimuli on taste cells in slices of vallate papilla from rat. From a subset of cells, we identified a hyperpolarization-activated current that was enhanced by sour stimulation at the taste pore. This current resembled Ih found in neurons and cardio-myocytes, a current carried by members of the family of hyperpolarization-activated and cyclic-nucleotide-gated (HCN) channels. We show by in situ hybridization and immunohistochemistry that HCN1 and HCN4 are expressed in a subset of taste cells. By contrast, gustducin, the G-protein involved in bitter and sweet taste, is not expressed in these cells. Lowering extracellular pH causes a dose-dependent flattening of the activation curve of HCN channels and a shift in the voltage of half-maximal activation to more positive voltages. Our results indicate that HCN channels are gated by extracellular protons and may act as receptors for sour taste.

Amiloride-blockable sodium currents in isolated taste receptor cells

SummaryIsolated taste receptor cells from the frog tongue were investigated under whole-cell patch-clamp conditions. With the cytosolic potential head at −80 mV, more than 50% of the cells had a stationary inward Na current of 10 to 700 pA in Ringers solution. This current was in some cells partially, in others completely, blockable by low concentrations of amiloride. With 110mm Na in the external and 10mm Na in the internal solution, the inhibition constant of amiloride was (at −80 mV) near 0.3 μm. In some cells the amiloride-sensitive conductance was Na specific; in others it passed both Na and K. The Na/K selectivity (estimated from reversal potentials) varied between 1 and 100. The blockability bysmall concentrations of amiloride resembled that of channels found in some Na-absorbing epithelia, but the channels of taste cells showed a surprisingly large range of ionic specificities. Receptor cells, whichin situ express these channels in their apical membrane, may be competent to detect the taste quality “salty.” The same cells also express TTX-blockable voltage-gated Na channels.

Patch-clamp study of isolated taste receptor cells of the frog

SummaryTaste discs were dissected from the tongue ofR. ridibunda and their cells dissociated by a collagenase/low Ca/mechanical agitation protocol. The resulting cell suspension contained globular epithelial cells and, in smaller number, taste receptor cells. These were identified by staining properties and by their preserved apical process, the tip of which often remained attached to an epithelial (associated) cell. When the patch pipette contained 110mm KCl and the cells were superfused with NaCl Ringers during whole-cell recording, the mean zero-current potential of 22 taste receptor cells was −65.2 mV and the slope resistance 150 to 750 MΩ. Pulse-depolarization from a holding voltage of −80 mV activated a transient TTX-blockable inward Na current. Activation became noticeable at −25 mV and was half-maximal at −8 mV. Steady-state inactivation was half-maximal at −67 mV and complete at −50 mV. Peak Na current averaged −0.5 nA/cell. The Ca-ionophore A23187 shifted the activation and inactivation curve to more negative voltages. Similar shifts occurred when the pipette Ca was raised. External Ni (5mm) shifted the activation curve towards positive voltages by 10 mV. Pulse depolarization also activated outward K currents. Activation was slower than that of Na current and inactivation slower still. External TEA (7.5mm) and 4-aminopyridine (1mm) did not block, but 5mm Ba blocked the K currents. K-tail currents were seen on termination of depolarizing voltage pulses. A23187 shifted theIK(V)-curve to more negative voltages. Action potentials were recorded when passing pulses of depolarizing outward current. Of the frog gustatory stimulants, 10mm Ca caused a reversible 5-to 10-mV depolarization in the current-clamp mode. Quinine (0.1mm, bitter) produced a reversible depolarization accompanied by a full block of Na current and, with slower time-course, a partial block of K currents. Cyclic AMP (5mm in the external solution or 0.5 μm in the pipette) caused reversible depolarization (to −40 to −20 mV) due to partial blockage of K currents, but only if ATP was added to the pipette solution. Similar responses were elicited by stimulating the adenylate cyclase with forskolin. Blockage of cAMP-phosphodiesterase enhanced the response to cAMP. These results suggest that cAMP may be one of the cytosolic messengers in taste receptor cells. Replacement of ATP by AMP-PNP in the pipette abolished the depolarizing response to cAMP. Inclusion of ATP-γ-S in the pipette caused slow depolarization to −40 to −20 mV, due to partial blockage of K currents. Subsequently, cAMP was without effect. The remaining K currents were blockable by Ba. These results suggest that cAMP initiates phosphorylation of one set of K channels to a nonconducting conformation.

The role of sodium-channel density in the natriferic response of the toad urinary bladder to an antidiuretic hormone.

SummaryUrinary bladders ofBufo marinus were depolarized, by raising the serosal K concentration, to facilitate voltage-clamping of the apical membrane. Passive Na transport across the apical membrane was then studied with near-instantaneous current-voltage curves obtained before and after eliciting a natriferic response with oxytocin. Fitting with the constant-field equation showed that the natriferic effect is accounted for by an increase in the apical Na permeability. It is accompanied by a small increase in cellular Na activity. Furthermore, fluctuation analysis of the amiloride-induced shot-noise component of the short-circuit current indicated that the permeability increase is not due to increased Na translocation through those Na channels which were already conducting prior to hormonal stimulation. Rather, the natriferic effects is found to be based on an increase in the population of transporting channels. It appears that, in response to the hormone, Na channels are rapidly “recruited” from a pool of electrically silent channels.

Noninvasive recording of receptor cell action potentials and sustained currents from single taste buds maintained in the tongue: the response to mucosal NaCl and amiloride.

SummaryApical membrane currents were recorded from the taste pore of single taste buds maintained in the tongue of the rat, using a novel approach. Under a dissection microscope, the 150-μm opening of a saline-filled glass pipette was positioned onto single fungiform papillae, while the mucosal surface outside the pipette was kept dry. Electrical responses of receptor cells to chemical stimuli, delivered from the pipette, were recorded through the pipette while the cells remained undamaged in their natural environment. We observed monophasic transient currents of 10-msec duration and 10–100 pA amplitude, apparently driven by action potentials arising spontaneously in the receptor cells. When perfusing the pipette with a solution of increased Na but unchanged Cl concentration, a stationary inward current (from pipette to taste cell) of 50–900 pA developed and the collective spike rate of the receptor cells increased. At a mucosal Na concentration of 250mm, the maximal collective spike rate of a bud was in the range of 6–10 sec−1. In a phasic/tonic response, the high initial rate was followed by an adaptive decrease to 0.5–2 sec−1. Buds of pure phasic response were also observed. Amiloride (30 μm) present in the pipette solution reversibly and completely blocked the increase in spike rate induced by mucosal Na. Amiloride also decreased reversibly the stationary current which depended on the presence of mucosal Na (inhibition constant near 1 μm). During washout of amiloride, spike amplitudes were first small, then increased, but always remained smaller than the amiloride-blockable stationary current of the bud. This is understandable since the stationary current of a bud arises from a multitude of taste cells, while each current spike is presumably generated by just one taste cell. We suggest that, in a Na-sensitive receptor cell, (i) the apical amiloride-blockable Na inward current serves as a generator current causing cell depolarization and firing of action potentials, and (ii) each current spike recorded from the taste pore arises mainly from a modulation of the apical Na inward current of this cell, because the action potential generated by the taste cell will transiently decrease or abolish the driving force for the apical Na inward current. The transients are indicators of receptor cell action potentials, which appear to be physiological responses of taste cellsin situ.

Current-voltage analysis of apical sodium transport in toad urinary bladder: Effects of inhibitors of transport and metabolism

SummaryThe basal-lateral surface of the epithelium of the urinary bladder of the toad (Bufo marinus) was depolarized by exposure of the serosal surface to 85mm KCL and 50mm sucrose. The extent of depolarization appeared to be virtually complete, as evaluated by the invariance in the transepithelial electrical potential difference and conductance on addition of nystatin (a monovalent cation ionophore) to the serosal medium. The Na-specific current (INa) was defined as the current sensitive to the removal of Na from the mucosal medium or inhibitable by addition of amiloride to this medium. In the presence of the high K-sucrose serosal medium, rapid, serial, stepwise clamping of the transepithelial voltage (V) yielded a curvilinear dependence ofINa onV; which is taken to represent theI–V curve of the apical Na channels. The constant field equation (Goldman, D.E. 1943;J. Gen. Physiol.27:37) fits theI–V data points closely, allowing estimates to be made of the permeability to Na of the apical membrane (PNa) and of the intracellular Na activity (Nac). Exposure of the apical surface to amiloride (5×10−7m) decreasedPNa in proportion to the decrease inINa (i.e., ∼70%) but decreased Nac only 25%. In contrast, an equivalent lent reduction inINa elicited by exposure of the basallateral surface to ouabain was accompanied by only a 20% decrease inPNa and a sixfold increase in Nac. The effects of amiloride onPNa and ouabain on Nac are consistent with the primary pharmacological actions of these drugs. In addition,PNa appears to be under metabolic control, in that 2-deoxyglucose, a specific inhibitor of glycolysis, decreasedINa andPNa proportionately, and lowered Nac marginally, effects indistinguishable from those obtained with amiloride.

Single chloride channels in colon mucosa and isolated colonic enterocytes of the rat

SummaryChloride channels from rat colonic enterocytes were studied using the patch-clamp technique. After removal of mucus, inside-out patches were excised from the apical membrane of intact epithelium located at the luminal surface. They contained spontaneously switching Cl− channels with a conductance of 35–40 pS. The channels were blocked reversibly by anthracene-9-carboxylic acid (1mm).In excised patches from single enterocytes, isolated by calcium removal, the Cl− channels were studied in more detail. TheI–V relation was linear between ±80 mV. The selectivity was I−>Br−>Cl−=NO3−>F−=HCO3−.Thirty pS Cl− channels were also found on the basolateral membrane of crypts isolated by brief calcium removal. TheI–V curve of these Cl− channels was also linear.The results provide direct evidence for the existence of Cl− channels in the apical membrane of surface cells in colonic mucosa. The properties of these channels are similar to those previously observed when incorporating membrane vesicles into planar lipid bilayers. Both results support the validity of the theoretical models describing intestinal secretion.

Odorant response of isolated olfactory receptor cells is blocked by amiloride.

SummaryOlfactory receptor cells were isolated from the nasal mucosa ofRana esculenta and patch clamped. Best results were obtained with free-floating cells showing ciliary movement. 1)On-cell mode: Current records were obtained for up to 50 min. Under control conditions they showed only occasional action potentials. The odorants cineole, amyl acetate and isobutyl methoxypyrazine were applied in saline by prolonged superfusion. At 500 nanomolar they elicited periodic bursts of current transients arising from cellular action potentials. The response was rapidly, fully and reversibly blocked by 50 μm amiloride added to the odorant solution. With 10 μm amiloride, the response to odorants was only partially abolished. 2)Whole-cell mode: Following breakage of the patch, the odorant response was lost within 5 to 15 min. Prior to this, odorants evoked a series of slow transient depolarizations (0.1/sec, 45 mV peak to peak) which reached threshold and thus elicited the periodic discharge of action potentials. These slow depolarizing waves were reversibly blocked by amiloride, which stabilized the membrane voltage between −80 and −90 mV. We conclude that amiloride inhibits chemosensory transduction of olfactory receptor cells, probably by blocking inward current pathways which open in response to odorants.

Chemoreception: Tasting the sweet and the bitter

Our understanding of the molecular basis of taste transduction has lagged behind that of other senses, but now a signalling protein-the G protein alpha subunit gustducin-has been shown to be taste-tissue specific and essential for both bitter and sweet tastes.