Point mutations in IIS4 alter activation and inactivation of rat brain IIA Na channels in Xenopus oocyte macropatches

Macroscopic currents of wild-type rat brain IIA (RBIIA) and mutant Na channels were recorded in excised patches from Xenopus oocytes. A charge deletion (K859Q) and an adjacent conservative mutation (L860F) in the second domain S4 membrane-spanning region differentially altered voltage sensitivity and kinetics. Analysis of voltage dependence was confined to Na currents with fast inactivation kinetics, although RBIIA and K859Q (but not L860F) also showed proportional shifts between at least two gating modes, rendering currents with fast or slow inactivation kinetics, respectively. Compared to RBIIA, the midpoint of the activation curve was shifted in both K859Q and L860F by 22 mV to more positive potentials, yet this shift was not associated with a corresponding change in the voltage dependence of time constants for activation ( _a) or inactivation ( _h1, _h2). L860F showed faster activation time constants _a than RBIIA, while K859Q was slower for both the activation ( _a) and the inactivation components ( _h1). Similarly, the steady-state inactivation curve of L860F but not K859Q shifted by 9 mV in the hyperpolarizing direction. Thus, the fourth charge in the IIS4 transmembrane segment exerts control over voltage sensitivity and kinetics of activation and may interact with structure that influence other aspects of channel gating.     

Gating currents of inactivating and non-inactivating potassium channels expressed in Xenopus oocytes

The Xenopus oocyte expression system in combination with patch-clamp techniques allows the measurement of ionic currents from a single class of genetically engineered ion channels. Ionic currents in the nanoampere range from oocytes injected with cRNA, corresponding to potassium channels, can be recorded in the inside-out patch configuration. These recordings have a high time resolution at low background noise. Substitution of impermeant ions for potassium and blocking of the channel conductance with tetraethylammonium allows the recording of potassium gating currents, I _g, which is hampered in natural excitable cells by the simultaneous presence of sodium channels and a variety of different potassium channels. The on transients, I _g ^on , are fast and can have amplitudes of up to several tens of pA. Upon repolarization to -100 mV after small depolarizations, off gating currents, I _g ^off , which reverse most of the on charge displacement, Q ^on, within 1 ms, are readily observed. However, this fast recovery of the gating charge is drastically reduced upon increasing the amplitude of the depolarizing pulse. In contrast to sodium channels, this temporary charge immobilization is complete within a few milliseconds at positive membrane potentials. Furthermore, there seems to be no direct correlation between charge immobilization and inactivation because the same phenomenon occurs for channels that do not inactivate.     

Kinetic properties of single sodium channels modified by fenvalerate in mouse neuroblastoma cells

(1) The kinetic properties of single sodium channels modified by the pyrethroid fenvalerate have been analyzed by patch clamp techniques using the cultured mouse neuroblastoma cells. (2) Fenvalerate drastically prolonged the open time of single sodium channels from the normal value of 5 ms to several hundred milliseconds during a depolarizing pulse. The channels remained open after termination of a depolarizing pulse for as long as several seconds. (3) The channel lifetime varied with the membrane potential, attained a maximum at –70 mV, and decreased with hyperpolarization and depolarization from –70 mV. (4) Prolonged openings of the modified channels allowed a current-voltage curve for a single channel to be plotted by sweeping a ramp pulse. The single channel conductance had a value of 11 pS and was linear over potentials ranging from 0 to –100 mV. (5) Power density spectral analysis of the open channel current noise indicated a single Lorentzian curve with a cut-off frequency at 90 Hz, indicating that the increase in noise during channel opening resulted from a relatively slow kinetic process. (6) The probability of the channel being modified by fenvalerate was independent of the length of time during which the channel was opened. This observation suggests that channel modification had taken place before the channel opened. This study of the prolonged opening at the single channel level provides a new insight into open channel properties and the kinetics of channel modification.     

Altered gating and conductance of Na+ channels in hyperkalemic periodic paralysis

Electrophysiological studies on muscle fibres from patients with hyperkalemic periodic paralysis with myotonia have shown that the episodes of weakness are caused by a sustained depolarization of the sarcolemma to potentials between -40 and -60 mV. In muscle fibre segments from three such patients this sustained depolarization was caused by noninactivating Na⁺ channels with reduced single-channel conductance blocked by TTX and procainamide. As the chloride conductance was normal, myotonia may be best explained with the abnormal reopenings of the Na⁺ channels. The recently described genetic linkage between hyperkalemic periodic paralysis with myotonia and the gene coding for the TTX-sensitive Na⁺ channel suggests an altered primary structure of this channel causing its abnormal function.     

Effects of veratridine on single neuronal sodium channels expressed inXenopus oocytes

(1) Chick neuronal Na⁺ channels were expressed inXenopus laevis oocytes after injection with total messenger ribonucleic acid (mRNA) isolated from chick brain. The currents were investigated with the whole cell voltage clamp and with the patch clamp technique. Activation and inactivation of the induced current, and its sensitivity towards tetrodotoxin (TTX) and veratridine were reminiscent of vertebrate neuronal Na⁺ channels. (2) In the presence of veratridine normal single channel openings often converted into small amplitude openings of long duration. These small amplitude openings persisted for hundreds of milliseconds after return to the holding potential. (3) The slope conductance of the veratridine modified open channel state was 5–6 pS as compared to the normal state with 21–25 pS in the voltage range between –35 and +5 mV. (4) The modified channel showed saturation behaviour towards Na⁺ ions. Half saturation of the single channel amplitude was observed at 330 mM Na⁺ at a membrane potential of –100 mV. (5) Final closure of the modified channel after return to the holding potential followed an exponential time course. Its potential dependence was similar to that of the time course of the veratridine induced tail currents in the whole cell configuration. (6) The properties of the Na⁺ channel derived from chick forebrain are compared with the properties of the same channel derived from chick skeletal muscle. Both were expressed in the same membrane environment, theXenopus oocyte plasma membrane. While earlier results with Na⁺ channels of muscle origin showed two channel populations, one with short and another with long mean open times, Na⁺ channels of neuronal origin were homogeneous and characterized by short open times.     

Single Ca2+-activated K+ channels in excised membrane patches of hamster oocytes

The properties of the Ca²⁺-activated K⁺ channel in unfertilized hamster oocytes were investigated at the single-channel level using inside-out excised membrane patches. The results indicate a new type of Ca²⁺-activated K⁺ channel which has the following characteristics: (1) single-channel conductance of 40–85 pS for outward currents in symmetrical K⁺ (150 mM) solutions, (2) inward currents of smaller conductance (10–50 pS) than outward currents, i.e. the channel is outwardly rectified in symmetrical K⁺ solutions, (3) channel activity dependent on the internal concentration of free Ca⁺ and the membrane potential, (4) modification of the channel activity by internal adenosine 5 diphosphate (0.1 mM) producing a high open probability regardless of membrane potential.     

Kinetics and distribution of voltage-gated Ca,Na and K channels on the somata of rat cerebellar Purkinje cells

Voltage gated ion channels on the somatic membrane of rat cerebellar Purkinje cells were studied in dissociated cell culture with the combination of cell-attached and whole-cell variation of patch clamp technique. The method enables us to record local somatic membrane current under an improved space clamp condition. Transient (fast-inactivating) and steady (slow inactivating) Ca channel currents, Na current, transient (fast-inactivating) and steady (slow-inactivating) K currents, were observed. Transient and steady Ca channel currents were activated at test potentials more positive than –40 mV and –20 mV, respectively (in 50 mM external Ba). The transient current inactivated with a half-decay time of 10–30 ms during maintained depolarizing pulses, while the steady current showed relatively little inactivation. Na current was activated at more positive potentials than –60 mV, and inactivated with a half-decay time of less than 5 ms. Transient and steady K outward currents were recorded at more positive potential than –20 mV and –40 mV, respectively. The transient current inactivated with a half-decay time of 2–8 ms. Ca, Na and K channels showed different patterns of distribution on the somatic membrane. Steady Ca channels tended to cluster compared with Na or K channels.     

Single-channel current/voltage relationships of two kinds of Na+ channel in vertebrate sensory neurons

The electrical signals of nerve and muscle are fundamentally dependent on the voltage-gated Na⁺ channel, which is responsible for the rising phase of the action potential. At least two kinds of Na⁺ channel are expressed in the membrane of frog dorsal root ganglion (DRG) cells: Na⁺ channels with fast kinetics that are blocked by tetrodotoxin (TTX) at high affinity, and Na⁺ channels with slower kinetics that are insensitive to TTX. Recordings of single-channel currents from frog DRG cells, under conditions favoring Na⁺ as the charge carrier, reveal two distinct amplitudes of single-channel events. With 300 mM external Na⁺, single-channel events that can be measured in the presence of 1 M TTX have a slope conductance 7.5 pS. In the absence of TTX, events with a slope conductance of 14.9 pS dominate. Ensemble averages of the smaller single-channel events display the slower kinetics characteristic of the macroscopic TTX-insensitive Na⁺ currents, and ensemble averages of the larger events display the faster kinetics characteristic of the TTX-sensitive currents. The results are consistent with the idea that the toxin-binding site is sufficiently close to the pore to influence ion permeation.     

Temperature-dependent subconducting states and kinetics of deltamethrin-modified sodium channels of neuroblastoma cells

The effects of temperature on the properties of sodium channels from mouse neuroblastoma cells modified by the pyrethroid insecticide deltamethrin were investigated using the patch-clamp technique. The study was aimed at determining various states of modified channels which were expected to be revealed by raising the temperature as a result of an increase in channel activity. After exposure to 10 M deltamethrin, the decay of whole cell sodium current at –30 mV was drastically slowed. It is expressed by two exponential functions at 11°C and by three exponential functions at room temperature (22±1° C). Thus, raising the temperature reveals a new process. Whole cell sodium tail currents associated with step repolarization from –30 mV to –100 mV were best fit by the sum of two exponential functions both at 11° C and at room temperature. The decay of the summed modified single sodium channel currents at –30 mV was expressed by a single exponential function at 11° C, and by two exponential functions at room temperature. In keeping with these results, the open time histograms show the single (11° C) and double (room temperature) exponential distributions. Thus, raising the temperature allows a new single channel process to be revealed. Other modified open states observed previously at 11° C were also found at room temperature including a flickering state and a subconducting state. In addition, several new subconducting states were found at room temperature. Furthermore, while at 11° C only a single state exists in which channels open with some delay at –100 mV after the termination of a depolarizing pulse, at room temperature, two such states having different amplitudes were found. The results show that more deltamethrin-modified channel states can be observed at room temperature than at 11° C. Even with this larger variety of channel states, similar states can be observed with normal channels or channels modified by other unrelated agents. This suggests that deltamethrin prolongs a variety of normal channel states.     

Characterization of Ca2+-activated K+ channels in excised patches of human T lymphocytes

Ca²⁺-activated K⁺ [K(Ca)] channels were studied in excised patches of resting and activated human peripheral blood T lymphocytes. The K(Ca) channel had a single-channel conductance of 50±6 pS in symmetrical high-K⁺ solutions in the potential range of –100 to –10 mV and was inwardly rectifying at more depolarized potentials. The channel was sensitive to block by charybdotoxin (10 nM) and insensitive to apamin (3 nM). Half-maximum activation occurred at an internal free Ca²⁺ concentration of 360±110 nM. The concentration-effect curve had a slope factor of 0.83±0.12, suggesting a 11 interaction of Ca²⁺ ions with the channel. Ca²⁺ affects the open time probability of the K(Ca) channels, mainly by modulating the frequency of channel opening. The open probability did not show voltage dependence. The kinetics of the channel could be described assuming one open state and two closed states. The time constant of the exponential describing the open time distribution amounted to 2.8±1.2 ms, whereas the closed time distribution could be described with two exponentials with time constants of 0.2±0.05 ms and 8.0±2.1 ms, respectively. Resting T lymphocytes expressed a low number of channels but the density of channels increased dramatically during chronic phytohaemagglutinin stimulation.     

Interaction of the antiepileptic drug lamotrigine with recombinant rat brain type IIA Na+ channels and with native Na+ channels in rat hippocampal neurones

Actions of the new antiepileptic drug lamotrigine (LTG, Lamictal) were characterised using recombinant rat brain type IIA Na⁺ channels expressed in Chinese hamster ovary (CHO) cells and native Na⁺ channels in rat hippocampal pyramidal neurones, using whole-cell recording and intracellular recording techniques. In CHO cells, LTG caused a tonic inhibition of Na⁺ currents in a concentration-dependent and voltage-dependent manner. The half-maximal inhibitory concentration (IC₅₀) of approximately 500 M was obtained at a holding potential (V _h) of –90 mV compared with an IC₅₀ of 100 M at a V _h of –60 mV. LTG (50 M) caused a 10–mV negative shift in the slow, steady-state inactivation curve and delayed considerably the recovery from inactivation, but had no significant effects on the voltage dependence of activation or fast inactivation, suggesting that LTG acts mainly on the slow inactivated state. The affinity for the inactivated channels was estimated at 12 M. The tonic inhibition was augmented by a use-dependent action in which a further inhibition by the drug developed during rapid repetitive stimulation using a train of 20-ms duration pulses (11 Hz). These results were consistent with the drug action being on firing properties of pyramidal neurones. Only in those epileptiform bursts which caused cumulative inactivation of Na⁺ spikes did LTG produce a potent inhibition. Our data suggest that the inactivated channel is a primary target for LTG action at therapeutic concentrations.     

cAMP-activation of amiloride-sensitive Na+ channels from guinea-pig colon expressed in Xenopus oocytes

Guinea-pig distal colonic mRNA injection into Xenopus laevis oocytes resulted in expression of functional active epithelial Na⁺ channels in the oocyte plasma membrane. Poly(A)⁺ RNA was extracted from distal colonic mucosa of animals fed either a high-salt (HS) or a low-salt (LS) diet. The electrophysiological properties of the expressed amiloride-sensitive Na⁺ conductances were investigated by conventional two-electrode voltage-clamp and patch-clamp measurements. Injection of poly(A)⁺ RNA from HS-fed animals [from hereon referred to as HS-poly(A)⁺ RNA] into oocytes induced the expression of amiloride-sensitive Na⁺ conductances. On the other hand, oocytes injected with poly(A)⁺ RNA from LS-fed animals [LS-poly(A)⁺ RNA] expressed a markedly larger amount of amiloride-blockable Na⁺ conductances. LS-poly(A)⁺ RNA-induced conductances were completely inhibitable by amiloride with a K _i of 77 nM, and were also blocked by benzamil with a K _i of 1.8 nM. 5-(N-Ethyl-N-isopropyl)-amiloride (EIPA), even in high doses (25 μM), had no detectable effect on the Na⁺ conductances. Expressed amiloride-sensitive Na⁺ channels could be further activated by cAMP leading to nearly doubled clamp currents. When Na⁺ was replaced by K⁺, amiloride (1 μM) showed no effect on the clamp current. Single-channel analysis revealed slow gating behaviour, open probabilities (P _o) between 0.4 and 0.9, and slope conductances of 3.8 pS for Na⁺ and 5.6 pS for Li⁺. The expressed channels showed to be highly selective for Na⁺ over K⁺ with a permeability ratio P _Na/P _K > 20. Amiloride (500 nM) reduced channel P _o to values < 0.05. All these features make the guinea-pig distal colon of LS-fed animals an interesting mRNA source for the expression of highly amiloride-sensitive Na⁺ channels in Xenopus oocytes, which could provide new insights in the regulatory mechanism of these channels. Received: 16 October 1995/Received after revision: 30 November 1995/Accepted: 12 December 1995     

Complete reversal of run-down in rabbit cardiac Ca2+ channels by patch-cramming in Xenopus oocytes; partial reversal by protein kinase A

 The rabbit cardiac Ca²⁺ channel (α_1C) expressed in Xenopus oocytes exhibited a complete run-down of ionic currents when cell-attached patches were excised. The α_1C channel was expressed alone or was coexpressed with the accessory β_2a or β_1b subunit. The catalytic subunit of protein kinase A (PKAc) and MgATP were capable of delaying the run-down of single-channel currents. In 33% of the α_1C patches, and 26% of the α_1C+β_2a patches, inclusion of PKAc in the bath solution delayed the run-down for a maximum of 20 min. In experiments where PKAc in the bath was not sufficient to delay the run-down of channel activity, insertion of the patch back into the oocyte (patch-cramming) could restore channel activity. Gating currents were also measured in the α_1C+β_1b channel and were not subject to any run-down, even after the complete run-down of ionic currents. The results presented here reveal that PKAc is capable of delaying the run-down of currents in a subset of patches. The patch-cramming results suggest that a cytoplasmic factor, in addition to phosphorylation of the channel (by PKAc), may be involved in the maintenance of channel activity. Received: 29 October 1998 / Accepted: 4 January 1999     

Regulation of chloride channels in the human colon carcinoma cell line HT29.cl19A

Chloride (Cl^–) channels are important in the regulation of salt and water transport in secretory epithelial cells. A disturbed Cl^– secretion is the most consistent characteristic in the genetic disease cystic fibrosis. An outwardly rectifying Cl^– channel (OR) with a conductance of 25–50 pS had been proposed to play a major role in Cl^– secretion. Activation by Ca²⁺ and the protein kinases (PK) A and C (at less than 10 nM Ca²⁺) as well as inhibition by PKC (at 1 M Ca²⁺) has been reported. In the present study, we have identified and characterized the OR in HT29.cl19A human colon carcinoma cells. The OR displayed a conductance of 31±4 pS (n=25). Its open probability in 10 nM Ca²⁺ was voltage-dependent in 50% of the patches, starting from 0.2 at -70 mV to 0.8 at 70 mV. The spontaneous activation in excised inside-out patches at –60 mV was Ca²⁺-dependent and decreased from 29% in 1 mM Ca²⁺ to 2% in 10 nM Ca²⁺. Active OR were found in (a) 25% of patches exposed to 10 nM Ca²⁺, ATP and cAMP only, (b) 42% of the patches exposed to 10 nM Ca²⁺, ATP and the catalytic subunit of PKA (C_AK) and (c) 67% of the patches exposed to 1 mM Ca²⁺, ATP plus C_AK. Inhibition of voltage-activated channels by addition of PKC in 1 M or 1 mM Ca²⁺ was not observed. Attempts to activate the OR in cell-attached patches by increasing cAMP levels under different experimental conditions were unsuccessful. Our data suggest that the OR may not be as important in Cl^– secretion as has been thought.     

Excision-activated chloride channels in cultured postnatal rat hippocampal neurons

Chloride permeable intermediate conductance single channel events activated on patch excision were found in outside-out patches from cultured postnatal hippocampal neurons. A majority of the channels had a conductance of 83 ± 2.1 pS when recorded in a symmetrical TEAC1 solution. Two other populations of channels with conductance values of 62 ± 2.1 pS and 145 ± 1.9 pS were also observed. The reversal potentials for these intermediate conductance Cl⁻ channels coincided with that of the GABA activated channels. The channels characteristically appeared 5–15 min after patch excision, suggesting that these channels may be blocked by some diffusible factors under physiological conditions. Based on the measurements of channel burst durations while the channel was partially blocked, and the channel open times after complete relief from the block, the mechanism of blockade does not appear to be a simple open channel blockade. The high prevalence and its potential regulation by cytosolic factors suggest an important physiological role for these Cl⁻ channels coupling neuronal excitability with cellular metabolism.     

Unitary delayed rectifier channels of rat hippocampal neurons: properties of block by external tetraethylammonium ions

Patch-clamp recording was used to characterise a delayed rectifier potassium channel and the effects of external tetraethylammonium (TEA) in neurons isolated from the CA1 region of cultured neonatal rat hippocampus. A preliminary kinetic analysis is presented. Very low concentrations of TEA included in the patch pipette solution had two effects on unitary currents: first unitary currents were reduced in amplitude, with an associated increase in open channel noise, and second channel mean open time was reduced. The reduction in unitary amplitude was consistent with a single TEA molecule blocking the channel with a voltage-independent K _d of 53.4 M. The blocking and unblocking rate constants, estimated using two independent methods, were approximately 350 mM^–1 ms^–1 and 20 ms^–1, both rate constants being independent of voltage. Channels blocked in this way appeared able to close normally without first having to become unblocked. The reduction in mean channel open time was probably due to a second, kinetically slower blocking reaction with a much lower K _d, probably between 300 and 800 M. The voltage-independent blocking rate constant of the slower block was at least 25 times slower than that of the faster block.     

TRESK-like potassium channels in leukemic T cells

In this study, we present patch-clamp characterization of the background potassium current in human lymphoma (Jurkat cells), generated by voltage-independent 16 pS channels with a high ( approximately 100-fold) K(+)/Na(+) selectivity. Depending on the background K(+) channels density, from few per cell up to approximately 1 open channel per mum(2), resting membrane potential was in the range of -40 to -83 mV, approaching E (K) = -88 mV. The background K(+) channels were insensitive to margotoxin (3 nM), apamine (3 nM), and clotrimazole (1 muM), high-affinity blockers of the lymphocyte Kv1.3, SKCa2, and IKCa1 channels. The current depended weakly on external pH. Arachidonic acid (20 muM) and Hg(2+) (0.3-10 muM) suppressed background K(+) current in Jurkat cells by 75-90%. Background K(+) current was weakly sensitive to TEA(+) (IC(50) = 14 mM), and was efficiently suppressed by externally applied bupivacaine (IC(50) = 5 muM), quinine (IC(50) = 16 muM), and Ba(2+) (2 mM). Our data, in particular strong inhibition by mercuric ions, suggest that background K(+) currents expressed in Jurkat cells are mediated by TWIK-related spinal cord K(+) (TRESK) channels belonging to the double-pore domain K(+) channel family. The presence of human TRESK in the membrane protein fraction was confirmed by Western blot analysis.     

Micromolar concentrations of veratridine activate sodium channels in embryonic cockroach neurones in culture

The mode of action of the alkaloid veratridine has been reinvestigated on cultured cockroach neurones, which are normally inexcitable and do not have a detectable fast sodium current. The whole-cell and cell-attached configurations of the patch-clamp technique were used to record the macroscopic and single channel currents, respectively. Concentrations of veratridine ranging from 10^–8 to 10^–5 M were found to induce a small tetrodotoxin (TTX)-sensitive inward current, which peaked around + 10 mV and reversed around + 55 mV. This current exhibited a pronounced plateau and was insensitive to changes in the holding potential. Bath application of veratridine induced typical TTX-sensitive inwardly-directed single-channel activity, falling into two (apparently coupled) categories of events: first, relatively large events (1 pA at a hyperpolarized potential of –125 mV relative to rest) of short duration and, second, small bursting events (0.4 pA under similar conditions) of slightly longer duration. Pipette application of similar concentrations of veratridine had similar effects in that two categories of events were observed: first, bursts of large events with multiple conductance states and, second, small events of very long duration. The current/ voltage relationship of these events was linear for the voltage range studied and the (extrapolated) reversal potential approximated + 110 mV. These results support the hypothesis that veratridine, in small concentrations, induces a slow voltage-dependent activation of TTX-sensitive sodium channels, independent of the fast activating and inactivating sodium channels involved in action potential generation.     

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.     

Calpastatin and nucleotides stabilize cardiac calcium channel activity in excised patches

The activity of single L-type Ca²⁺ channels is rapidly lost (run-down) when contact between the membrane and cytosol is interrupted. We have now achieved the stabilization of cardiac Ca²⁺ channel activity of guinea-pig ventricular myocytes by using either cytosol or defined components added to excised patches. The endogenous protease inhibitor, calpastatin, together with nucleotides, ATP + GTP, was found to prevent rundown as effectively as cardiac cytosolic solution. These results suggest the involvement of proteolysis by calpain in run-down of channel activity and enable the study of cardiac Ca²⁺ channel regulation with free access to both sides of the membrane.