Membrane properties of isolated mudpuppy taste cells

The voltage-dependent currents of isolated Necturus lingual cells were studied using the whole-cell configuration of the patch-clamp technique. Nongustatory surface epithelial cells had only passive membrane properties. Small, spherical cells resembling basal cells responded to depolarizing voltage steps with predominantly outward K+ currents. Taste receptor cells generated both outward and inward currents in response to depolarizing voltage steps. Outward K+ currents activated at approximately 0 mV and increased almost linearly with increasing depolarization. The K+ current did not inactivate and was partially Ca++ dependent. One inward current activated at -40 mV, reached a peak at -20 mV, and rapidly inactivated. This transient inward current was blocked by tetrodotoxin (TTX), which indicates that it is an Na+ current. The other inward current activated at 0 mV, peaked at 30 mV, and slowly inactivated. This more sustained inward current had the kinetic and pharmacological properties of a slow Ca++ current. In addition, most taste cells had inwardly rectifying K+ currents. Sour taste stimuli (weak acids) decreased outward K+ currents and slightly reduced inward currents; bitter taste stimuli (quinine) reduced inward currents to a greater extent than outward currents. It is concluded that sour and bitter taste stimuli produce depolarizing receptor potentials, at least in part, by reducing the voltage-dependent K+ conductance.     

Voltage gated ionic channels in rat cultured astrocytes, reactive astrocytes and an astrocyte-oligodendrocyte progenitor cell

Astrocytes (both type 1 and type 2), cultured from the central nervous system of newborn or 7 day old rats show voltage gated sodium and potassium channels that are activated when the membrane is depolarized to greater than -40 mV. The sodium channels in these cells have an h-infinity curve similar to that of nodal membranes but the activation (peak current-voltage) curves are shifted along the voltage axis by about +30 mV. These sodium currents are blocked only by high concentrations of tetrodotoxin. The voltage activated potassium currents in both types of astrocyte show at least two components; an inactivating component that is suppressed at holding potentials of greater than -40 mV and a persistent, non-inactivating current. Several types of single channel currents were observed in outside-out membrane patches from type 2 astrocytes. One type of potassium channel showed inactivation on depolarization and may contribute to the whole-cell inactivating current. In contrast, oligodendrocytes showed no obvious voltage gated membrane channels. The properties of the type 2 astrocyte-oligodendrocyte progenitor cell were investigated in two ways: 1) by examination of cells just beginning to differentiate along the "electrically silent" oligodendrocyte pathway or 2) by recording from progenitor cells cultured for 24 hours in the presence of cycloheximide to block the appearance of new membrane channels. In both cases, voltage gated inward (sodium) and outward (potassium) currents were noted. The outward current response showed both an inactivating and a non-inactivating component. Similar voltage activated inward and outward membrane currents were noted in reactive astrocytes freshly isolated (3-6 hours) from lesioned areas of adult rat brains.(ABSTRACT TRUNCATED AT 250 WORDS)     

Membrane currents and pharmacology of retinal bipolar cells: a comparative study on goldfish and mouse.

We obtained solitary bipolar cells using enzymatic (papain) dissociation of the goldfish and mouse (C57BL/6J, adult) retinae and measured the membrane currents of these cells by whole-cell patch clamp. Bipolar cells of these two species showed two main differences. A. Ca current 1. In the mouse, depolarization evoked a transient Ca current that had maximal amplitude at about -30 mV. 2. The Ca conductance was activated by voltage steps to potentials greater than -60 mV and inactivated fully at potentials greater than -20 mV. 3. The mouse Ca current was insensitive to Cd2+ or dihydropyridine. 4. Contrary to mouse, goldfish bipolar cells had a sustained Ca current, which was activated over a more positive potential range (greater than -30 mV), blocked by either 50 microM Cd2+ or 10 microM nifedipine, and markedly augmented by 10 microM Bay K8644. 5. The transient character of the Ca current in mouse bipolar cells may help to shape phasic responses of ganglion cells, while in goldfish the sustained nature of Ca current may contribute to shape tonic responses of ganglion cells. B. Pharmacology 1. We examined the effects of the inhibitory transmitters, glycine and GABA, on bipolar cells. 2. GABA produced strong inhibitory effects on bipolar cells of both goldfish and mouse. 3. The highest GABA sensitivity was found at the bipolar cell axon terminal, the site of reciprocal connection with amacrine cells. 4. GABA increased the Cl conductance. 5. Unlike GABA, glycine was effective only on the mouse bipolar cells. Axon terminals showed the highest glycine sensitivity. 6. Glycine-induced currents were also carried by Cl ions. 7. Since ECl in intact cells is assumed to be -55 mV, both GABA and glycine are thought to generate hyperpolarizing responses in cells maintained at their resting potential (ca. -45 mV). 8. The present study suggests that inhibition from amacrine cells to bipolar cells, found in both species, is mediated by different transmitters.     

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.     

Two light-dependent conductances in Lima rhabdomeric photoreceptors

Light-dependent membrane currents were recorded from solitary Lima photoreceptors with the whole-cell clamp technique. Light stimulation from a holding voltage near the cell's resting potential evokes a transient inward current graded with light intensity, accompanied by an increase in membrane conductance. While the photocurrent elicited by dim flashes decays smoothly, at higher stimulus intensities two kinetically distinct components become visible. Superfusion with TEA or intracellular perfusion with Cs do not eliminate this phenomenon, indicating that it is not due to the activation of the Ca-sensitive K channels that are present in these cells. The relative amplitude of the late component vs. the early peak of the light response is significantly more pronounced at -60 mV than at -40 mV. At low light intensities the reversal potential of the photocurrent is around 0 mV, but with brighter lights no single reversal potential is found; rather, a biphasic response with an inward and an outward component can be seen within a certain range of membrane voltages. Light adaptation through repetitive stimulation with bright flashes diminishes the amplitude of the early but not the late phase of the photocurrent. These observations can be accounted for by postulating two separate light-dependent conductances with different ionic selectivity, kinetics, and light sensitivity. The light response is also shown to interact with some of the voltage-sensitive conductances: activation of the Ca current by a brief conditioning prepulse is capable of attenuating the photocurrent evoked by a subsequent test flash. Thus, Ca channels in these cells may not only shape the photoresponse, but also participate in the process of light adaptation.     

Voltage-dependent ionic currents in taste receptor cells of the larval tiger salamander

Voltage-dependent membrane currents of cells dissociated from tongues of larval tiger salamanders (Ambystoma tigrinum) were studied using whole-cell and single-channel patch-clamp techniques. Nongustatory epithelial cells displayed only passive membrane properties. Cells dissociated from taste buds, presumed to be gustatory receptor cells, generated both inward and outward currents in response to depolarizing voltage steps from a holding potential of -60 or -80 mV. Almost all taste cells displayed a transient inward current that activated at -30 mV, reached a peak between 0 and +10 mV and rapidly inactivated. This inward current was blocked by tetrodotoxin (TTX) or by substitution of choline for Na+ in the bath solution, indicating that it was a Na+ current. Approximately 60% of the taste cells also displayed a sustained inward current which activated slowly at about -30 mV and reached a peak at 0 to +10 mV. The amplitude of the slow inward current was larger when Ca2+ was replaced by Ba2+ and it was blocked by bath applied CO2+, indicating it was a Ca2+ current. Delayed outward K+ currents were observed in all taste cells although in about 10% of the cells, they were small and activated only at voltages more depolarized than +10 mV. Normally, K+ currents activated at -40 mV and usually showed some inactivation during a 25-ms voltage step. The inactivating component of outward current was not observed at holding potentials more depolarized -40 mV. The outward currents were blocked by tetraethylammonium chloride (TEA) and BaCl2 in the bath or by substitution of Cs+ for K+ in the pipette solution. Both transient and noninactivating components of outward current were partially suppressed by CO2+, suggesting the presence of a Ca2(+)-activated K+ current component. Single-channel currents were recorded in cell-attached and outside-out patches of taste cell membranes. Two types of K+ channels were partially characterized, one having a mean unitary conductance of 21 pS, and the other, a conductance of 148 pS. These experiments demonstrate that tiger salamander taste cells have a variety of voltage- and ion-dependent currents including Na+ currents, Ca2+ currents and three types of K+ currents. One or more of these conductances may be modulated either directly by taste stimuli or indirectly by stimulus-regulated second messenger systems to give rise to stimulus-activated receptor potentials. Others may play a role in modulation of neurotransmitter release at synapses with taste nerve fibers.(ABSTRACT TRUNCATED AT 400 WORDS)     

Voltage-dependent and odorant-regulated currents in isolated olfactory receptor neurons of the channel catfish.

The electrical properties of olfactory receptor neurons, enzymatically dissociated from the channel catfish (Ictalurus punctatus), were studied using the whole-cell patch-clamp technique. Six voltage-dependent ionic currents were isolated. Transient inward currents (0.1-1.7 nA) were observed in response to depolarizing voltage steps from a holding potential of -80 mV in all neurons examined. They activated between -70 and -50 mV and were blocked by addition of 1 microM tetrodotoxin (TTX) to the bath or by replacing Na+ in the bath with N-methyl-D-glucamine and were classified as Na+ currents. Sustained inward currents, observed in most neurons examined when Na+ inward currents were blocked with TTX and outward currents were blocked by replacing K+ in the pipette solution with Cs+ and by addition of 10 mM Ba2+ to the bath, activated between -40 and -30 mV, reached a peak at 0 mV, and were blocked by 5 microM nimodipine. These currents were classified as L-type Ca2+ currents. Large, slowly activating outward currents that were blocked by simultaneous replacement of K+ in the pipette with Cs+ and addition of Ba2+ to the bath were observed in all olfactory neurons examined. The outward K+ currents activated over approximately the same range as the Na+ currents (-60 to -50 mV), but the Na+ currents were larger at the normal resting potential of the neurons (-45 +/- 11 mV, mean +/- SD, n = 52). Four different types of K+ currents could be differentiated: a Ca(2+)-activated K+ current, a transient K+ current, a delayed rectifier K+ current, and an inward rectifier K+ current. Spontaneous action potentials of varying amplitude were sometimes observed in the cell-attached recording configuration. Action potentials were not observed in whole-cell recordings with normal internal solution (K+ = 100 mM) in the pipette, but frequently appeared when K+ was reduced to 85 mM. These observations suggest that the membrane potential and action potential amplitude of catfish olfactory neurons are significantly affected by the activity of single channels due to the high input resistance (6.6 +/- 5.2 G omega, n = 20) and low membrane capacitance (2.1 +/- 1.1 pF, n = 46) of the cells.(ABSTRACT TRUNCATED AT 400 WORDS)     

Characterization of two distinct depolarization-activated K+ currents in isolated adult rat ventricular myocytes

Depolarization-activated outward K+ currents in isolated adult rat ventricular myocytes were characterized using the whole-cell variation of the patch-clamp recording technique. During brief depolarizations to potentials positive to -40 mV, Ca(2+)-independent outward K+ currents in these cells rise to a transient peak, followed by a slower decay to an apparent plateau. The analyses completed here reveal that the observed outward current waveforms result from the activation of two kinetically distinct voltage-dependent K+ currents: one that activates and inactivates rapidly, and one that activates and inactivates slowly, on membrane depolarization. These currents are referred to here as Ito (transient outward) and IK (delayed rectifier), respectively, because their properties are similar (although not identical) to these K+ current types in other cells. Although the voltage dependences of Ito and IK activation are similar, Ito activates approximately 10-fold and inactivates approximately 30-fold more rapidly than IK at all test potentials. In the composite current waveforms measured during brief depolarizations, therefore, the peak current predominantly reflects Ito, whereas IK is the primary determinant of the plateau. There are also marked differences in the voltage dependences of steady-state inactivation of these two K+ currents: IK undergoes steady-state inactivation at all potentials positive to -120 mV, and is 50% inactivated at -69 mV; Ito, in contrast, is insensitive to steady-state inactivation at membrane potentials negative to -50 mV. In addition, Ito recovers from steady-state inactivation faster than IK: at -90 mV, for example, approximately 70% recovery from the inactivation produced at -20 mV is observed within 20 ms for Ito; IK recovers approximately 25-fold more slowly. The pharmacological properties of Ito and IK are also distinct: 4-aminopyridine preferentially attenuates Ito, and tetraethylammonium suppresses predominantly IK. The voltage- and time-dependent properties of these currents are interpreted here in terms of a model in which Ito underlies the initial, rapid repolarization phase of the action potential (AP), and IK is responsible for the slower phase of AP repolarization back to the resting membrane potential, in adult rat ventricular myocytes.     

Excitatory amino acid receptor-channels in Purkinje cells in thin cerebellar slices.

Glutamate receptors of the N-methyl-D-aspartate (NMDA) and non-NMDA type serve different functions during excitatory synaptic transmission. Although many central neurons bear both types of receptor, the evidence concerning the sensitivity of cerebellar Purkinje cells to NMDA is contradictory. To investigate the receptor types present in Purkinje cells, we have used whole-cell and outside-out patch-clamp methods to record from cells in thin cerebellar slices from young rats. At a holding potential of -70 mV (in nominally Mg(2+)-free medium, with added glycine) NMDA caused a whole-cell current response which consisted of a dramatic increase in the frequency of synaptic currents. In the presence of tetrodotoxin (TTX) and the gamma-aminobutyric acidA (GABAA) receptor antagonist bicuculline, spontaneous synaptic currents and responses to NMDA were inhibited. In a proportion of cells a small polysynaptic response to NMDA persisted, which was further reduced by the non-NMDA receptor antagonist 6-cyano-2,3-dihydro-7-nitroquinoxalinedione (CNQX). The non-NMDA glutamate receptor agonists kainate (KA), quisqualate (QA) and s-alpha-amino-3-hydroxy-5-methyl-4-isoazolepropionic acid (s-AMPA), evoked large inward currents due to the direct activation of receptors in Purkinje cells. NMDA applied to excised membrane patches failed to evoke any single-channel currents, whereas s-AMPA and QA caused small inward currents accompanied by marked increases in current noise. Spectral analysis of the s-AMPA noise in patches gave an estimated mean channel conductance of approximately 4 pS.(ABSTRACT TRUNCATED AT 250 WORDS)     

Voltage-activated Ca2+ currents in insulin-secreting cells

Membrane voltage and voltage-clamped membrane currents have been investigated with the whole-cell patch clamp method in the insulin-secreting cell line RINm5F. The mean resting membrane potential of RINm5F cells was found to be -52 mV. Overshooting spike potentials could be evoked by depolarising voltage steps in the absence of a secretagogue. Inward membrane currents evoked by depolarising voltage steps were dependent upon extracellular Ca2+ and blocked by Co2+, nifedipine and verapamil. Outward membrane currents which were evoked by depolarising voltage steps to positive membrane potentials were reduced when Ca2+ entry was prevented. It is concluded that the voltage-activated Ca2+ currents underlie the voltage-activated spike potentials recorded from insulin-secreting cells.     

Voltage- and time-dependent K+ channel currents in the basolateral membrane of villus enterocytes isolated from guinea pig small intestine

Patch-clamp studies were carried out in villus enterocytes isolated from the guinea pig proximal small intestine. In the whole-cell mode, outward K+ currents were found to be activated by depolarizing command pulses to -45 mV. The activation followed fourth order kinetics. The time constant of K+ current activation was voltage-dependent, decreasing from approximately 3 ms at -10 mV to 1 ms at +50 mV. The K+ current inactivated during maintained depolarizations by a voltage- independent, monoexponential process with a time constant of approximately 470 ms. If the interpulse interval was shorter than 30 s, cumulative inactivation was observed upon repeated stimulations. The steady state inactivation was voltage-dependent over the voltage range from -70 to -30 mV with a half inactivation voltage of -46 mV. The steady state activation was also voltage-dependent with a half- activation voltage of -22 mV. The K+ current profiles were not affected by chelation of cytosolic Ca2+. The K+ current induced by a depolarizing pulse was suppressed by extracellular application of TEA+, Ba2+, 4-aminopyridine or quinine with half-maximal inhibitory concentrations of 8.9 mM, 4.6 mM, 86 microM and 26 microM, respectively. The inactivation time course was accelerated by quinine but decelerated by TEA+, when applied to the extracellular (but not the intracellular) solution. Extracellular (but not intracellular) applications of verapamil and nifedipine also quickened the inactivation time course with 50% effective concentrations of 3 and 17 microM, respectively. Quinine, verapamil and nifedipine shifted the steady state inactivation curve towards more negative potentials. Outward single K+ channel events with a unitary conductance of approximately 8.4 pS were observed in excised inside-out patches of the basolateral membrane, when the patch was depolarized to -40 mV. The ensemble current rapidly activated and thereafter slowly inactivated with similar time constants to those of whole-cell K+ currents. It is concluded that the basolateral membrane of guinea pig villus enterocytes has a voltage-gated, time-dependent, Ca(2+)-insensitive, small-conductance K+ channel. Quinine, verapamil, and nifedipine accelerate the inactivation time course by affecting the inactivation gate from the external side of the cell membrane.     

Ca-dependent-chloride and potassium currents in rat Leydig cells

Voltage-clamped membrane currents have been investigated from whole-cell patch-clamp recordings performed on single Leydig cells isolated from the adult rat testis. Two outward membrane currents were evoked by depolarizing voltage steps. A potassium current was recorded in cells dialyzed with low (10(-9)-10(-8) M) calcium media. This current was decreased by TEA (10 mM). A chloride current was recorded in cells dialyzed with high (10(-7)-10(-6) M) calcium media. This current was decreased by an external exposure to glutamate. Comparison of the currents at low and high internal calcium concentrations suggests that an increase of the intracellular calcium activates a chloride current.     

成年蜜蜂脑神经细胞的培养和电生理特征

为了研究杀虫剂等对蜜蜂毒性作用的神经机制,需在体外建立成年蜜蜂脑神经细胞的分离培养和电生理记录技术并研究其正常电生理特征,而对成年蜜蜂脑神经细胞的分离培养和电生理特性的研究报道甚少。我们采用酶解和机械吹打相结合的方法获得了数量较多且活力较好的成年意大利蜜蜂Apis mellifera脑神经细胞,并用全细胞膜片钳技术研究了成年意大利蜜蜂脑神经细胞对电流和电压刺激的反应,获得了成年意蜂脑神经细胞的基本电生理特征以及钠电流和钾电流的特性。全细胞电流钳的记录结果表明,在体外培养条件下,细胞无自发放电发生,注射电流后仅引起细胞单次放电,引起细胞放电的阈电流平均为60.8±63 pA; 细胞动作电位产生的阈电位平均为−27.4±2.3 mV。用全细胞电压钳记录了神经细胞的钠电流和钾电流。钠电流的分离是在电压刺激下通过阻断钾通道和钙通道实现。细胞的内向钠电流在指令电压为−40～−30 mV左右激活,−10 mV达峰值,钠通道的稳态失活电压V_1/2为−58.4 mV; 外向钾电流成份至少包括较小的快速失活钾电流和和较大的缓慢失活钾电流（占总钾电流的80%）,其半激活膜电位V_1/2为3.86 mV,无明显的稳态失活。结果提示缓慢失活钾电流的特征可能是细胞单次放电的机制之一。     

Ionic mechanisms of two types of on-center bipolar cells in the carp retina. I. The responses to central illumination.

Properties of the depolarizing response of on-center bipolar cells to a light spot stimulus were studied in the carp retina. On-center bipolar cells were classified into two types, cone-dominant and rod-dominant, according to their major input from cones and rods. Cone-dominant bipolar cells responded to spectral light with the maximum amplitude near 625 nm, suggesting major input from red cones. The response was accompanied by a resistance increase and showed a reversal potential at -63 +/- 21 mV when the membrane was hyperpolarized by current. The results suggest that the photoresponse of cone-dominant cells is due to a decrease of gK and/or gCl, membrane conductances to potassium and chloride, respectively. Rod-dominant bipolar cells responded to spectral light with the maximum amplitude near 525 nm under scotopic conditions and near 625 nm under photopic conditions, providing evidence that they receive input from rods and red cones. In the scoptopic condition their response was accompanied by a resistance decrease and showed a reversal potential at 29 +/- 13 mV, whereas in the photopic condition the response in most of them was accompanied by a resistance increase, at least in their part and showed a reversal at -53 +/- 11 mV. The results suggest that the photoresponse activated by rod input is due to an increase in gNa. In the mesopic condition rod-dominant cells showed complex electrical membrane properties as the result of electric interaction between the above two differnt ionic mechanisms activated by rod and cone inputs.     

Odorant receptors activated by amino acids in sensory neurons of the channel catfish Ictalurus punctatus

Odorant receptors activated by amino acids were investigated with patch- clamp techniques in olfactory receptor neurons of the channel catfish, Ictalurus punctatus. The L-isomers of alanine, norvaline, arginine, and glutamate, known to act predominantly on different olfactory receptor sites, activated nondesensitizing inward currents with amplitudes of - 2.5 to -280 pA in olfactory neurons voltage-clamped at membrane potentials of -72 or -82 mV. Different amino acids were shown to induce responses in the same sensory neurons; however, the amplitude and the kinetics of the observed whole cell currents differed among the stimuli and may therefore reflect activation of different amino acid receptor types or combinations of receptor types in these cells. Amino acid- induced currents appeared to have diverse voltage dependence and could also be classified according to the amplitude of the spontaneous channel fluctuations underlying the macroscopic currents. A mean single- channel conductance (gamma) of 360 fS was estimated from small noise whole-cell currents evoked by arginine within the same olfactory neuron in which a mean gamma value of 23.6 pS was estimated from ''large noise'' response to norvaline. Quiescent olfactory neurons fired bursts of action potentials in response to either amino acid stimulation or application of 8-Br-cyclic GMP (100 microM), and voltage-gated channels underlying generation of action potentials were similar in these neurons. However, in whole-cell voltage-clamp, 8-Br-cyclic GMP evoked large rectangular current pulses, and single-channel conductances of 275, 220, and 110 pS were obtained from the discrete current levels. These results suggest that in addition to the cyclic nucleotide-gated transduction channels, olfactory neurons of the channel catfish possess a variety of odor receptors coupled to different types of transduction channels.     

Whole-cell currents in single and confluent M-1 mouse cortical collecting duct cells

M-1 cells, derived from a microdissected cortical collecting duct of a transgenic mouse, grown to confluence on a permeable support, develop a lumen-negative amiloride-sensitive transepithelial potential, reabsorb sodium, and secrete potassium. Electron micrographs show morphological features typical of principal cells in vivo. Using the patch clamp technique distinct differences are detected in whole-cell membrane current and voltage (Vm) between single M-1 cells 24 h after seeding vs cells grown to confluence. (a) Under control conditions (pipette: KCl- Ringer; bath: NaCl-Ringer) Vm averages -42.7 +/- 3.4 mV in single cells vs -16.8 +/- 4.1 mV in confluent cells. Whole-cell conductance (Gcell) in confluent cells is 2.6 times higher than in single cells. Cell capacitance values are not significantly different in single vs confluent M-1 cells, arguing against electrical coupling of confluent M- 1 cells. (b) In confluent cells, 10(-4)-10(-5) M amiloride hyperpolarizes Vm to -39.7 +/- 3.0 mV and the amiloride-sensitive fractional conductance of 0.31 shows a sodium to potassium selectivity ratio of approximately 15. In contrast, single cells express no significant amiloride-sensitive conductance. (c) In single M-1 cells, Gcell is dominated by an inwardly rectifying K-conductance, as exposure to high bath K causes a large depolarization and doubling of Gcell. The barium-sensitive fraction of Gcell in symmetrical KCl-Ringer is 0.49 and voltage dependent. (d) In contrast, neither high K nor barium in the apical bath affect confluent M-1 cells, showing that confluent cells lack a significant apical K conductance. (e) Application of 500 microM glibenclamide reduces whole-cell currents in both single and confluent M-1 cells with a glibenclamide-sensitive fractional conductance of 0.71 and 0.83 in single and confluent cells, respectively. Glibenclamide inhibition occurs slower in confluent M-1 cells than in single cells, suggesting a basolateral action of this lipophilic drug on ATP-sensitive basolateral K channels in M-1 cells. (f) A component of the whole-cell conductance in M-1 cells appears as a deactivating outward current during large depolarizing voltage pulses and is abolished by extracellular chloride removal. The deactivating chloride current averages 103.6 +/- 16.1 pA/cell, comprises 24% of the outward current, and decays with a time constant of 179 +/- 13 ms. The outward to inward conductance ratio obtained from deactivating currents and tail currents is 2.4, indicating an outwardly rectifying chloride conductance.     

Cyclic GMP regulation of a voltage-activated K channel in dissociated Enterocytes

Summary Enterrocytes from the intestinal epithelium of the winter flounder were isolated by collagenase digestion and incubated in flounder Ringer solution. Conventional whole-cell and amphotericin-perforated whole-cell recording techniques were used to characterize the properties of a voltage-activated K current present in dissociated cells. Resting membrane potentials and series resistances were significantly lower (from –23 to – 39 mV and 29 to 13 M, respectively) when amphotericin was used to achieve the whole-cell configuration. When cells were placed in flounder Ringer solution, held at –80 mV and subsequently stepped to a series of depolarizing voltages (from–70 to 0 mV), an outward current was observed that exhibited inactivation at voltages above –20 mV. This current was sensitive to holding potential and was not activated when the cells were held at –40 mV or above. When cells were bathed in symmetric K Ringer solution and the same voltage protocol was applied to the cell, inward currents were observed in response to the negative intracellular potentials. Reversal potentials at two different extracellular K concentrations were consistent with K as the currentcarrying ion. BaCl₂ (2 mM) and CsCl (0.5 mM) both produced voltage-dependent blockade of the current when added to the bathing solution. Charybdotoxin (300 nM extracellular concentration) completely blocked the current. The IC₅₀ for charybdotoxin was 50 nM. Cyclic. GMP inhibited the voltage-activated current in flounder Ringer and in symmetric K Ringer solution. The cyclic GMP analog, 8-Br cGMP, lowered the threshold for voltage activation and potentiated inactivation of the current at voltages above–40 mV. Previous studies with intact flounder epithelium showed that K recycling and net K secretion were inhibited by Ba²⁺ and by cGMP. We suggest that the channel responsible for the whole-cell current described in this study may be important in K recycling and secretion.     

The light-sensitive conductance of hyperpolarizing invertebrate photoreceptors: a patch-clamp study

Tight-seal recording was employed to investigate membrane currents in hyperpolarizing ciliary photoreceptors enzymatically isolated from the eyes of the file clam (Lima scabra) and the bay scallop (Pecten irradians). These two organisms are unusual in that their double retinas also possess a layer of depolarizing rhabdomeric cells. Ciliary photoreceptors from Lima have a rounded soma, 15-20 microns diam, and display a prominent bundle of fine processes up to 30 microns long. The cell body of scallop cells is similar in size, but the ciliary appendages are modified, forming small spherical structures that protrude from the cell. In both species light stimulation at a voltage near the resting potential gives rise to a graded outward current several hundred pA in amplitude, accompanied by an increase in membrane conductance. The reversal potential of the photocurrent is approximately -80 mV, and shifts in the positive direction by approximately 39 mV when the concentration of extracellular K is increased from 10 to 50 mM, consistent with the notion that light activates K-selective channels. The light-activated conductance increases with depolarization in the physiological range of membrane voltages (-30 to -70 mV). Such outward rectification is greatly reduced after removal of divalent cations from the superfusate. In Pecten, cell- attached recordings were also obtained; in some patches outwardly directed single-channel currents could be activated by light but not by voltage. The unitary conductance of these channels was approximately 26 pS. Solitary ciliary cells also gave evidence of the post stimulus rebound, which is presumably responsible for initiating the "off" discharge of action potentials at the termination of a light stimulus: in patches containing only voltage-dependent channels, light stimulation suppressed depolarization-induced activity, and was followed by a strong burst of openings, directly related to the intensity of the preceding photostimulation.     

Outward currents in olfactory receptor neurons activated by odorants and by elevation of cyclic AMP

We studied the outward currents elicited by an odorous compound, isoamyl acetate, in isolated olfactory receptor neurons of the grass frog under whole-cell perforated-patch voltage-clamp recording. Odorant-induced outward currents were relatively rare, occurring in about 16% of the responding cells. Responses had smaller amplitudes and shorter time courses when compared to the more commonly found odorant-induced inward currents. There was a high correlation between odorant-induced outward current and responses evoked by either 8-(4-chlorophenylthio) adenosine 3':5'-cyclic monophosphate, a membrane-permeant cyclic adenosine monophosphate analog, or 3-isobutyl-1-methylxanthine, a phosphodiesterase inhibitor. The outward current responses to all three substances increased in amplitude when the membrane potential was more negative than -60 mV and decreased in amplitude when the membrane potential was more positive. Responses were still present when the potential was held at -100 mV, indicating that the responses are not the result of a potassium conductance. Removal of external calcium from the perfusion medium abolished the outward currents. Our results indicate that the odorant-induced outward current is a calcium-dependent event that may be mediated by cyclic adenosine monophosphate.     

新鲜分离小鼠胃ICC样细胞的鉴定及其电生理学特性

将免疫荧光及传统全细胞膜片钳技术应用于新鲜分离的小鼠胃Cajal 间质细胞样细胞上,探讨了小鼠胃Cajal 间质细胞样细胞形态和电生理学特性。经胶原酶消化得到的Cajal间质细胞样细胞胞体呈短梭形,且自胞体发出多个较短的毛刺状突起。免疫细胞化学结果表明,Cajal 间质细胞样细胞胞体和突起酪氨酸激酶受体c-kit表达呈阳性。在传统全细胞记录模式、膜电位钳制在-60 mV 的条件下,可以记录到自发、节律性内向电流,即起搏电流。钙调蛋白抑制剂W-7 (50µmol/L）明显增强了起搏电流幅度并引发明显的内向钳制电流。当电极内液中EGTA 的浓度由0.1 mmol/L增加到10 mmol/L时,也明显增强了起搏电流幅度并引发明显的内向钳制电流。实验结果提示,新鲜分离的小鼠胃Cajal 间质细胞样细胞可以产生自发、自律性内向电流,而这种电流对胞内低钙或钙调蛋白抑制剂敏感。这种具有自发性电活动的Cajal 间质细胞样细胞可能就是胃Cajal 间质细胞。