Alterations in activation gating of single Shaker A-type potassium channels by the Sh5 mutation

Alterations in voltage-dependent gating of A-type potassium channels by the Sh5 mutation were studied using macroscopic and single-channel recording techniques on cultured embryonic myotubes from Drosophila. The mutation increases the voltage required to activate and inactivate the A1 channel by approximately 20 mV and decreases the steepness of the voltage dependence of steady-state inactivation. Boltzmann fits to the prepulse inactivation curves have slopes of e-fold/3.5 mV for wild type and e-fold/5.0 mV for Sh5. A kinetic analysis of single Sh5 A1 channels revealed that the transition rates into and out of the open state are not significantly affected by the mutation. In contrast, the latencies until the channel opens following a voltage step are increased at low voltages. These alterations probably do not result from an offset of the transmembrane voltage sensed by the channel as might be expected for an alteration in the surface charge of the protein. By using a kinetic model developed for wild-type A1 channels, we show that an alteration in the amplitude and voltage dependence of the deactivation rate for each subunit (beta) can account for all of the alterations observed.     

Voltage clamp analysis of membrane currents in larval muscle fibers of Drosophila: alteration of potassium currents in Shaker mutants

The body wall muscles in Drosophila larvae are suitable for voltage clamp analysis of changes in membrane excitability caused by mutations. Both inward and outward ionic currents are present in these muscle fibers. The inward current is mediated by voltage-dependent Ca2+ channels. In Ca2+-free saline, the inward current is eliminated. The remaining outward K+ currents consist of two distinct components, an early transient IA and a delayed steady IK, which are separable by differences in the rate and voltage dependence of activation and inactivation. The steady-state and kinetic properties of the activation and inactivation processes of these two currents are analyzed. The results provide a basis for quantitative analysis of altered membrane currents in behavioral mutants of Drosophila. Previous studies indicate that mutations in the Shaker (Sh) locus alter excitability in both nerve and muscle in Drosophila. Our results support the idea that the channels mediating IA are molecularly distinct from those mediating IK. All Sh mutations studied specifically affect IA without changing the properties of the calcium current and IK. In certain alleles (ShKS133, Sh102, and ShM) IA is eliminated, permitting detailed studies of IK in isolation of IA. Studies of the alleles that do not eliminate IA provide additional information of the channels. In one such allele, Sh5, voltage dependence of IA activation is shifted to more positive potentials. This is accompanied by a less pronounced shift in the voltage dependence of inactivation. These results suggest that Sh5 mutation affects the voltage-sensitive mechanism of both activation and inactivation processes and that these two processes are not controlled by independent parts of the channel. Furthermore, the differential effects of these alleles on different excitable membranes imply that other genes take part in the control of IA. The effects of Sh5 on muscle depend on developmental stage. In larval muscle, Sh5 reduces the amplitude of IA because of the shift in the current-voltage (I-V) relation. In contrast, in adult Sh5 muscles, IA is reported to be normal in amplitude but shows abnormally rapid inactivation (Salkoff, L., and R. Wyman (1981) Nature 293: 228-230). A different allele, ShrK0120, causes a clear defect in nerve excitability, but analysis of IA in ShrK0120 larval muscle reveals I-V relations, inactivation, and recovery from inactivation similar to those seen in normal fibers. We suggest a possible mechanism of combinations of multiple interacting genes participating in the control of potassium channels to account for the presence of a variety of potassium channels in different excitable membranes.     

Postdecentralization plasticity of voltage-gated K+ currents in glandular sympathetic neurons in rats

This paper presents the kinetic and pharmacological properties of voltage-gated K(+) currents in anatomically identified glandular postganglionic sympathetic neurons isolated from the superior cervical ganglia in rats. The neurons were labelled by injecting the fluorescent tracer Fast Blue into the submandibular gland. The first group of neurons remained intact, i.e. innervated by the preganglionic axons until the day of current recordings (control neurons). The second group of neurons was denervated by severing the superior cervical trunk 4-6 weeks prior to current recordings (decentralized neurons). In every control and decentralized neuron three categories of voltage-dependent K(+) currents were found. (i) The I(Af) K(+) current, steady state, inactivated at hyperpolarized membrane potentials. This current was fast activated and fast time-dependently inactivated, insensitive to TEA and partially depressed by 4-AP. (ii) The I(As) K(+) current, which was steady-state inactivated at less hyperpolarized membrane potentials than I(Af). The current activation and time-dependent inactivation kinetics were slower than those of I(Af). I(As) was blocked by TEA and partially inhibited by 4-AP. (iii) The IK K(+) current did not undergo steady-state inactivation. In decentralized compared to control neurons the maximum I(Af) K(+) current density (at +50 mV) increased from 116.9 +/- 8.2 to 189.0 +/- 11.5 pA/pF, the 10-90% current rise time decreased from 2.3 to 0.7 ms and the recovery from inactivation was faster. Similarly, in decentralized compared to control neurons the maximum I(As) K(+) current density (at +50 mV) increased from 49.9 +/- 3.5 to 74.3 +/- 5.0 pA/pF, the 10-90% current rise time shortened from 29 to 16 ms and the recovery from inactivation of the current was also faster. The maximum density (at +50 mV) of I(K) in decentralized compared to control neurons decreased from 76.6 +/- 3.9 to 60.7 +/- 6.3 pA/pF. We suggest that the upregulation of voltage-gated time-dependently-inactivated K(+) currents and their faster recovery from inactivation serve to restrain the activity of glandular sympathetic neurons after decentralization.     

The effects of pumiliotoxin-B on sodium currents in guinea pig hippocampal neurons

The actions of pumiliotoxin-B, extracted from the skin of the frog Dendrobates pumilio, were examined on hippocampal slices and on acutely dissociated hippocampal neurons from the adult guinea pig. Application of 0.5-1 microM pumiliotoxin-B to hippocampal slices caused spontaneous, repetitive field discharges in the CA3 subfield. In whole-cell patch-clamp recordings of isolated CA1 and CA3 neurons, 1-2 microM pumiliotoxin-B shifted the midpoint of Na+ current activation by -11.4 +/- 1.1 mV. This shift was not dependent upon prior activation of the sodium channel. Pumiliotoxin-B did not block macroscopic Na+ inactivation but did reduce the apparent voltage-dependence of inactivation such that currents decayed faster at membrane potentials more negative than -30 mV. Single-channel recordings of sodium currents from excised membrane patches indicated that pumiliotoxin-B had little or no effect on channel closings due to entry into inactivated state(s) but did increase the rate of channel closings due to reversal of channel opening. The increase in the channel closing rate was consistent with a +8.7 mV shift in voltage sensitivity. Negative shifts in activation and positive shifts in closing rates implied a negative shift in the voltage-dependence of channel opening, suggesting that pumiliotoxin-B increases the rate of Na+ channel opening and closing in cells at rest, which could result in spontaneous activity in the neurons.     

Single K+ channel properties in cultured mouse Schwann cells: conductance and kinetics

Cultured Schwann cells are characterized by a strong outward rectification of the membrane; the threshold of the outward currents is close to the resting membrane potential of about -50 mV (Gray et al.: In Ritchie, Keynes (eds): Ion Channels in Neural Membranes. New York: Alan R. Liss, Inc., pp 145-157, 1986). These outward currents show up a heterogeneity among the cultured Schwann cells: some cells displayed inactivating, others non-inactivating outward currents (Hoppe et al.: Pflügers Arch 415:22-28, 1989). In this study we characterized the single channel currents using the patch-clamp technique in the intact patch recording configuration. The conductance of all recorded channels was 10-12 pS (5.6 mM [K+]o). These channels were K+ selective since changes in extracellular [K+] resulted in changes of the reversal potential as predicted for an exclusively K+ selective pore. The reversal potentials also predicted an intracellular [K+] of 60 mM indicating that the K+ equilibrium potential is slightly negative to the membrane potential. Analysis of the kinetic behavior of the channels resolved two different types of behaviour: 40% inactivated during a depolarizing voltage step, the others showing no sign of inactivation. The analysis of open probability and gating properties in the steady state showed up more differences between these two channel types: mean open probability peaked at about 10 mV for inactivating channels, while it continuously increased for non-inactivating channels. The inactivation time constants of averaged single channel and whole cell currents were similar and showed both a similar voltage dependency. We conclude that cultured Schwann cells express either two types of K+ channels with similar conductance or a channel which can acquire two functional states and that these channels can account for the different types of K+ currents observed in these cells.     

Comparison of the Ca2 + currents induced by expression of three cloned alpha1 subunits, alpha1G, alpha1H and alpha1I, of low-voltage-activated T-type Ca2 + channels

Expression of rat alpha1G, human alpha1H and rat alpha1I subunits of voltage-activated Ca2 + channels in HEK-293 cells yields robust Ca2 + inward currents with 1.25 mM Ca2 + as the charge carrier. Both similarities and marked differences are found between their biophysical properties. Currents induced by expression of alpha1G show the fastest activation and inactivation kinetics. The alpha1H and alpha1I currents activate and inactivate up to 1.5- and 5-fold slower, respectively. No differences in the voltage dependence of steady state inactivation are detected. Currents induced by expression of alpha1G and alpha1H deactivate with time constants of up to 6 ms at a test potential of - 80 mV, but currents induced by alpha1I deactivate about three-fold faster. Recovery from short-term inactivation is more than three-fold slower for currents induced by alpha1H and alpha1I in comparison to alpha1G. In contrast to these characteristics, reactivation after long-term inactivation was fastest for currents arising from expression of alpha1I and slowest in cells expressing alpha1H calcium channels. The calcium inward current induced by expression of alpha1I is increased by positive prepulses while currents induced by alpha1H and alpha1G show little ( < 5%) or no facilitation. The data thus provide a characteristic fingerprint of each channel's activity, which may allow correlation of the alpha1G, alpha1H and alpha1I induced currents with their in vivo counterparts.     

A mouse brain homolog of the Drosophila Shab K+ channel with conserved delayed-rectifier properties

We have cloned and expressed a mouse brain K+ channel that is the homolog of the Drosophila Shab K+ channel. Mouse and Drosophila Shab K+ channels (mShab and fShab, respectively) represent an instance of K+ channels and structurally related species that are both functionally and structurally conserved; most kinetic, voltage-sensitive, and pharmacological properties are similar for the 2 channels. The greatest functional difference between the currents is recovery from inactivation, which is several times slower for mShab than for fShab currents. In addition to conserved structure, the mShab polypeptide has an unusually long nonconserved region at the carboxyl end of the protein. Truncation of 293 residues from the carboxyl end produced no noticeable change in voltage-sensitive, kinetic, or pharmacological properties. Thus, the measured functional properties of mShab are determined by the remaining 564 residues, most of which are conserved. The mShab and fShab channels are naturally occurring structural variants having substitutions in conserved portions that appear relatively neutral with respect to all measured properties except for, possibly, the rate of recovery from inactivation. The mShab current closely resembles a native delayed-rectifier-type potassium current, IK, in hippocampal neurons.     

Anticonvulsant phenytoin affects voltage-gated potassium currents in cerebellar granule cells

The anticonvulsant drug phenytoin (diphenylhydantoin, DPH) was examined for its action on potassium currents in cerebellar granule cells using the whole-cell patch-clamp technique. Granular cells expressed two main types of voltage-dependent potassium currents: the first, sensitive to Tetraethylammonium ion (TEA), resembles a delayed rectifier K(+) channel (I(d)); the second shows biophysical and pharmacological properties similar to an I(A)-type potassium current. Phenytoin blocks the I(A) current in a dose-dependent manner, with an apparent dissociation constant K(d) of (73+/-7) microM. The drug shifts the steady-state inactivation curves towards a more negative potential, stabilizing the inactivated state, while the activation kinetics remain unaffected. The estimated K(d) when the cell is held to -100 mV (closed state of the channel) is 145+/-8 microM which decreases to 35+/-10 microM at -80 mV holding potential (partial inactivation of the channel). Phenytoin shows a discriminant behaviour between the two different types of potassium channels because at high concentration the effect of the drug on the delayed rectifier K(+) channel is negligible.     

Ca(2+)- and voltage-dependent inactivation of Ca2+ currents in rat intermediate pituitary.

We used single electrode voltage-clamp methods to investigate the inactivation of Ca2+ currents in melanotrophs of the intermediate lobe of the pituitary. The low threshold transient current was inactivated by brief prepulses to potentials above -30 mV and inhibition remained complete as prepulse potential was increased from 0 to +70 mV. Both the high threshold transient and sustained currents, however, were inhibited to the greatest extent (60%) by prepulses to 0 mV. Prepulses to more positive potentials close to the Ca2+ reversal potential produced much less (15%) inactivation. Buffering intracellular Ca2+ by including BAPTA in the recording electrode or replacing extracellular Ca2+ with Ba2+ reduced the effect of prepulses. Slowing Ca2+ extrusion by reducing the Na+ gradient across the cell increased the duration of the effect of prepulses. We conclude that the low threshold, transient current is inactivated primarily by membrane voltage while both the high threshold currents are inhibited by elevation of intracellular Ca2+ although the two currents display different sensitivities to Ca2+ concentration. Inhibition of the high threshold transient current by the neurotransmitter dopamine, however, acts by a different mechanism not mediated by Ca(2+)-dependent inactivation.     

Cell-specific posttranslational events affect functional expression at the plasma membrane but not tetrodotoxin sensitivity of the rat brain IIA sodium channel alpha-subunit expressed in mammalian cells.

The rat brain IIA Na+ channel alpha-subunit was expressed and studied in mammalian cells. Cells were infected with a recombinant vaccinia virus (VV) carrying the bacteriophage T7 RNA polymerase gene and were transfected with cDNA encoding the IIA Na+ channel alpha-subunit under control of a T7 promoter. Whole-cell patch-clamp recording showed that functional IIA channels were expressed efficiently (approximately 10 channels/microns2 in approximately 60% of cells) in Chinese hamster ovary (CHO) cells and in neonatal rat ventricular myocytes but were expressed poorly in undifferentiated BC3H1 cells and failed to express in Ltk- cells. However, voltage-dependent Drosophila Shaker H4 K+ channels and Escherichia coli beta-galactosidase were expressed efficiently in all four cell types with VV vectors. Because RNA synthesis probably occurs without major differences in the cytoplasm of all infected cell types under the control of the T7 promoter and T7 polymerase, we conclude that cell type-specific expression of the Na+ channel probably reflects differences at posttranslational steps. The gating properties of the IIA Na+ currents expressed in cardiac myocytes differed from those expressed in CHO cells; most noticeably, the IIA Na+ currents displayed more rapid macroscopic inactivation when expressed in cardiac myocytes. These differences also suggest cell-specific posttranslational modifications. IIA channels were blocked by approximately 90% by 90 nM TTX when expressed either in CHO cells or in cardiac myocytes; the latter also continued to display endogenous TTX-resistant Na+ currents. Therefore, the TTX binding site of the channel is not affected by cell-specific modifications and is encoded by the primary amino acid sequence.     

Spatial distribution of Ca currents in molluscan neuron cell bodies and regional differences in the strength of inactivation

The spatial distribution of Ca current in molluscan neuron cell bodies was studied using a large patch method in combination with 2-microelectrode voltage clamp. The method has a spatial resolution equal to about 0.1% of the cell body area. Ca current is not uniformly distributed. The current density varies between patches, changing by as much as a factor of 2.5 over a distance of 20 micron, and there is evidence that Ca current occurs in "hot spots" involving a few hundred channels. The current density increases in a moderately steep gradient from the soma cap, opposite the axon, toward the axon hillock. Ca currents in patches from different regions of the soma are qualitatively different. Currents near the soma cap do not inactivate or inactivate weakly during depolarization, while currents of equal density nearer the axon hillock exhibit pronounced inactivation. The strength of inactivation increases in parallel with the gradient in current density, but local differences in current density, or in the number of active Ca channels, do not explain the variability in inactivation. Inactivating and noninactivating Ca currents could not be distinguished on the basis of activation or deactivation kinetics, voltage dependence of activation, or sensitivity to hyperpolarizing conditioning pulses. Also, the amplitude of noninactivating current near the soma cap is reduced by intracellular Ca injection showing that, like the whole-cell current, Ca current in this region is subject to Ca-dependent inactivation. The data favor the hypothesis that these cells express only one type of Ca channel. Differences in the strength of inactivation may result from local differences in cytoplasmic Ca buffering, local modification of Ca channels in a way that changes their sensitivity to Ca-dependent inactivation, or local differences in the availability of cytoplasmic factors or enzymes that are necessary for inactivation.     

Transplanting the N-terminus from Kv1.4 to Kv1.1 generates an inwardly rectifying K+ channel

A chimeric channel, 4N/1, was generated from two outwardly rectifying K+ channels by linking the N-terminal cytoplasmic domain of hKv1.4 (N terminus ball and chain of hKv1.4) with the transmembrane body of hKv1.1 (delta78N1 construct of hKv1.1). The recombinant channel has properties similar to the six transmembrane inward rectifiers and opens on hyperpolarization with a threshold of activation at -90 mV. Outward currents are seen on depolarization provided the channel is first exposed to a hyperpolarizing pulse of -100 mV or more. Hyperpolarization at and beyond -130 mV provides evidence of channel deactivation. Delta78N1 does not show inward currents on hyperpolarization but does open on depolarizing from -80 mV with characteristics similar to native hKv1.1. The outward currents seen in both delta78N1 and 4N/1 inactivate slowly at rates consistent with C-type inactivation. The inward rectification of the 4N/1 chimera is consistent with the inactivation gating mechanism. This implies that the addition of the N-terminus from hKv1.4 to hKv1.1 shifts channel activation to hyperpolarizing potentials. These results suggest a mechanism involving the N-terminal cytoplasmic domain for conversion of outward rectifiers to inward rectifiers.     

The sensitivity of sodium channels in immature and mature rat CA1 neurones to the local anaesthetics procaine and lidocaine

Sodium currents were recorded in CA1 hippocampal cells from new-born (P(4-10)) and older (P(>22)) rats, using whole-cell voltage clamp techniques. The effects of local anaesthetics (procaine and lidocaine) were studied in both cell populations. Parameters defining steady-state inactivation, removal of inactivation and the affinity of the anaesthetic molecules to the inactivated state were determined at both stages of maturation. Procaine and lidocaine induced a hyperpolarizing shift in steady-state inactivation curves, and slowed the rate of recovery from the inactivated state. Procaine disclosed differences between immature and older cells in what concerns block of the closed (resting) channels, drug affinity and binding to the inactivated state, i.e. the binding rate of procaine was found higher and the affinity lower in younger cells. The characteristics of procaine and lidocaine block on CA1 sodium currents differed in some particular aspects: magnitude of block on resting channels, shift in the voltage dependence and voltage sensitivity of steady-state inactivation, slow recovery from inactivation and use-dependent block.     

Voltage-activated K+ currents in acutely isolated hippocampal astrocytes.

Hippocampal astrocytes were acutely isolated by papain treatment and mechanical trituration. Astrocytes were identified by their distinctive stellate morphology and immunocytochemical staining for glial fibrillary acidic protein. The electrophysiological properties of these cells were investigated using whole-cell voltage-clamp techniques. Three kinetically and pharmacologically distinct voltage-activated K+ currents were identified in most cells; they resembled the neuronal A-current, delayed rectifier, and inward rectifier. The activation threshold of the A-current was -40 mV with a time to peak that ranged from 10 msec at -20 mV to 6 msec at 100 mV. Steady-state inactivation was observed when the holding potential was positive to -100 mV. The current was half-inactivated at -60 mV and totally inactivated at -20 mV. The A-current was suppressed by 4-aminopyridine (4-AP). The delayed rectifier was activated by depolarizing pulses more positive than -40 mV and had a half time of activation that ranged from 18 msec at -20 mV to 10 msec at potentials more positive than 40 mV. This current did not inactivate during a 100 msec pulse and was suppressed by extracellular tetraethylammonium (TEA). An inwardly rectifying current was elicited by hyperpolarizing pulses more negative than -80 mV. This current was not blocked by extracellular TEA or 4-AP and was never observed in the presence of external Ba2+. Voltage-activated inward Na+ currents were never observed. Voltage-activated K+ channels may enhance the local K+ spatial buffering capabilities of the astrocyte syncytium when extracellular [K+] increases during neuronal activity.     

Different Types of Potassium Outward Current in Relay Neurons Acutely Isolated from the Rat Lateral Geniculate Nucleus

Different classes of potassium (K+) outward current activated by depolarization were characterized in relay neurons acutely isolated from the rat lateral geniculate nucleus (LGN), using the whole-cell version of the patch-clamp technique. A fast-transient current (IA), activated at around - 70 mV, declined rapidly with a voltage-dependent time constant (tau=6 ms at + 45 mV), was 50% steady-state inactivated at - 70 mV, and rapidly recovered from inactivation with a monoexponential time course (tau=21 ms). IA was blocked by 4-aminopyridine (4-AP, 2 - 8 mM) and was relatively insensitive to tetraethylammonium (TEA, 2 - 10 mM). After elimination of IA by a conditioning prepulse (30 ms to - 50 mV), a slow-transient K+ current could be studied in isolation, and was separated into three components, IKm, IKs and a calcium (Ca2+)-dependent current, IK[Ca]. The slow-transient current was not consistently affected by 4-AP (up to 8 mM), while TEA (2 - 10 mM) predominantly blocked IKs and IK[Ca]. The component IKm persisted in a solution containing TEA and 4-AP, activated at around - 55 mV, declined monoexponentially during maintained depolarization (tau=98 ms at + 45 mV), was 50% inactivated at - 39 mV, and recovered with tau=128 ms from inactivation. IKs activated at a similar threshold, but declined much slower with tau=2662 ms at + 45 mV. Steady-state inactivation of IKs was half-maximal at - 49 mV, and recovery from inactivation occurred relatively fast with tau=116 ms. From these data and additional current-clamp recordings it is concluded that the K+ currents, due to their wide range of kinetics and dependence on membrane voltage or internal Ca2+ concentration, are capable of cooperatively controlling the firing threshold and of shaping the different states of electrophysiological behaviour in LGN relay cells.     

Voltage-gated potassium channels in larval CNS neurons of Drosophila

The availability of genetic, molecular, and biophysical techniques makes Drosophila an ideal system for the study of ion channel function. We have used the patch-clamp technique to characterize voltage-gated K+ channels in cultured larval Drosophila CNS neurons. Whole-cell currents from different cells vary in current kinetics and magnitude. Most of the cells contain a transient A-type 4-AP-sensitive current. In addition, many cells also have a more slowly inactivating TEA-sensitive component and/or a sustained component. No clear correlation between cell morphology and whole-cell current kinetics was observed. Single-channel analysis in cell-free patches revealed that 3 types of channels, named A2, KD, and K1 can account for the whole-cell currents. None of these channels requires elevated intracellular calcium concentration for activation. The A2 channels have a conductance of 6-8 pS and underlie the whole-cell A current. They turn on rapidly, inactivate in response to depolarizing voltage steps, and are completely inactivated by prepulses to -50 mV. The KD (delayed) channels have a conductance of 10-16 pS and can account, in part, for the more slowly inactivating component of whole-cell current. They have longer open times and activate and inactivate more slowly than the A2 channels. The K1 channels have a slope conductance, measured between 0 and +40 mV, of 20-40 pS. These channels do not inactivate during 500 msec voltage steps and thus can contribute to the sustained component of current. They exhibit complex gating behavior with increased probability of being open at higher voltages. Although the K1 channels are sufficient to account for the noninactivating component of whole-cell current, we have observed several other channel types that have a similar voltage dependence and average kinetics.     

Ethanol inhibits voltage-gated sodium channels in cultured superior cervical ganglion neurons

To determine whether actions on sodium channels contribute to ethanol's depressant effects on the autonomic nervous system, the acute effects of ethanol on Na+ currents in primary cultured superior cervical ganglion were examined by whole-cell patch clamp recordings. Ethanol inhibited Na+ currents concentration dependently, and decreased action potential firing. Ethanol (100 mM) did not affect activation curve, but resulted in a left shift of the inactivation curve and prolonged the recovery from inactivation. This finding indicates that the channels in the inactivated state are more susceptible to ethanol than those in the resting state. For the first time, this study demonstrates acute inhibitory effects of ethanol on sodium channel gating in sympathetic neurons.     

Evidence for voltage-activated outward currents in the neuropilar membrane of locust nonspiking local interneurons

Outward currents activated by depolarization were studied in the neuropilar membrane of locust nonspiking local interneurons, using the single-electrode voltage-clamp technique in situ. Preliminary observation of these currents in 272 neurons revealed two families. The first and most commonly observed (85% of recordings) showed a large transient current followed by a slowly decaying/late current. The second (15% of recordings) showed an additional outward current with a slow rate of activation, a peak within 100-150 msec, and a slow rate of inactivation. Only neurons of the first type were studied further. The transient current was activated by depolarization around -60 mV, with a time to peak of approximately 11 msec at -50 mV and less than 3 msec at -20 mV. This current decayed exponentially, with a time constant of 8.1 +/- 1.6 msec (n = 8 interneurons) at -30 mV. This time constant of inactivation did not appear to depend strongly on membrane voltage, in the range in which it was studied. A second and longer time constant of inactivation of 50-400 msec could not be assigned to either of the transient and late components of the outward current. The ratio of transient-to-late current varied between 1.6 and 5.4, with a mean of about 2.5. The reversal potential for the transient current could, on average, be shifted by 14 mV by a threefold increase in the bath K+ concentration, indicating that K+ is a charge carrier for the current. The transient current became inactivated with maintained depolarization and appeared half-inactivated at about -60 mV (slope factor k1/2 = 8 mV). This current was thus not fully inactivated at "resting" potential (average, -58 mV). Recovery from inactivation followed a single exponential time course, with a time constant of approximately 100 msec at -80 mV. The time course of recovery from inactivation of the transient current was well correlated with that of the recovery of transient outward rectification, as measured in current-clamp recording. Tetraethylammonium, at a bath concentration of 10 mM reduced the transient current by 70% and the delayed current by 60%. 4-Aminopyridine, at a bath concentration of 5 mM, had a significant effect in only two of five interneurons, reducing the transient current by approximately 85% and the late current by approximately 15%. Quinidine at a bath concentration of 100 microM was ineffective. Although these blockers did not allow a clear pharmacological separation of the currents, they were effective in reducing the outward rectification observed in current clamp during step depolarization.(ABSTRACT TRUNCATED AT 400 WORDS)     

Modification of Kv2.1 K+ currents by the silent Kv10 subunits

Human and rat Kv10.1a and b cDNAs encode silent K+ channel pore-forming subunits that modify the electrophysiological properties of Kv2.1. These alternatively spliced variants arise by the usage of an alternative site of splicing in exon 1 producing an 11-amino acid insertion in the linker between the first and second transmembrane domains in Kv10.1b. In human, the Kv10s mRNA were detected by Northern blot in brain kidney lung and pancreas. In brain, they were expressed in cortex, hippocampus, caudate, putamen, amygdala and weakly in substantia nigra. In rat, Kv10.1 products were detected in brain and weakly in testes. In situ hybridization in rat brain shows that Kv10.1 mRNAs are expressed in cortex, olfactory cortical structures, basal ganglia/striatal structures, hippocampus and in many nuclei of the amygdala complex. The CA3 and dentate gyrus of the hippocampus present a gradient that show a progression from high level of expression in the caudo-ventro-medial area to a weak level in the dorso-rostral area. The CA1 and CA2 areas had low levels throughout the hippocampus. Several small nuclei were also labeled in the thalamus, hypothalamus, pons, midbrain, and medulla oblongata. Co-injection of Kv2.1 and Kv10.1a or b mRNAs in Xenopus oocytes produced smaller currents that in the Kv2.1 injected oocytes and a moderate reduction of the inactivation rate without any appreciable change in recovery from inactivation or voltage dependence of activation or inactivation. At higher concentration, Kv10.1a also reduces the activation rate and a more important reduction in the inactivation rate. The gene that encodes for Kv10.1 mRNAs maps to chromosome 2p22.1 in human, 6q12 in rat and 17E4 in mouse, locations consistent with the known systeny for human, rat and mouse chromosomes.