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.     

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)     

Kinetic properties of single sodium channels in rat heart and rat brain

Single Na channel currents were compared in ventricular myocytes and cortical neurons of neonatal rats using the gigaseal patch-clamp method to determine whether tissue-specific differences in gating can be detected at the single-channel level. Single-channel currents were recorded in cell-attached and excised membrane patches at test potentials of -70 to -20 mV and at 9-11 degrees C. In both cell-attached and excised patches brain Na channel mean open time progressively increased from less than 1 ms at -70 mV to approximately 2 ms at -20 mV. Near threshold, single openings with dispersed latencies were observed. By contrast, in cell-attached patches, heart Na channel mean open time peaked near -50 mV, was three times brain Na channel mean open time, and declined continuously to approximately 2 ms at -20 mV. Near threshold, openings occurred frequently usually as brief bursts lasting several milliseconds and rarely as prolonged bursts lasting tens of milliseconds. Unlike what occurs in brain tissue where excision did not change gating, in excised heart patches both the frequency of prolonged bursting and the mean open time of single units increased markedly. Brain and cardiac Na channels can therefore be distinguished on the basis of their mean open times and bursting characteristics.     

The feasibility and limitation of patch-clamp recordings from neonatal rat cardiac ventricular slices

Examine the feasibility of whole-cell patch-clamp recordings from the cardiac ventricular slices of newborn (P(3)-P(7)) Sprague-Dawley rats to identify a better substitute for single cardiac myocytes prepared using enzymatic treatment. High resistance seals (>1 G?) were obtained from cardiac ventricle tissues prepared without using enzymatic treatment. Thereafter, cell-attached and whole-cell patch-clamp techniques were used on thin cardiac slices (200 μm thick) in 2009 in the Institute of Molecular Medicine of Peking University. An averaged sodium current (n=11 cells) was recorded in the cell-attached mode, and this displayed features similar to those previously reported for isolated rat ventricular myocytes. The outward potassium current, hyperpolarization-activated cation channel or I (f) channel (HCN channel), and action potential were recorded in the whole-cell mode (n=2 cells), and the identical properties were observed from the cardiac slices. The cell-attached mode is stable and reliable for recording the ion current. The resting potential for cardiac slices measured using current-clamp recording in the whole-cell mode was -50 to -70 mV. The resting potential of cardiac slices has properties similar to those of enzyme-prepared cardiomyocytes, with the exception that it is positive. We achieved whole-cell patch-clamp recordings from cardiac slices and affirmed the feasibility and values of both cell-attached and whole-cell recording modes using this technique. Nevertheless, there remain difficulties and limitations associated with the application of whole-cell patch-clamping to cardiac slices, due primarily to the existence of large amounts of connective tissue even in newborn rats.     

Single Ionic Channels of Two Caenorhabditis elegans Chemosensory Neurons in Native Membrane

The genome of Caenorhabditis elegans contains representatives of the channel families found in both vertebrate and invertebrate nervous systems. However, it lacks the ubiquitous Hodgkin-Huxley Na+ channel that is integral to long-distance signaling in other animals. Nematode neurons are presumed to communicate by electrotonic conduction and graded depolarizations. This fundamental difference in operating principle may require different channel populations to regulate transmission and transmitter release. We have sampled ionic channels from the somata of two chemosensory neurons (AWA and AWC) of C. elegans. A Ca2+-activated, outwardly rectifying channel has a conductance of 67 pS and a reversal potential indicating selectivity for K+. An inwardly rectifying channel is active at potentials more negative than -50 mV. The inward channel is notably flickery even in the absence of divalent cations; this prevented determination of its conductance and reversal potential. Both of these channels were inactive over a range of membrane potentials near the likely cell resting potential; this would account for the region of very high membrane resistance observed in whole-cell recordings. A very-large-conductance (> 100 pS), inwardly rectifying channel may account for channel-like fluctuations seen in whole-cell recordings.     

GABA action in immature neocortical neurons directly depends on the availability of ketone bodies

In the early postnatal period, energy metabolism in the suckling rodent brain relies to a large extent on metabolic pathways alternate to glucose such as the utilization of ketone bodies (KBs). However, how KBs affect neuronal excitability is not known. Using recordings of single NMDA and GABA-activated channels in neocortical pyramidal cells we studied the effects of KBs on the resting membrane potential ( E _m) and reversal potential of GABA-induced anionic currents ( E _GABA), respectively. We show that during postnatal development (P3–P19) if neocortical brain slices are adequately supplied with KBs, E _m and E _GABA are both maintained at negative levels of about −83 and −80 mV, respectively. Conversely, a KB deficiency causes a significant depolarization of both E _m (>5 mV) and E _GABA (>15 mV). The KB-mediated shift in E _GABA is largely determined by the interaction of the NKCC1 cotransporter and Cl⁻/HCO₃ transporter(s). Therefore, by inducing a hyperpolarizing shift in E _m and modulating GABA signaling mode, KBs can efficiently control the excitability of neonatal cortical neurons.     

The Ca2+-activated K+ channel and its functional roles in smooth muscle cells of guinea pig taenia coli

Currents through single potassium channels were studied in cell-attached or inside-out patches from collagenase-dispersed smooth muscle cells of the guinea pig taenia coli. Under conditions mimicking the physiological state with [K+]i = 135 mM: [K+]o = 5.4 mM, three distinct types of K+ channel were identified with conductances around 0 mV of 147, 94, and 63 pS. The activities of the 94- and 63-pS channel were observed infrequently. The 147-pS channel was most abundant. It has a reversal potential of approximately -75 mV. It is sensitive to [Ca2+]i and to membrane potential. At -30 mV, the probability of a channel being open is at a minimum. At more positive voltages, the probability follows Boltzman distribution. A 10-fold change in [Ca2+]i causes a 25-mV negative shift of the voltage where half of the channels are open; an 11.3-mV change in membrane potential produces an e-fold increase in the probability of the channel being open when P is low. At voltages between -30 and -50 mV, the open probability increases in an anomalous manner because of a large decrease of the channel closed time without much change in the channel open time. This anomalous activity may play a regulatory role in maintaining the resting potential. The histograms of channel open and closed time fit well, respectively, with single and double exponential distributions. Upon step depolarizations by 100-ms pulses, the 147-pS channel opens with a brief delay. The delay shortens and both the number of open channels and the open time increase with increasing positivity of the potential. The averaged currents during the step depolarizations closely resemble the delayed rectifying outward K+ currents in whole-cell recordings.     

Large-conductance calcium-activated anion channel characteristics in neuroblastoma cells

Large-conductance anion channel characteristics were investigated in neuroblastoma cells (N2A) by using different configurations of the patch-clamp technique. In excised patches, the channel was induced by depolarising potentials in 90% of experiments, had a conductance of 340 pS in symmetrical 135 mmol/l NaCl and exhibited the typical bell-shape activity. Neither the channel induction nor the channel activity was affected by rising the Ca2+ concentration on the cytopasmic side of membranes. In cell-attached configuration the maximal channel activity was shifted towards more positive potentials in comparison to that of excised patches and an increase in intracellular Ca2+, obtained by extracellular application of the Ca2+-ionophore A23187 in the presence of 0.2 micromol/l Ca2+, induced single-channel currents in 80% of patches compared to 31% of cell-attached experiments showing channel activity in normal conditions. In turn, application of 2 micromol/l Ca2+ induced channel activity in 100% of patches. The reversal potential of the channel in cell-attached patches was around -10 mV as the resting potential of cells eliciting channel activity. For cells where channel activity was not detected in cell-attached mode, the resting potential was around -45 mV. Channel activity could be restored in most whole-cell recordings in the presence of 2 micromol/l or more intracellular Ca2+ concentrations. The Ca2+-induction and the relation between channel activity and cell resting potential seem to suggest a role of the large-conductance anion channel in resting potential modulation during some basic functions of the neuroblastoma cell proliferation.     

Na channel kinetics during the spontaneous heart beat in embryonic chick ventricle cells.

Using cell-attached and whole-cell recording techniques simultaneously allows the measurement of Na currents during action potentials in beating heart cells. In 7-d chick ventricle, the dynamic reversal potential for Na is 30 mV, which is 20 mV less than the reversal potential in nonbeating cells. This result implies that the spontaneous activity of heart cells raises the Na concentration at the internal face of the membrane to nearly 40 mM. Fitting the Na action currents to the Hodgkin and Huxley equations shows that patches may contain from 50 to 700 Na channels, with an average density of 23 +/- 21 per micron2. Only approximately 2% of the available Na channels are open at the peak of the Na action current. This low probability is a consequence of the channels' continual inactivation during the diastolic depolarization phase.     

Functional evidence for Na+-activated K+ channels in circular smooth muscle of the opossum lower esophageal sphincter

Na(+) reduction induces contraction of opossum lower esophageal sphincter (LES) circular smooth muscle strips in vitro; however, the mechanism(s) by which this occurs is unknown. The purpose of the present study was to investigate the electrophysiological effects of low Na(+) on opossum LES circular smooth muscle. In the presence of atropine, quanethidine, nifedipine, and substance P, conventional intracellular electrodes recorded a resting membrane potential (RMP) of -37.5 +/- 0.9 mV (n = 4). Decreasing [Na(+)] from 144.1 to 26.1 mM by substitution of equimolar NaCl with choline Cl depolarized the RMP by 7.1 +/- 1.1 mV. Whole cell patch-clamp recordings revealed outward K(+) currents that began to activate at -60 mV using 400-ms stepped test pulses (-120 to +100 mV) with increments of 20 mV from holding potential of -80 mV. Reduction of [Na(+)] in the bath solution inhibited K(+) currents in a concentration-dependent manner. Single channels with conductance of 49-60 pS were recorded using cell-attached patch-clamp configurations. The channel open probability was significantly decreased by substitution of bath Na(+) with equimolar choline. A 10-fold increase of [K(+)] in the pipette shifted the reversal potential of the single channels to the positive by -50 mV. These data suggest that Na(+)-activated K(+) channels exist in the circular smooth muscle of the opossum LES.     

Charybdotoxin selectively blocks small Ca-activated K channels in Aplysia neurons

The action of charybdotoxin (ChTX), a peptide component isolated from the venom of the scorpion Leiurus quinquestriatus, was investigated on membrane currents of identified neurons from the marine mollusk, Aplysia californica. Macroscopic current recordings showed that the external application of ChTX blocks the Ca-activated K current in a dose- and voltage-dependent manner. The apparent dissociation constant is 30 nM at V = -30 mV and increases e-fold for a +50- to +70-mV change in membrane potential, which indicates that the toxin molecule is sensitive to approximately 35% of the transmembrane electric field. The toxin is bound to the receptor with a 1:1 stoichiometry and its effect is reversible after washout. The toxin also suppresses the membrane leakage conductance and a resting K conductance activated by internal Ca ions. The toxin has no significant effect on the inward Na or Ca currents, the transient K current, or the delayed rectifier K current. Records from Ca-activated K channels revealed a single channel conductance of 35 +/- 5 pS at V = 0 mV in asymmetrical K solution. The channel open probability increased with the internal Ca concentration and with membrane voltage. The K channels were blocked by submillimolar concentrations of tetraethylammonium ions and by nanomolar concentrations of ChTX, but were not blocked by 4-aminopyridine if applied externally on outside-out patches. From the effects of ChTX on K current and on bursting pacemaker activity, it is concluded that the termination of bursts is in part controlled by a Ca-activated K conductance.     

Satellite glial cells In Situ within mammalian prevertebral ganglia express K+ channels active at rest potential

Patch-clamp experiments were performed on satellite glial cells wrapped around sympathetic neurons in the rabbit coeliac ganglion. With the cleaning method used, the glial cells could be kept in place and were directly accessible to the patch-clamp pipettes. Whole-cell recordings showed that glial cells had almost ohmic properties. Their resting potential (–79.1±1.2 mV) was found to be very nearly the same as the K⁺ reversal potential and 20 mV more negative than that of the neurons they encapsulated. Unitary currents from ionic channels present in the glial membrane were recorded in the cell-attached configuration with pipettes filled with various amounts of K⁺, Na⁺ and gluconate. Only K⁺-selective channels with slight inwardly rectifying properties (in the presence of 150 mM [K⁺]₀) were detected. These channels were active (P ₀=0.7–0.8) at the cell resting potential. The channel conductance, but not its opening probability, was dependent on the [K⁺] in the pipette. Cl^–-selective channels (outwardly rectifying and large conductance channels) were detected in excised patches.The properties of the K⁺ channels (increased inward current with [K⁺] and detectable outward current at low [K⁺]) are well suited for siphoning the K⁺ released by active neurons.     

Regulation of Cl- channels by multifunctional CaM kinase.

cAMP kinase has been shown to mediate the cAMP pathway for regulation of Cl- channels in lymphocytes, but the mediator of an alternative, Ca2+ pathway has not been identified. We show here that Ca2+ ionophore activates Cl- currents in cell-attached and whole-cell patch-clamp recordings of Jurkat T lymphocytes, but this activation is not direct. The effect of Ca2+ ionophore on whole-cell Cl- currents is inhibited by a specific peptide inhibitor of multifunctional Ca2+/calmodulin-dependent protein kinase (CaM kinase). Furthermore, Cl- channels are activated in excised patches by purified CaM kinase in a fashion that mimics the effect of Ca2+ ionophore in cell-attached recordings. These results suggest that CaM kinase mediates the Ca2+ pathway of Cl- channel activation.     

A slow voltage-activated potassium current in rat vagal neurons.

Potassium currents play a key role in controlling the excitability of neurons. In this paper we describe the properties of a novel voltage-activated potassium current in neurons of the rat dorsal motor nucleus of the vagus (DMV). Intracellular recordings were made from DMV neurons in transverse slices of the medulla. Under voltage clamp, depolarization of these neurons from hyperpolarized membrane potentials (more negative than -80 mV) activated two transient outward currents. One had fast kinetics and had properties similar to A-currents. The other current had an activation threshold of around -95 mV (from a holding potential -110 mV) and inactivated with a time constant of about 3s. It had a reversal potential close to the potassium equilibrium potential. This current was not calcium dependent and was not blocked by 4-aminopyridine (5 mM), catechol (5 mM) or tetraethylammonium (20 mM). It was completely inactivated at the resting membrane potential. This current therefore represents a new type of voltage-activated potassium current. It is suggested that this current might act as a brake to repetitive firing when the neuron is depolarized from membrane potentials negative to the resting potential.     

Mediation by calcium/calmodulin-dependent protein kinase II of suppression of GABAA receptors by NMDA

Using nystatin-perforated whole-cell recording configuration, the modulatory effect of N-methyl-D-aspartate (NMDA) on -aminobutyric acid (GABA)-activated whole-cell currents was investigated in neurons freshly dissociated from the rat sacral dorsal commissural nucleus (SDCN). The results showed that: (I) NMDA suppressed GABA- and muscimol (Mus)-activated currents (IGABA and Imus), respectively in the Mg2+-free external solution containing 1 mol/L glycine at a holding potential (VH) of 40 mV in SDCN neurons. The selective NMDA receptor antagonist, D-2-amino-5-phosphonovaleric acid (APV, 100 mol/L), inhibited the NMDA-evoked currents and blocked the NMDA-induced suppression of IGABA; (ii) when the neurons were incubated in a Ca2+-free bath or pre-loaded with a membrane-permeable Ca2+ chelator, BAPTA AM (10 mol/L), the inhibitory effect of NMDA on IGABA disappeared. Cd2+ (10 mol/L) or La3+ (30 mol/L), the non-selective blockers of voltage-dependent calcium channels, did not affect the suppression of IGABA by NMDA application; (iii) the suppression of IGABA by NMDA was inhibited by KN-62, a calcium/calmodulin-dependent protein kinase II (CaMKII) inhibitor. These results indicated that the inhibition of GABA response by NMDA is Ca2+-dependent and CaMKII is involved in the process of the Ca2+-dependent inhibition.     

The actions of intermediate and long-chain n-alkanols on unitary NMDA currents in hippocampal neurons.

The actions of the n-alkanols butanol, pentanol, and octanol on unitary currents passing through N-methyl-D-aspartate (NMDA) ion channels have been studied in cultured CA1 hippocampal neurons. The cell-attached patch clamp method, with L-homocysteic acid included in the patch pipette, was used to record single channel NMDA currents at the cell resting potential or for hyperpolarizing patch potentials. With the n-alkanols added to the bath solution, the mean open times for the NMDA channel were diminished and the channel conductance was unchanged. A decrease in mean open time to about 70% of control value was found with butanol (3 mM), pentanol (1 mM), and octanol (0.02 mM). In addition the n-alkanols had small effects to decrease the frequency of channel openings and to increase the amplitude of the unitary currents. The effects of the alcohols on intracellular calcium levels, during NMDA applications, were also measured using the fluorescent dye FURA II.     

The effect of leu-enkephalin on membrane potential and activity of the rat respiratory center neurons in vitro

In frontal brainstem slices of Wistar rats, the whole-cell patch-clamp recordings showed the effect of opioid peptide leu-enkephalin (10 nM-1 microM) on membrane potential and spontaneous activity pattern of neurons in two divisions of the respiratory center, ventro-lateral area of the solitary tract nucleus, and the pre-B?tzinger complex. Leu-enkephalin induced a membrane hyperpolarization of the respiratory centre neurons and reduction of the spike activity level in spontaneously active units. After administration of leu-enkephalin, a decrease in frequency of bursts was found in bursting cells of the pro-B?tzinger complex; in two cases, there was a transition of bursting activity to tonic one. The data suggest that the mechanism of the central respiratory activity of leu-enkephalin is based on its direct action at the level of membrane of the respiratory centre neurons.     

The use of thin slices of myocardium for recording the currents across single ion channels]

Thin cardiac slices (100-200 microns) from newborn (1-14 days old) rat heart ventricles were used for patch clamp recordings. High resistance seals (10-50 GOhms) between patch-clamp pipettes and the membrane of cardiac cells as well as classical patch-clamp configurations can be achieved on this preparation without any enzymatic treatment of tissue. Resisting potential for cardiac cells measured in whole-cell configuration ranged between -30 and -65 mV. Averaged sodium currents and single inward rectifying potassium elementary currents recorded in cell-attached mode displayed basic features similar to those previously reported for isolated rat ventricular cells. Application of the method described here in cardiac electrophysiology will allow patch-clamp studies on heart cells without the complicated procedures of cell isolation. In addition, the uncertainty associated with enzyme treatment can be avoided. In future, this technique could be a new tool for studying electrophysiological properties of heart cells in situ.     

Voltage-gated and Ca(2+)-activated K+ channels in intact human T lymphocytes. Noninvasive measurements of membrane currents, membrane potential, and intracellular calcium

Voltage-gated n-type K(V) and Ca(2+)-activated K+ [K(Ca)] channels were studied in cell-attached patches of activated human T lymphocytes. The single-channel conductance of the K(V) channel near the resting membrane potential (Vm) was 10 pS with low K+ solution in the pipette, and 33 pS with high K+ solution in the pipette. With high K+ pipette solution, the channel showed inward rectification at positive potentials. K(V) channels in cell-attached patches of T lymphocytes inactivated more slowly than K(V) channels in the whole-cell configuration. In intact cells, steady state inactivation at the resting membrane potential was incomplete, and the threshold for activation was close to Vm. This indicates that the K(V) channel is active in the physiological Vm range. An accurate, quantitative measure for Vm was obtained from the reversal potential of the K(V) current evoked by ramp stimulation in cell-attached patches, with high K+ solution in the pipette. This method yielded an average resting Vm for activated human T lymphocytes of -59 mV. Fluctuations in Vm were detected from changes in the reversal potential. Ionomycin activates K(Ca) channels and hyperpolarizes Vm to the Nernst potential for K+. Elevating intracellular Ca2+ concentration ([Ca2+]i) by ionomycin opened a 33-50-pS channel, identified kinetically as the CTX-sensitive IK-type K(Ca) channel. The Ca2+ sensitivity of the K(Ca) channel in intact cells was determined by measuring [Ca2+]i and the activity of single K(Ca) channels simultaneously. The threshold for activation was between 100 and 200 nM; half-maximal activation occurred at 450 nM. At concentrations > 1 microM, channel activity decreased. Stimulation of the T-cell receptor/CD3 complex using the mitogenic lectin, PHA, increased [Ca2+]i, and increased channel activity and current amplitude resulting from membrane hyperpolarization.