Dependence of single channel properties of the N-methyl-d-aspartate ion channel on stereoisomer agonists

The cell-attached patch-clamp configuration has been used to determine the single channel properties of the N-methyl-D-aspartate (NMDA) ion channel with activation of the NMDA receptor by stereoisomer agonists. All of the agonists studied, including the L and d forms of N-methyl-aspartate and the L and d forms of homocysteate, activated a 42-pS conductance channel in cultured hippocampal neurons. For all agonists, the mean open times of the channel were diminished with increased patch hyperpolarization and exhibited an exponential dependence on potential over the range -40 mV to -120 mV. The mean open times, for patch potentials close to resting potential, and the mean frequencies of channel openings, at all patch potentials, were significantly different between each member of the stereoisomer pairs. For both L-homocysteate and NMLA, a fourfold increase in the patch pipette concentration caused an approximate quadrupling in the frequency of unitary events, with no significant change in mean open time. The open channel probability was used as a measure of agonist potency, and, at a concentration of 30 M, NMDA and L-homocysteate were significantly more potent (P open in excess of 1.5%) than the corresponding stereoisomer compounds NMLA and D-homocysteate (P open near 0.3%). The relative potencies of the stereoisomer pairs were in reasonable agreement with the potency ratios measured in binding studies.     

Quinolinate activation of N-methyl-D-aspartate ion channels in rat hippocampal neurons.

The cell attached configuration of the patch clamp method has been used to determine the single channel properties of the ion channel coupled to activation of the N-methyl-D-aspartate (NMDA) receptor by the endogenous NMDA agonist quinolinate. Openings of the NMDA channel were recorded from cultured CA1 hippocampal neurons over a hyperpolarizing potential range from cell resting potential. The slope conductance of the channel was 39 pS with 75 microM quinolinate, 1.8 mM Ca2+ and no Mg2+ in the patch pipette. The mean channel open times were decreased with hyperpolarization in an exponential manner with a mean slope of 0.6 ms/20 mV. Addition of Mg2+ to the pipette (at 30 microM) caused the mean open time, at a potential of -100 mV, to be decreased to a value about one-third that of control. The mean open times with quinolinate as the agonist were shorter for all potentials studied compared with activation of the NMDA receptor with NMDA or D-cis-1-amino-1,3-cyclopentanedicarboxylic acid (ACPD). Both the mean open times and the channel amplitudes were significantly altered when the bath temperature was decreased; the Q10 values for both quantities were in excess of 2.8.     

Properties of the bursting Na channel in the presence of DPI 201-106 in guinea-pig ventricular myocytes

Single Na channel currents were measured in cellattached patches of guinea-pig ventricular myocytes in the presence of the S-enantiomer of DPI 201-106. DPI changes the kinetic pattern of channel activity from short living openings at the beginning of a depolarizing pulse (voltageindependent mean open time about 0.4 ms between –60 and –20 mV), into longlasting bursts of openings. The single channel current-voltage relation can be approximated by a straight line with a single channel conductance of 15 pS, which is the same as in the absence of DPI, and a reversal potential near the estimated Na equilibrium potential (+ 74 mV). The ensemble averaged Na current shows a fast peak of inward current, which partially decays within less than 10 ms, but which shows a large component which decays very slowly with a time constant of the order of 1s (1.31±0.6 s at –30 mV, 19 measurements in 12 cell-attached patches). The slowly decaying component activates with a half-maximum potential at –55.4±2.3 mV and a slope parameter s of 4.9±1.9 mV. The half-maximum potential of the steady-state inactivation is –115.6±1.8 mV, and the slope parameter is 9.1±1.5 mV. The open time distribution can be fitted by a single exponential only at potentials negative to –40 mV. The time constant is 1.3±0.14 ms at –50 mV (7 patches). At more positive potentials a slower second component is present (14.4±7.1 ms at –30mV, 7 patches), in addition to a fast component with a time constant between 2 and 3 ms (2.2±1.9 ms at –30 mV, 7 patches). The closed time distribution contains two exponential components. The time constant of the fast component decreases from about 1.3 ms at –70 mV to 0.3 ms at –20 mV (0.3±0.17 ms at –30 mV, 10 patches). The time constant of the second component decreases from about 4.0 ms at –70 mV to 1.5 ms at –20 mV (2.2±1.8 ms at –30 mV, 10 patches). At potentials positive to –50 mV also a small very slow component is present (8.8±5.6 ms at –30 mV). The contribution of the slow component to the closed time distribution increases for stronger depolarizations from 2.2±2.1% at –40 mV to 64±22% at –20 mV. The contribution of short closings to the total number of closings is increased from 51±4% at –70 mV to 83±12% at –20 mV. Openings occur clearly in bursts and the burst duration shows a very good correlation with the time constant of the decay of the ensemble average current in a large number of different experimental conditions. The channels are blocked by application of TTX. With 10 and 30 M TTX in the patch pipette (1 s pacing interval) the probability of the channel being open is decreased by about 90%. The long mean open time is significantly reduced when TTX is present in the patch pipette (5.7±2.6 ms at –30 mV with 5–30 M TTX, 4 patches,n=11). No significant differences are obtained for both closed times (_c1 = 0.26±0.07 ms, _c2 = 1.9±0.7 ms, 4 patches,n=9). The steady-state inactivation is not significantly changed in the presence of TTX (V_H=–112±2.9 mV, s=9.0±2.8 mV). The open state probability of the bursting Na channel is also reduced by more than 80% when 200 M Cd is present in the pipette solution (all mean ±SD).     

Single channel analysis of the inward rectifier K current in the rabbit ventricular cells

The inward rectifier K channel in rabbit ventricular cells was studied by the patch-clamp method. Single channel currents were recorded in giga-sealed cell-attached patches with 150 mM K+ in the pipette. The slope conductance in the membrane potential range from -140 to -40 mV was 46.6 +/- 6.7 pS (mean +/- S.D., n = 16), and was reduced by decreasing [K+] in the pipette (20 or 50 mM). The channel was blocked by an application of Cs+ or Ba2+ (0.04-1 mM) in the pipette. Outwardly directed current, recorded with 50 mM K+ in the pipette, revealed the inward rectification of the single channel current. The probability of the channel being open was 0.33 +/- 0.05 (n = 10) at the resting potential (RP=-81.7 +/- 1.7 mV, n = 16) with 150 mM K+ in the pipette, and it decreased with hyperpolarization. The mean open time of the channel was 178 +/- 25 msec (n = 6) at RP. The closed time of the channel seemed to have two exponential components, with time constants of 11.0 +/- 2.0 msec and 1.92 +/- 0.52 sec (n = 6) at RP. The slower time constant was increased with hyperpolarization. The averaged patch current recorded upon hyperpolarizing pulses demonstrated a time-dependent current decay as expected from the single channel kinetics. The results indicated that the inward rectifier K+ current has time- and voltage-dependent kinetics.     

Two types of transient outward currents in cardiac ventricular cells of mice

Ventricular cells of adult mice were prepared by an enzyme digestion procedure. Single channel currents were recorded by a conventional patch clamp technique from cell attached patches. Voltage steps from the holding potential of –80 mV to test potentials between –35 and +50 mV caused openings of two types of outward currents through single channels with the conductances of 27 and 12 pS, respectively. The averaged currents reveal transient time courses for both channel types. The current-voltage relations of both single channel currents were linear over the tested voltage range and intersected the voltage axis at –70 mV. This indicates that both single channel currents are mainly carried by potassium ions. All open and closed times were found to be voltage independent. The 27 pS channel had a mean open time of 3.9±1.0 ms (n=8). The closed time consisted of two components with ₁ = 2.1 ± 0.2 ms and ₂ = 50 ± 19 ms (n=8). The 12 pS channel had a mean open time of 34.0±5.2 ms (n=3) and the two components of the mean closed time have been calculated as ₁ = 8.3 ± 2.1 ms and ₂ = 120 ± 50 ms (n=3; all mean ±SD).     

Permeation properties of a non-selective cation channel in human vascular endothelial cells

Summary Endothelial cells obtained from human umbilical chord have been studied by the patch clamp method. An ion channel is described that is activated by M concentrations of histamine and shows a slow run-down in cell-attached patches. After excision, channel activity quickly runs down to zero open probability. In symmetrical potassium concentrations (140 mM K in the bath and the pipette), the single channel conductance is 28±2 pS and the reversal potential is 0.3±0.8 mV (mean ± SEM, n=4). With 140 mM Na in the pipette, the conductance is 26±2 pS. A reversal potential of -1.5±0.9 mV (n=7) was measured. With 60 mM Ca and 70 mM Na in the pipette, 140 mM K in the bath, the reversal potential was -11±3 mV, the single channel conductance 16±3 pS (n=5). The single channel conductance in 110 mM Ca (pipette) and 140 mM K (bath) is 8±2 pS and the reversal potential is –18±6 mV (n=3). From analysis of the reversal potentials, a permeation ratio of KNaCa=10.90.2 was calculated. This ligand-gated non-selective cation channel in human endothelial cells is Ca permeable and could induce a sustained agonist mediated Ca influx.     

Single glutamate-gated synaptic channels at the crayfish neuromuscular junction

Single, glutamate activated ionic channel currents were recorded from crayfish muscle in the cell attached mode. Different concentrations of glutamate were present in the patch clamp pipette. Bursts of openings were observed with a concentration dependent number of short gaps per burst. Also the mean burst length was concentration dependent and varied between 0.3 ms (100 microM) and 1.3 ms (20 mM). Even with the highest concentrations of glutamate the channel activations were well separated and the beginning and the end of a burst could be defined. The distributions of open times and of burst lengths could be fitted well with a single exponential component for all studied concentrations of glutamate. The distributions of closed times were composed of two or three exponential components (with possibly more than one channel contributing). The mean burst length was compared with the time constants of decay of synaptic currents (0.8-3.0 ms at 19 degrees C) which were measured either with the same pipette as the single channel currents or with a macro patch technique. An estimation of the glutamate concentration at the receptors during synaptic transmission gave values in the millimolar range. The most simple model of glutamate-receptor interaction contains two binding sites for glutamate but no singly liganded open states. Rate constants were estimated for this model.     

The luminal K+ channel of the thick ascending limb of Henle's loop

In vitro perfused rat thick ascending limbs of Henle's loop (TAL) were used (n=260) to analyse the conductance properties of the luminal membrane applying the patch-clamp technique. Medullary (mTAL) and cortical (cTAL) tubule segments were dissected and perfused in vitro. The free end of the tubule was held and immobilized at one edge by a holding pipette kept under continuous suction. A micropositioner was used to insert a patch pipette into the lumen, and a gigaohm seal with the luminal membrane was achieved in 455 instances out of considerably more trials. In approximately 20% of all gigaohm seals recordings of single ionic channels were obtained. We have identified only one single type of K⁺ channel in these cell-attached and cell-excised recordings. In the cell-attached configuration with KCl or NaCl in the pipette, the channel had a conductance of 60±6 pS (n=24) and 31±7 pS (n=4) respectively. In cell-free patches with KCl either in the patch pipette or in the bath and with a Ringer-type solution (NaCl) on the opposite side the conductance was 72±4 pS (n=37) at a clamp voltage of 0 mV. The permeability was 0.33±0.02 · 10^±12 cm³/s. The selectivity sequence für this channel was: K⁺=Rb⁺=NH ₄ ⁺ =Cs⁺>Li⁺Na⁺=0; the conductance sequence was K⁺Li⁺Rb⁺=Cs⁺= NH ₄ ⁺ =Na⁺=0. In excised patches Rb⁺, Cs⁺ and NH ₄ ⁺ when present in the bath at 145 mmol/l all inhibited K⁺ currents out of the pipette. The channel kinetics were described by one open (9.5±1.5 ms, n=18) and by two closed (1.4±0.1 and 14±2 ms) time constants. The open probability of this channel was increased by depolarization. The channel open probability was reduced voltage dependently by Ba²⁺ (half maximal inhibition at 0 mV: 0.07 mmol/l) from the cytosolic side. Verapamil, diltiazem, quinine and quinidine inhibited at approximately 1 mol/l ±0.1 mmol/l from either side. Similarly, the amino cations lidocaine, tetraethylammonium and choline inhibited at 10–100 mmol/l. The channel was downregulated in its open probability by cytosolic Ca²⁺ activities > 10^±7 mol/l and by adenosine triphosphate 10^±4 mol/l. The open probability was downregulated by decreasing cytosolic pH (2-fold by a decrease in pH by 0.2 units). The described channel differs in several properties from the K⁺ channels of other epithelia and of renal cells and TAL cells in culture. It appears to be responsible for K⁺ recycling in the TAL segment.Preliminary reports of the present study have been given at the following conferences: Tagung der Deutschen Physiologischen Gesellschaft, Würzburg, October 1988; Membranforum, Frankfurt, April 1989; 3rd Int. Conf. Diur., Mexico City, April 1989; 3rd Nephrology Forefront Symposium, Arrola, July, 1989; IUPS meeting, Helsinki, July 1989. This study has been supported by Deutsche Forschungsgemeinschaft Grant No. Gr 480/9     

Calcium channels in smooth muscle cells from cerebral precapillary arterioles activate at more negative potentials than those from basilar artery

We compared Ca²⁺ channels in cell-attached patches of smooth muscle cells from cerebral precapillary arterioles and basilar artery of guinea pig. Patches were studied without Ca²⁺ channel activators in the pipette solution. In both preparations, a 23 pS channel (40 mM Ba²⁺) sensitive to block by nifedipine was identified. In arteriolar but not in basilar artery patches, channel activity was recorded without apparent inactivation at potentials of –40 to –20 mV. Values for the number of channels in a patch x probability of channel opening (n·P_o) at various potentials were fit to a Boltzmann function. For the arteriolar patches (n=5) and for patches from basilar artery (n=5), the midpoint potentials for the voltage dependence of n·P_o were –9.3 mV and +8.9 mV, and maximum values of n·P_o at positive potentials were 1.23 and 0.33. At potentials 0 mV, the average for the maximum number of superimposed openings in basilar artery patches was 1.7 (n=17) and in arteriolar patches was 6.5 (n=6). For both preparations, histograms of channel open times at –10 mV required two time constants, 0.48 and 3.95 ms, and the shorter open state accounted for 88% of openings. Our data indicate that Ca²⁺ channel activity is likely to be more prominent near resting membrane potentials in arteriolar cells than in basilar artery cells.     

Potentiation of a metabotropic glutamatergic response following NMDA receptor activation in rat hippocampus

Interactions between metabotropic glutamate andN-methyl-D-aspartate (NMDA) receptor-mediated responses were investigated in hippocampal CA3 cells using the single-electrode voltage-clamp method. Bath application (2.5–10 M, 30 s) or iontophoresis of 1-amino-cyclopentyl-trans-1S, 3R-dicarboxylate (ACPD), a selective agonist for metabotropic glutamate receptors, resulted in an inward current associated with a decrease in membrane conductance. Following transient bath application of NMDA (5–10 M, 30–60 s), the ACPD-induced inward current was potentiated for a period of up to 25 min (by 61±8% with bath application, by 32±15% with iontophoresis). Transient application of NMDA did not result in a potentiation of ionotropic RS--amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) or metabotropic muscarinic responses. ACPD responses were not potentiated following transient AMPA application. Intracellular buffering of calcium with tetrapotassium bis(O-aminophenoxy)-ethane-N,N,N,N-tetraacetic acid (BAPTA) prevented potentiation by NMDA in all cells. Bath application of arachidonic acid did not mimic the NMDA-induced potentiation. These results demonstrate that activation of NMDA receptors can specifically induce a long-lasting potentiation of a metabotropic glutamatergic response in hippocampal CA3 pyramidal cells. The characterization of this interaction may contribute to the elucidation of the physiological significance of metabotropic glutamate receptors.     

Cation channel blocked by extracellular Ca2+ in the apical membrane of the chick embryonic ectoderm

In the chick embryo (20 h incubation, gastrula stage), the apical membrane of the ectodermal cells shows a high density of a non-selective cation channel which is blocked by very low extracellular Ca²⁺ concentrations. Properties of this channel were studied at the single-channel level using the patch-clamp technique in the cell-attached mode.With 1 mmol/l Ca²⁺ in the pipette, only outward current was present and the channel conductance measured at +120 mV was 25.5 pS. In the absence of Ca²⁺, also inward current through the channel was observed. The conductances measured at –50 mV were 49.5 pS with Na⁺ as the charge carrier, 72.5 pS with K⁺, 49.1 pS with Cs⁺, and 18.5 pS with Li⁺. The conductance measured at +80 mV was around 23 pS in all four cases. The reversal potential was similar (around 25 mV) for all four ions, which indicates a poor selectivity of the channel. In the absence of Ca²⁺ and the presence of 1 mmol/l ethylene-bis(oxonitrilo) tetraacetate (EGTA), the kinetics of the channel were characterized by bursts of the order of seconds. During a burst, the channel flickered between one open and one closed level. The open time was constant between –30 mV and –80 mV, while the closed time decreased with hyperpolarization. The open time varied according to the permeant ion (K⁺+=Cs⁺+).Extracellular Ca²⁺ blocked the inward current in a voltage-dependent manner. The K _d values, 1 mol/l at –30 mV and 3.2 mol/l at –80 mV, indicate that Ca²⁺ ions exit the channel toward the intracellular side. A weak voltage dependency of the association rate constant suggests that the Ca²⁺-binding site is close to the outside mouth. Extracellular Ca²⁺ was much less efficient at blocking the outward current (K _d about 1 mmol/l at 80 mV). Tetracaine, but not uraniumdioxide, decreased the opening probability of the channel. The embryonic channel shows similarities with the Ca²⁺-blockable, poorly selective channel described in the epithelium of toad urinary bladder.     

Characteristics of single Na+ channels of adult human skeletal muscle

The patch-clamp technique was used to study Na⁺ channels of human skeletal muscle. Preparations were from biopsies of quadriceps muscle from adults who were not suffering from neuromuscular diseases. Activity of Na⁺ channels was recorded from inside-out patches when the membrane potential was stepped from a holding potential of ±110mV to potential above a threshold of about ±65 mV. Single channel activity increased within minutes after hyperpolarizing the patch due to recovery from ultraslow inactivation. Up to ten Na⁺ channels were active in individual patches. Macroscopic currents were reconstructed by averaging single channel currents. The time-to-peak current declined from 1.6 ms at ±60 mV to 0.5 ms at +10 mV. The currents decayed mono-exponentially with time constants between 12.1 ms at ±60 mV and 0.4 ms at +10 mV (21°C). The conductance of single Na⁺ channels was 1.65 pS and the mean open time was voltage-dependent. At ±50 mV, the mean open time was 0.4 ms, while positive to ±10 mV it increased to values above 1 ms. In the threshold potential range, the number of openings per depolarizing pulse was larger than the number of channels under the patch-clamp pipette, indicating reopening of Na⁺ channels at this potential. Openings could be observed only rarely 10 ms after onset of depolarization and the macroscopic current produced by late openings was less than 0.1% of the peak current. Human skeletal muscle is thus suitable for investigation with the patch-clamp technique and the determination of properties of Na⁺ channels with this technique could be the basis for an assessment of possible defects of these channels in diseased muscle.     

Giga-seal formation alters properties of sodium channels of human myoballs

The influence of giga-seal formation on the properties of the Na⁺ channels within the covered membrane patch was investigated with a whole-cell pipette and a patch pipette applied to the same cell. Current kinetics, current/voltage relation and channel densities were determined in three combinations: (i) voltage-clamping and current recording with the whole-cell pipette, (ii) voltage-clamping with the whole-cell pipette and current recording with the patch pipette and, (iii) voltage-clamping and current recording with the patch pipette. The Hodgkin-Huxley (1952) parameters _m and _h were smaller for the patch currents than for the whole cell, and the h curve was shifted in the negative direction. The channel density was of the order of 10 times smaller. All effects were independent of the extracellular Ca²⁺ concentration. The capacitive current generated in the patch by the whole-cell Na⁺ current and its effect on the transmembrane voltage of the patch were evaluated. The kinetic parameters of the Na⁺ channels in the patch did not depend on whether the voltage was clamped with the whole-cell pipette or the patch pipette. Thus, the results are not due to spurious voltage.     

Intracellular calcium ions activate a low-conductance chloride channel in smooth-muscle cells isolated from human mesenteric artery

Calcium-activated chloride currents were studied by the patch-clamp technique in vascular smooth muscle cells (VSMC) isolated from human mesenteric arteries. Bath application of 20 mM caffeine caused the cell membrane to depolarize by a calcium-activated inward current that peaked to –654±230 pA (holding potential –50 mV). Cell-attached, at the same time inwardly directed single-channel currents were detected with an amplitude of –0.22 pA. In open-cell-attached patches channel activity was triggered by elevating [Ca²⁺]_i to 10 M. At –60 mV the mean amplitude of the current was –0.24 pA and the mean open time of the channels was 28 ms. Plotting the amplitude of the current versus the test potential yielded a single-channel conductance of 2.8±0.5 pS. The currents disappeared when [Cl^–] was reduced from 150 mM to 5 mM at the cytosolic side of the inside-out patch at a holding potential of -60 mV (calculated reversal potential –58 mV) suggesting that the calcium-activated current was a chloride current. This suggests that, in human mesenteric VSMC, elevation of [Ca²⁺]_i activates a low-conductance chloride channel, which may mediate the agonist-induced depolarization of the cell membrane.     

Some properties of single potassium channels in cultured oligodendrocytes

K⁺ channels were studied in oligodendrocytes in cultures of mouse spinal cord. Single channel currents were measured using the gigaseal technique. The conductance of the channels varied greatly i.e. from 6 to 125 pS (38±28 SD,N=21). In some patches there were up to three current levels of the same size. At –70 mV the open state probability was 0.51±0.17 and the average duration of an opening 70±20 ms for 4 channels with conductance from 16–57 pS. These analyses exclude brief flickering (less than 2 ms) or long closed periods (seconds to minutes). These times were not markedly affected by pulling the patch off the cell or by superfusing the isolated patch with media containing 10 mmol×1^–1 TEA or EGTA without Ca²⁺. At membrane potentials between –90 and –30 mV there was a small but consistent effect of depolarization to increase the open state probability. Large positive or negative voltage steps decreased the open state probability. Current voltage measurements on intact cells showed a striking decrease in membrane conductance at these large membrane potentials. The leakage conductance of the patch also exhibited some K⁺ selectivity. The oligodendrocyte membrane appears to contain about one K⁺ channel per 5 m². The known electrical properties of cultured oligodendrocytes can essentially be explained by the distribution and properties of these K⁺ channels.     

Blocker Studies of the Functional Architecture of the NMDA Receptor Channel

Blockade of ion channels passing through the NMDA receptors of isolated rat hippocampus pyramidal neurons with tetraalkylammonium compounds, 9-aminoacridine, and Mg²⁺was studied using patch-clamp methods in the whole-cell configuration. Currents through NMDA channels were evoked by application of 100 M aspartate in magnesium-free medium containing glycine (3 M) to neurons. Analysis of the kinetics, charge transfer, and relationships between the extent of suppression of stationary currents on the one hand and membrane potential, agonist concentration, and blocker concentration on the other showed that blockers had different effects on the closing, desensitization, and agonist dissociation of NMDA channels. The size of the blocker was found to be the decisive factor determining its action on the gating functions of NMDA channels: larger blockers prevented closure and/or desensitization of the channel; smaller blockers only had partial effects on these processes, while the smallest blockers had no effect at all. These experiments showed that the apparent affinity of the blocker for the channel (1/IC₅₀) depended not only on the microscopic equilibrium dissociation constant (K _d), but also on the number of blocker binding sites, their mutual influences, and, of particular importance, the interaction of the blocker with the gating structures of the channel. These data led us to propose hypotheses relating to the geometry of the NMDA channel and the structure of its gating mechanism. The channel diameter at the level of activated gates was estimated to be 11 .     

The T-type Ca channel in guinea-pig ventricular myocytes is insensitive to isoproterenol

Whole cell clamp experiments and single channel measurements were used to study whether the T-type Ca. channel in guinea-pig ventricular myocytes is sensitive to -adrenergic stimulation. In whole cell clamp experiments we determined the T-type Ca current by measuring the difference between the currents at a test potential of –30 mV in Na-free solution, starting from a holding potential elther at –90 or at –50 mV. Application of 1 M isoproterenol (ISO) did not change the difference current, while the L-type Ca current was enhanced during application of the drug. In cell attached patch experiments with 110 mM CaCl₂ in the pipette solution, single channel currents were observed which were due to opening of the T-type Ca channel. Application of 4 M ISO to the bath solution did neither change the appearance of the channel openings, nor the ensemble averaged currents. It is concluded that the T-type Ca channel in guinea-pig ventricular cells is insensitive to ISO.     

A new non-voltage-dependent,epithelial-like Na+ channel in vascular smooth muscle cells

A new type of Na⁺ channel was identified in smooth muscle cells of the rat aortic cell line A7r5, and in smooth muscle cells cultured from rat aorta and rat portal vein. The channel is highly selective for Na⁺ (P _Na/P _K>11). It is active in cell-attached patches, and independent of the trans-patch membrane potential. The single channel conductance is low (10.7 pS). Two substates were identified. This channel is insensitive to effectors of other types of Na⁺ channels, such as amiloride (100 M) or tetrodotoxin (100 M). It is inhibited by phenamil at high concentrations (>10 M). The mean open state probability P(O) varied from patch to patch (0.05–0.88). Kinetics analysis reveals a complex behaviour: open times separate in short (₁ = 84 ms) and long (₂ = 845 ms) openings and closed times separate into short (₁ = 60 ms) and long closures (₂ = 272 –3130 ms). Short openings and long closures are preponderant at a low P(O). Long openings are absent in the presence of phenamil (50 M) and are unaffected by amiloride (100 M). Fluctuations of the channel activity in cell-attached patches and the fast disappearance after excision suggest that this channel is under metabolic control. This vascular smooth muscle channel appears to be a potentially important Na⁺ entry pathway for vascular cells and an amiloride-resistant homologue of the epithelial Na⁺ channel.     

Tetraethylammonium block of Slowpoke calcium-activated potassium channels expressed in Xenopus oocytes: Evidence for tetrameric channel formation

Unitary currents were recorded from insideout membrane patches pulled from Xenopus oocytes that had been injected with RNA transcribed from a cDNA encoding the Drosophila maxi-K channel (Slowpoke). Site-directed mutagenesis was used to make cDNAs encoding channel subunits with single amino acid substitutions (Y308V and C309P). The extracellular side of the patch was exposed to tetraethylammonium (TEA) in the pipette solution; unitary currents in the presence of TEA were compared with currents in the absence of TEA to compute the inhibition. Amplitude distributions were fit by functions to estimate the blocking and unblocking rate constants. For wild-type channels, TEA blocked with an apparent K _d of 80 M at 0 mV and sensed 0.18 of the membrane electric field; the voltage dependence lay entirely in the blocking rate constant. TEA blocked currents through C309P channels with a similar affinity to wild-type at 0 mV, but this was not voltage-dependent. Currents through Y308V channels were very insensitive to any block by TEA; the apparent K _d at 0 mV was 26 mM and the blockade sensed 0.18 of the electric field. Oocytes injected with a mixture of RNAs encoding wild-type and Y308V channels showed unitary currents of four discrete amplitudes in the presence of 3 mM TEA; at 40 mV these corresponded to inhibitions of approximately 80%, 55%, 25% and 10%. These values agreed well with those expected for inhibition by TEA of currents through channels containing 3, 2, 1 and 0 tyrosine residues at the channel mouth, assuming that a tyrosine residue from each of four subunits contributes equally to the binding of the TEA ion. This indicates that Slowpoke channels form as tetramers.     

The effect of external potassium on the elementary conductance of the ACh-induced potassium channel in the sino-atrial node

The mechanism underlying the increase in the potassium conductance of the acetylcholine (ACh)-induced channel on increasing the extracellular potassium concentration (|K|₀) was studied. The relaxation as well as the current fluctuations of the drug-induced current were measured at 3 and 12 mM |K|₀ by conducting voltage clamp experiments in the rabbit sinoatrial node. The following results were obtained:

1. The time constant of relaxation (τ_relax) was not affected by changing |K|₀. In both cases τ_relax was about 130 ms at ?80 mV, 100 ms at ?40 mV and 60 ms at + 20 mV. It is, therefore, unlikely that the increase of the ACh-induced potassium conductance is due to a longer average open time of the drug-operated potassium channels.
2. The chord conductance of the ACh-induced K current was increased by a factor of 1.7 at ?40 mV and by 1.5 at +10 mV on elevation of |K|₀ from 3 to 12 mM.
3. From the relation between the variance of the current fluctuations and the mean amplitude of the current the single channel conductance was determined to be 3.3 pS at 3 mM |K|₀ and 5.9 pS at 12 mM |K|₀, thus γ was increased by a factor of 1.7.
4. From the power density spectrum an increase in the single channel conductance by a factor of 1.8 (at ?40 mV) could be calculated. The corner frequency was not affected.
5. The increase in the potassium conductance, expected from the constant field equation when |K|₀ is increased, agrees well with the experimental results.