A novel voltage clamp technique for mapping ionic currents from cultured skeletal myotubes.

The biophysical properties and cellular distribution of ion channels largely determine the input/output relationships of electrically excitable cells. A variety of patch pipette voltage clamp techniques are available to characterize ionic currents. However, when used by themselves, such techniques are not well suited to the task of mapping low-density channel distributions. We describe here a new voltage clamp method (the whole cell loose patch (WCLP) method) that combines whole-cell recording through a tight-seal pipette with focal extracellular stimulation through a loose-seal pipette. By moving the stimulation pipette across the cell surface and using a stationary whole-cell pipette to record the evoked patch currents, this method should be suitable for mapping channel distributions, even on large cells possessing low channel densities. When we applied this method to the study of currents in cultured chick myotubes, we found that the cell cable properties and the series resistance of the recording pipette caused significant filtering of the membrane currents, and that the filter characteristics depended in part upon the distance between the stimulating and recording pipettes. We describe here how we determined the filter impulse response for each loose-seal pipette placement and subsequently recovered accurate estimates of patch membrane current through deconvolution.     

Odorant-induced currents in intact patches from rat olfactory receptor neurons: theory and experiment.

Odorant-induced currents in mammalian olfactory receptor neurons have proved difficult to obtain reliably using conventional whole-cell recording. By using a mathematical model of the electrical circuit of the patch and rest-of-cell, we demonstrate how cell-attached patch measurements can be used to quantitatively analyze responses to odorants or a high (100 mM) K+ solution. High K+ induced an immediate current flux from cell to pipette, which was modeled as a depolarization of approximately 52 mV, close to that expected from the Nernst equation (56 mV), and no change in the patch conductance. By contrast, a cocktail of cAMP-stimulating odorants induced a current flux from pipette into cell following a significant (4-10 s) delay. This was modeled as an average patch conductance increase of 36 pS and a depolarization of 13 mV. Odorant-induced single channels had a conductance of 16 pS. In cells bathed with no Mg2+ and 0.25 mM Ca2+, odorants induced a current flow from cell to pipette, which was modeled as a patch conductance increase of approximately 115 pS and depolarization of approximately 32 mV. All these results are consistent with cAMP-gated cation channels dominating the odorant response. This approach, which provides useful estimates of odorant-induced voltage and conductance changes, is applicable to similar measurements in any small cells.     

Leukotriene C4 modulation of muscarinic K+ current activation in bullfrog atrial myocytes

The effects of leukotriene C4 (LTC4) on activation of muscarinic acetylcholine receptor (mAChR)-stimulated, inwardly rectifying K+ current (IK[ACh]) were examined in single bullfrog atrial myocytes using the whole-cell patch clamp technique. LTC4 produced a reversible, concentration-dependent increase in steady-state, guanosine-gamma- thiotriphosphate (GTP gamma S)-activated IK[ACh], with a K0.5 of 3.1 microM. LTC4 also increased the rate of GTP gamma S-mediated IK[ACh] activation, both in the absence and presence of 1 nM ACh, with comparable K0.5 values of 4.7 microM under basal conditions and 4.9 microM in the presence of 1 nM ACh. LTC4 did not alter the relative affinities of the G protein, Gk, for GTP gamma S and GTP. We hypothesize that all of the effects of LTC4 on the kinetics of Gk- mediated IK[ACh] activation are produced at a common site with a K0.5 of 3-5 microM. The effects of LTC4 on IK[ACh] activation are fully reversible in the presence of GTP gamma S. Under physiological conditions (i.e., intracellular GTP), 10 microM LTC4 increased the ACh- activated peak IK[ACh]. Inhibitors of cellular LTC4 production, including 5,8,11,14-eicosatetraynoic acid, baicalein, cinnamyl-3,4- dihydroxy-alpha-cyanocinnamate, and alpha-pentyl-4-(2- quinolinylmethoxy)-benzene methanol, greatly attenuated ACh-dependent IK[ACh] activation, preventing activation of peak, and producing a lower steady-state IK[ACh] (when compared with the control response in the same cell). Addition of exogenous LTC4 was able to overcome the effects of LTC4 synthesis inhibitors, restoring both the peak and steady-state IK[ACh] responses. Although the mechanism of LTC4-mediated modulation of IK[ACh] activation is not known, our results suggest that endogenously produced lipoxygenase metabolites of arachidonic acid, specifically LTC4, are involved in the physiological process of IK[ACh] activation.     

Rundown of GH3 cell K+ conductance response to TRH following patch recording can be obviated with GH3 cell extract

GH3/B6 pituitary cells release prolactin (PRL) in response to thyrotropin releasing hormone (TRH). Electrophysiological assays of individual GH3 cells with sharp high-resistance microelectrodes have revealed complex effects of TRH on membrane excitability consisting of a transient hyperpolarization (1), which is thought to result from activation of Ca-dependent K+ conductance (2), followed by a prolonged phase of spontaneous, Ca-dependent action potential activity (3). Using the whole-cell patch recording (WCR) technique (4), we have found that these TRH actions on GH3 excitability rapidly rundown following patch recording. When the supernatant from osmotically lysed GH3 cells was added to the WCR patch pipette, the K+ conductance response was not only promoted but well-maintained. The results indicate that diffusible factors mediate these TRH actions and further, that the WCR technique should be useful in identifying different second messengers and elucidating their roles in membrane excitability and PRL secretion.     

Currents recorded through small areas of squid axon membrane with an internal virtual ground voltage clamp.

A new voltage-clamp apparatus for the squid axon has been implemented to enable recording of currents through small areas of axon membrane. The performance of this clamp was tested by recording total sodium currents from perfused axons (I total) and sodium currents from small membrane patches (I patch), which were recorded from inside the axon with an L-shaped pipette. The I patch records, although four orders of magnitude smaller than I total, were stable and showed normal kinetics and voltage dependence, and appeared to reflect the activation of a small population of normal sodium channels. The size of the current recorded from the patch was mainly a function of the tip diameter of the L-shaped pipette and of the shunt resistance between inside the pipette and the axoplasm.     

Apparent absence of a redox requirement for blue light activation of pump current in broad bean guard cells

In guard cells, membrane hyperpolarization in response to a blue light (BL) stimulus is achieved by the activation of a plasma membrane H(+)-ATPase. Using the patch clamp technique on broad bean (Vicia faba) guard cells we demonstrate that both steady-state- and BL-induced pump currents require ATP and are blocked by vanadate perfused into the guard cell during patch clamp recording. Background-pump current and BL-activated currents are voltage independent over a wide range of membrane potentials. During BL-activated responses significant hyperpolarization is achieved that is sufficient to promote K(+) uptake. BL activation of pump current becomes desensitized by three or four pulses of 30 s x 100 micromol m(-2) s(-1) BL. This desensitization is not a result of pump inhibition as maximal responses to fusicoccin are observed after full BL desensitization. BL treatments prior to whole cell recording show that BL desensitization is not due to washout of a secondary messenger by whole cell perfusion, but appears to be an important feature of the BL-stimulated pump response. We found no evidence for an electrogenic BL-stimulated redox chain in the plasma membrane of guard cells as no steady-state- or BL-activated currents are detected with NADH or NADPH added to the cytosol in the absence of ATP. Steady-state- nor BL-activated currents are affected by the inclusion along with ATP of 1 mM NADH in the pipette under saturating red light or by including NADPH in the pipette under darkness or saturating red light. These data suggest that reduced products of photosynthesis do not significantly modulate plasma membrane pump currents and are unlikely to be critical regulators in BL-stimulation of the plasma membrane H(+)-ATPase in guard cells.     

Arachidonic acid metabolites alter G protein-mediated signal transduction in heart. Effects on muscarinic K+ channels

The muscarinic acetylcholine receptor (mAChR)-stimulated, inwardly rectifying K+ current (IK [ACh]) was examined in single bullfrog atrial cells using the whole-cell patch clamp technique. IK[ACh] was activated either by bath addition of 1 microM ACh or via activation of the G protein, Gk, with guanosine-gamma-thiotriphosphate (GTP gamma S). Arachidonic acid (AA) modulated IK[ACh] under both conditions. AA decreased mAChR-stimulated IK[ACh] and increased the rate of decay from the peak current (desensitization). In addition, AA affected GTP gamma S-activated IK[ACh] by modulation of Gk. The effects of AA and its metabolites on Gk were assessed by examining their effects on both the basal rate of Gk activation by GTP gamma S, and the mAChR-mediated increase in activation rate produced by nanomolar ACh. AA increased the basal rate of GTP gamma S-mediated IK[ACh] activation, but reduced the ACh-induced augmentation of this rate. All of the effects of AA on GTP gamma S-mediated IK[ACh] activation were produced by metabolites. A lipoxygenase inhibitor, nordihydroguaiaretic acid (NDGA), decreased the basal and ACh-enhanced rate of IK[ACh] activation in both the presence and absence of exogenous AA. In contrast, indomethacin (INDO), a cyclooxygenase inhibitor, increased the basal rate of IK[ACh] activation by GTP gamma S in both the presence and absence of exogenous AA, and reversed the effects of AA on the ACh-augmented basal rate. AA metabolites produced via lipoxygenase and cyclooxygenase pathways thus have opposing effects on the signal transduction pathway from mAChR to IK[ACh]. We directly tested a lipoxygenase pathway metabolite, LTC4, on GTP gamma S-mediated IK[ACh] activation and found that it not only overcame the inhibitory effects of NDGA, but also increased both the basal and ACh-augmented rate of IK[ACh] activation. From these data, we propose that AA metabolites modulate the function of Gk by altering its kinetic properties.     

Properties of calcium and potassium currents of clonal adrenocortical cells

The ionic currents of clonal Y-1 adrenocortical cells were studied using the whole-cell variant of the patch-clamp technique. These cells had two major current components: a large outward current carried by K ions, and a small inward Ca current. The Ca current depended on the activity of two populations of Ca channels, slow (SD) and fast (FD) deactivating, that could be separated by their different closing time constants (at -80 mV, SD, 3.8 ms, and FD, 0.13 ms). These two kinds of channels also differed in (a) activation threshold (SD, approximately -50 mV; FD, approximately -20 mV), (b) half-maximal activation (SD, between -15 and -10 mV; FD between +10 and +15 mV), and (c) inactivation time course (SD, fast; FD, slow). The total amplitude of the Ca current and the proportion of SD and FD channels varied from cell to cell. The amplitude of the K current was strongly dependent on the internal [Ca2+] and was almost abolished when internal [Ca2+] was less than 0.001 microM. The K current appeared to be independent, or only slightly dependent, of Ca influx. With an internal [Ca2+] of 0.1 microM, the activation threshold was -20 mV, and at +40 mV the half-time of activation was 9 ms. With 73 mM external K the closing time constant at -70 mV was approximately 3 ms. The outward current was also modulated by internal pH and Mg. At a constant pCa gamma a decrease of pH reduced the current amplitude, whereas the activation kinetics were not much altered. Removal of internal Mg produced a drastic decrease in the amplitude of the Ca-activated K current. It was also found that with internal [Ca2+] over 0.1 microM the K current underwent a time-dependent transformation characterized by a large increase in amplitude and in activation kinetics.     

Ion permeation through light-activated channels in rhabdomeric photoreceptors. Role of divalent cations

The receptor potential of rhabdomeric photoreceptors is mediated primarily by a Na influx, but other ions must also permeate through light-dependent channels to account for some properties of the photoresponse. We examined ion conduction in macroscopic and single- channel light-induced currents of Lima and Pecten photoreceptors. In the absence of Na, a fivefold change in extracellular K shifted the reversal voltage of the photocurrent (Vrev) by approximately 27 mV. Because the dependency of Vrev on [K]o was sub-Nernstian, and Vrev in each condition was more positive than Ek, some other ion(s) with a positive equilibrium potential must be implicated, in addition to K. We assessed the participation of calcium, an important candidate because of its involvement in light adaptation. Three strategies were adopted to minimize the impairments to cytosolic Ca homeostasis and loss of responsiveness that normally result from the required ionic manipulations: (a) Internal dialysis with Na-free solutions, to prevent reverse operation of the Na/Ca exchanger. (b) Rapid solution changes, temporally limiting exposure to potentially detrimental ionic conditions. (c) Single-channel recording, exposing only the cell- attached patch of membrane to the test solutions. An inward whole-cell photocurrent could be measured with Ca as the only extracellular charge carrier. Decreasing the [Ca]o to 0.5 mM reduced the response by 43% and displaced the reversal potential by -4.3 mV; the shift was larger (delta Vrev = -44 mV) when intracellular permeant cations were also removed. In all cases, however, the current carried by Ca was < 5% of that measured with normal [Na]o. Unitary light-activated currents were reduced in a similar way when the pipette contained only divalent cations, indicating a substantial selectivity for Na over Ca. The fall kinetics of the photoresponse was slower when external Ca was replaced by Ba, or when the membrane was depolarized; however, dialysis with 10 mM BAPTA failed to antagonize this effect, suggesting that mechanisms other than the Ca influx participate in the modulation of the time course of the photocurrent.     

A comparison of calcium-activated potassium channel currents in cell-attached and excised patches

Single channel currents from Ca-activated K channels were recorded from cell-attached patches, which were then excised from 1321N1 human astrocytoma cells. Cells were depolarized with K (110 mM) so that the membrane potential was known in both patch configurations, and the Ca ionophore A23187 or ionomycin (20-100 microM) was used to equilibrate intracellular and extracellular [Ca] (0.3 or 1 microM). Measurements of intracellular [Ca] with the fluorescent Ca indicator quin2 verified that [Ca] equilibration apparently occurred in our experiments. Under these conditions, where both membrane potential and intracellular [Ca] were known, we found that the dependence of the channel percent open time on membrane potential and [Ca] was similar in both the cell-attached and excised patch configuration for several minutes after excision. Current-voltage relations were also similar, and autocorrelation functions constructed from the single channel currents revealed no obvious change in channel gating upon patch excision. These findings suggest that the results of studies that use excised membrane patches can be extrapolated to the K-depolarized cell-attached configuration, and that the relation between [Ca] and channel activity can be used to obtain a quantitative measure of [Ca] near the membrane intracellular surface.     

[Na] and [K] dependence of the Na/K pump current-voltage relationship in guinea pig ventricular myocytes

Na/K pump current was determined between -140 and +60 mV as steady-state, strophanthidin-sensitive, whole-cell current in guinea pig ventricular myocytes, voltage-clamped and internally dialyzed via wide-tipped pipettes. Solutions were designed to minimize all other components of membrane current. A device for exchanging the solution inside the pipette permitted investigation of Na/K pump current-voltage (I-V) relationships at several levels of pipette [Na] [( Na]pip) in a single cell; the effects of changes in external [Na] [( Na]o) or external [K] [( K]o) were also studied. At 50 mM [Na]pip, 5.4 mM [K]o, and approximately 150 mM [Na]o, Na/K pump current was steeply voltage dependent at negative potentials but was approximately constant at positive potentials. Under those conditions, reduction of [Na]o enhanced pump current at negative potentials but had little effect at positive potentials: at zero [Na]o, pump current was only weakly voltage dependent. At 5.4 mM [K]o and approximately 150 mM [Na]o, reduction of [Na]pip from 50 mM scaled down the sigmoid pump I-V relationship and shifted it slightly to the right (toward more positive potentials). Pump current at 0 mV was activated by [Na]pip according to the Hill equation with best-fit K0.5 approximately equal to 11 mM and Hill coefficient nH approximately equal to 1.4. At zero [Na]o, reduction of [Na]pip seemed to simply scale down the relatively flat pump I-V relationship: Hill fit parameters for pump activation by [Na]pip at 0 mV were K0.5 approximately equal to 10 mM, nH approximately equal to 1.4. At 50 mM [Na]pip and high [Na]o, reduction of [K]o from 5.4 mM scaled down the sigmoid I-V relationship and shifted it slightly to the right: at 0 mV, K0.5 approximately equal to 1.5 mM and nH approximately equal to 1.0. At zero [Na]o, lowering [K]o simply scaled down the flat pump I-V relationships yielding, at 0 mV, K0.5 approximately equal to 0.2 mM, nH approximately equal to 1.1. The voltage-independent activation of Na/K pump current by both intracellular Na ions and extracellular K ions, at zero [Na]o, suggests that neither ion binds within the membrane field. Extracellular Na ions, however, seem to have both a voltage-dependent and a voltage-independent influence on the Na/K pump: they inhibit outward Na/K pump current in a strongly voltage-dependent fashion, with higher apparent affinity at more negative potentials (K0.5 approximately equal to 90 mM at -120 mV, and approximately 170 mM at -80 mV), and they compete with extracellular K ions in a seemingly voltage-independent manner.(ABSTRACT TRUNCATED AT 400 WORDS)     

Apical K+ channels in Necturus taste cells. Modulation by intracellular factors and taste stimuli

The apically restricted, voltage-dependent K+ conductance of Necturus taste receptor cells was studied using cell-attached, inside-out and outside-out configurations of the patch-clamp recording technique. Patches from the apical membrane typically contained many channels with unitary conductances ranging from 30 to 175 pS in symmetrical K+ solutions. Channel density was so high that unitary currents could be resolved only at negative voltages; at positive voltages patch recordings resembled whole-cell recordings. These multi-channel patches had a small but significant resting conductance that was strongly activated by depolarization. Patch current was highly K+ selective, with a PK/PNa ratio of 28. Patches containing single K+ channels were obtained by allowing the apical membrane to redistribute into the basolateral membrane with time. Two types of K+ channels were observed in isolation. Ca(2+)-dependent channels of large conductance (135-175 pS) were activated in cell-attached patches by strong depolarization, with a half-activation voltage of approximately -10 mV. An ATP-blocked K+ channel of 100 pS was activated in cell-attached patches by weak depolarization, with a half-activation voltage of approximately -47 mV. All apical K+ channels were blocked by the sour taste stimulus citric acid directly applied to outside-out and perfused cell-attached patches. The bitter stimulus quinine also blocked all channels when applied directly by altering channel gating to reduce the open probability. When quinine was applied extracellularly only to the membrane outside the patch pipette and also to inside-out patches, it produced a flickery block. Thus, sour and bitter taste stimuli appear to block the same apical K+ channels via different mechanisms to produce depolarizing receptor potentials.     

A whole-cell patch clamp technique which minimizes cell dialysis

A variant of the whole-cell patch clamp technique is described which allows measurement of whole-cell ionic currents in small cells while minimizing cell dialysis with the pipette solution. The technique involves the application of negative pressure to the inside of small (less than 1 micron) tip diameter pipettes placed on the cell surface to achieve high resistance seals and membrane rupture. The technique has been used successfully in a variety of different types of cells to study membrane currents carried by Ca and K, currents generated by exchange carriers as well as electrical coupling between cells. Overall, the technique seems well suited for the study of ionic currents in small cells, and provides an alternative to conventional patch clamping techniques which necessitate intracellular dialysis.     

Membrane currents in taste cells of the rat fungiform papilla. Evidence for two types of Ca currents and inhibition of K currents by saccharin

Taste buds were isolated from the fungiform papilla of the rat tongue and the receptor cells (TRCs) were patch clamped. Seals were obtained on the basolateral membrane of 281 TRCs, protruding from the intact taste buds or isolated by micro-dissection. In whole-cell configuration 72% of the cells had a TTX blockable transient Na inward current (mean peak amplitude 0.74 nA). All cells had outward K currents. Their activation was slower than for the Na current and a slow inactivation was also noticeable. The K currents were blocked by tetraethylammonium, Ba, and 4-aminopyridine, and were absent when the pipette contained Cs instead of K. With 100 mM Ba or 100 mM Ca in the bath, two types of inward current were observed. An L-type Ca current (ICaL) activated at -20 mV had a mean peak amplitude of 440 pA and inactivated very slowly. At 3 mM Ca the activation threshold of ICaL was near -40 mV. A transient T-type current (ICaT) activated at -50 mV had an average peak amplitude of 53 pA and inactivated with a time constant of 36 ms at -30 mV. ICaL was blocked more efficiently by Cd and D600 than ICaT. ICaT was blocked by 0.2 mM Ni and half blocked by 200 microM amiloride. In whole-cell voltage clamp, Na-saccharin caused (in 34% of 55 cells tested) a decrease in outward K currents by 21%, which may be expected to depolarize the TRCs. Also, Na-saccharin caused some taste cells to fire action potentials (on-cell, 7 out of 24 cells; whole-cell, 2 out of 38 cells responding to saccharin) of amplitudes sufficient to activate ICaL. Thus the action potentials will cause Ca inflow, which may trigger release of transmitter.     

A whole-cell patch clamp technique which minimizes cell dialysis

Summary A variant of the whole-cell patch clamp technique is described which allows measurement of whole-cell ionic currents in small cells while minimizing cell dialysis with the pipette solution. The technique involves the application of negative pressure to the inside of small (< 1 µm) tip diameter pipettes placed on the cell surface to achieve high resistance seals and membrane rupture. The technique has been used successfully in a variety of different types of cells to study membrane currents carried by Ca and K, currents generated by exchange carriers as well as electrical coupling between cells. Overall, the technique seems well suited for the study of ionic currents in small cells, and provides an alternative to conventional patch clamping techniques which necessitate intracellular dialysis.     

Diffusion of nystatin in plasma membrane is inhibited by a glass-membrane seal.

In perforated patch recording, the pore former nystatin is incorporated into a cell-attached patch, to increase its conductance. The possibility of lateral diffusion of nystatin through the membrane and under the glass-membrane seal was examined by reversing the nystatin gradient. Namely, a cell-attached patch on a cell was examined while placing nystatin into the bath. The reversal potential and current-voltage relationship of single Ca2+ activated K+ channels in the patch were readily changed by varying the K+ concentration in the bath, showing that nystatin was active in the cell membrane outside of the patch. However, the patch itself did not become leaky. The absence of a conductance induced in the patch by the nystatin in the rest of the plasma membrane of the cell suggests that the lateral diffusion of nystatin is inhibited by the glass-membrane seal.     

A quinine-activated cationic conductance in vertebrate taste receptor cells

The chincona alkaloid quinine is known to be a bitter tasting substance for various vertebrates. We examined the effects of quinine on isolated taste receptor cells from the bullfrog (Rana catesbeiana). Membrane currents were recorded by whole-cell recording, while quinine hydrochloride was applied extracellularly from a puffer pipette. At the resting potential (-77 +/- 9 mV, mean +/- SD, n = 49 cells), taste cells generated inward currents in response to quinine stimulation (> 1 mM), indicating a depolarizing response in the taste cells. Two types of current responses were observed; a newly found quinine-activated cationic conductance and a previously reported blocking effect of quinine on K+ conductances. The cationic current was isolated from the K+ current by using a Cs(+)-containing patch pipette. The relative permeabilities (Pion) of the quinine-activated cationic conductance were: PNa/PK/PCs = 1:0.5:0.42. The quinine dose-response relation was described by the Hill equation with the K1/2 of 3.6 mM and Hill coefficient of 5.3. When extracellular [Ca2+] (1.8 mM) was reduced to nominally free, the conductance was enhanced by about sixfold. This property is consistent with observations on quinine responses recorded from the gustatory nerve, in vivo. The quinine-induced cationic current was decreased with an application of 8-bromo-cAMP. We conclude that the bitter substance quinine activates a cation channel in taste receptor cells and this channel plays an important role in bitter taste transduction.     

Activation of Ca(2+)-dependent K+ current by acetylcholine and histamine in a human gastric epithelial cell line

The effects of acetylcholine (ACh) and histamine (His) on the membrane potential and current were examined in JR-1 cells, a mucin-producing epithelial cell line derived from human gastric signet ring cell carcinoma. The tight-seal, whole cell clamp technique was used. The resting membrane potential, the input resistance, and the capacitance of the cells were approximately -12 mV, 1.4 G ohms, and 50 pF, respectively. Under the voltage-clamp condition, no voltage-dependent currents were evoked. ACh or His added to the bathing solution hyperpolarized the membrane by activating a time- and voltage- independent K+ current. The ACh-induced hyperpolarization and K+ current persisted, while the His response desensitized quickly (< 1 min). These effects of ACh and His were mediated predominantly by m3- muscarinic and H1-His receptors, respectively. The K+ current induced by ACh and His was inhibited by charybdotoxin, suggesting that it is a Ca(2+)-activated K+ channel current (IK.Ca). The measurement of intracellular Ca2+ ([Ca2+]i) using Indo-1 revealed that both agents increased [Ca2+]i with similar time courses as they increased IK.Ca. When EGTA in the pipette solution was increased from 0.15 to 10 mM, the induction of IK.Ca by ACh and His was abolished. Thus, both ACh and His activate IK.Ca by increasing [Ca2+]i in JR-1 cells. In the Ca(2+)-free bathing solution (0.15 mM EGTA in the pipette), ACh evoked IK.Ca transiently. Addition of Ca2+ (1.8 mM) to the bath immediately restored the sustained IK.Ca. These results suggest that the ACh response is due to at least two different mechanisms; i.e., the Ca2+ release-related initial transient activation and the Ca2+ influx-related sustained activation of IK.Ca. Probably because of desensitization, the Ca2+ influx-related component of the His response could not be identified. Intracellularly applied inositol 1,4,5-trisphosphate (IP3), with and without inositol 1,3,4,5-tetrakisphosphate (IP4), mimicked the ACh response. IP4 alone did not affect the membrane current. Under the steady effect of IP3 or IP3 plus IP4, neither ACh nor His further evoked IK.Ca. Intracellular application of heparin or of the monoclonal antibody against the IP3 receptor, mAb18A10, inhibited the ACh and His responses in a concentration-dependent fashion. Neomycin, a phospholipase C (PLC) inhibitor, also inhibited the agonist-induced response in a concentration-dependent fashion. Although neither pertussis toxin (PTX) nor N-ethylmaleimide affected the ACh or His activation of IK,Ca, GDP beta S attenuated and GTP gamma S enhanced the agonist response.(ABSTRACT TRUNCATED AT 400 WORDS)     

Localization of cholinergic and purinergic receptors on outer hair cells isolated from the guinea-pig cochlea.

Acetylcholine (ACh) and adenosine 5'-triphosphate (ATP) are shown to act in opposing fashion on guinea-pig cochlear outer hair cells (OHCS) via receptors localized within different fluid compartments of the organ of Corti. The cholinergic (efferent) receptors localized at the basal (perilymphatic) region of these cells activated a rapidly desensitizing hyperpolarizing K+ current. In contrast, purinergic (ATP) receptors were localized at the apical (endolymphatic) surface of OHCS and activated a depolarizing nonselective cation current which exhibited inward rectification and lacked desensitization. Localization of the receptors was determined by using whole-cell patch-clamp, by recording onset latencies and response amplitudes to pulses of either ACh or ATP pressure-applied at selected sites along the length of isolated OHCS. Under voltage-clamp at -60 mV, the largest ACh-induced (outward) currents were recorded when ACh was directed at the basal region of the cells. Conversely, the maximum (inward) ATP currents were obtained when ATP was directed toward the apical surface of these cells. Onset latencies increased rapidly from a minimum of approximately 10 ms for either ACh or ATP as the drug pipette was moved away from these optimal sites. The ATP response was antagonized by amiloride in a dose-dependent manner with a KD of approximately 400 microM. The localization of P2-type purinoceptors to the endolymphatic surface of OHCS suggests that ATP mediates a humoral modulation of the mechano-electrical transduction process.     

Nonselective cationic currents activated by acetylcholine in swine tracheal smooth muscle cells

Membrane currents in isolated swine tracheal smooth muscle cells were investigated using a pipette solution containing BAPTA-Ca2+ buffer and Cs+ as the major cation. With a pipette solution containing 100 nM free Ca2+, acetylcholine (ACh; 1-100 microM), in a concentration-dependent manner, activated a current without inducing shortening of cells, although neither 1 mM histamine nor 1 microM leukotriene D4 activated the current (n = 7, n is the number of cells). The effect of 100 microM ACh was suppressed by pretreatment with 100 microM atropine (n = 6) or intracellular application of preactivated pertussis toxin at a concentration of 0.1 microg x mL(-1) (n = 8). Genistein (0.1-100 microM), in a concentration-dependent manner, suppressed the activation of the inward current by 100 microM ACh, whereas it did not significantly suppress that of the outward current (n = 6-8). With a pipette solution containing 50 nM free Ca2+, outward current, but not inward current, was activated by 100 microM ACh (n = 10). When the pipette solution had free Ca2+ concentrations greater than 50 nM, the inward current together with the outward current was activated. The ratio between the amplitude of the inward and outward currents was significantly increased as the free Ca2+ concentration in the pipette solution increased. The steady-state activation curve of the ACh-activated current with the 50 nM free Ca2+ pipette solution was fitted by a single Boltzmann distribution (Vh = +69.8 mV, k = -11.9 mV, n = 10). The activation time constant became smaller as the membrane potential was more depolarized (164.3+/-5.9 ms at +40 mV to 92.4+/-6.3 ms at +120 mV, n = 10). The reversal potential was not significantly changed by reducing extracellular Cl- concentration to one-tenth of the control (n = 8), suggesting that the current is a nonselective cationic current. These results suggest that ACh activates an outward nonselective cationic current via pertussis toxin-sensitive G-protein(s) coupled with muscarinic receptors. Involvement of genistein-sensitive tyrosine kinase in the activation process of the current is unlikely.