Expression and biophysical properties of Kv1 channels in supragranular neocortical pyramidal neurones

Potassium channels are extremely diverse regulators of neuronal excitability. As part of an investigation into how this molecular diversity is utilized by neurones, we examined the expression and biophysical properties of native Kv1 channels in layer II/III pyramidal neurones from somatosensory and motor cortex. Single-cell RT-PCR, immunocytochemistry, and whole cell recordings with specific peptide toxins revealed that individual pyramidal cells express multiple Kv1 α-subunits. The most abundant subunit mRNAs were Kv1.1 > 1.2 > 1.4 > 1.3. All of these subunits were localized to somatodendritic as well as axonal cell compartments. These data suggest variability in the subunit complexion of Kv1 channels in these cells. The α-dendrotoxin (α-DTX)-sensitive current activated more rapidly and at more negative potentials than the α-DTX-insensitive current, was first observed at voltages near action potential threshold, and was relatively insensitive to holding potential. The α-DTX-sensitive current comprised about 10% of outward current at steady-state, in response to steps from −70 mV. From −50 mV, this percentage increased to ∼20%. All cells expressed an α-DTX-sensitive current with slow inactivation kinetics. In some cells a transient component was also present. Deactivation kinetics were voltage dependent, such that deactivation was slow at potentials traversed by interspike intervals during repetitive firing. Because of its kinetics and voltage dependence, the α-DTX-sensitive current should be most important at physiological resting potentials and in response to brief stimuli. Kv1 channels should also be important at voltages near threshold and corresponding to interspike intervals.     

Kv3.1-Kv3.2 channels underlie a high-voltage-activating component of the delayed rectifier K+ current in projecting neurons from the globus pallidus.

The globus pallidus plays central roles in the basal ganglia circuitry involved in movement control as well as in cognitive and emotional functions. There is therefore great interest in the anatomic and electrophysiological characterization of this nucleus. Most pallidal neurons are GABAergic projecting cells, a large fraction of which express the calcium binding protein parvalbumin (PV). Here we show that PV-containing pallidal neurons coexpress Kv3. 1 and Kv3.2 K+ channel proteins and that both Kv3.1 and Kv3.2 antibodies coprecipitate both channel proteins from pallidal membrane extracts solubilized with nondenaturing detergents, suggesting that the two channel subunits are forming heteromeric channels. Kv3.1 and Kv3.2 channels have several unusual electrophysiological properties when expressed in heterologous expression systems and are thought to play special roles in neuronal excitability including facilitating sustained high-frequency firing in fast-spiking neurons such as interneurons in the cortex and the hippocampus. Electrophysiological analysis of freshly dissociated pallidal neurons demonstrates that these cells have a current that is nearly identical to the currents expressed by Kv3.1 and Kv3.2 proteins in heterologous expression systems, including activation at very depolarized membrane potentials (more positive than -10 mV) and very fast deactivation rates. These results suggest that the electrophysiological properties of native channels containing Kv3.1 and Kv3.2 proteins in pallidal neurons are not significantly affected by factors such as associated subunits or postranslational modifications that result in channels having different properties in heterologous expression systems and native neurons. Most neurons in the globus pallidus have been reported to fire sustained trains of action potentials at high-frequency. Kv3.1-Kv3.2 voltage-gated K+ channels may play a role in helping maintain sustained high-frequency repetitive firing as they probably do in other neurons.     

Kv1.1-containing channels are critical for temporal precision during spike initiation

Low threshold, voltage-gated potassium currents (Ikl) are widely expressed in auditory neurons that can fire temporally precise action potentials (APs). In the medial nucleus of the trapezoid body (MNTB), channels containing the Kv1.1 subunit (encoded by the Kcna1 gene) underlie Ikl. Using pharmacology, genetics and whole cell patch-clamp recordings in mouse brain slices, we tested the role of Ikl in limiting AP latency-variability (jitter) in response to trains of single inputs at moderate to high stimulation rates. With dendrotoxin-K (DTX-K, a selective blocker of Kv1.1-containing channels), we blocked Ikl maximally (approximately 80% with 100 nM DTX-K) or partially (approximately 50% with 1-h incubation in 3 nM DTX-K). Ikl was similar in 3 nM DTX-K-treated cells and cells from Kcna1(-/-) mice, allowing a comparison of these two different methods of Ikl reduction. In response to current injection, Ikl reduction increased the temporal window for AP initiation and increased jitter in response to the smallest currents that were able to drive APs. While 100 nM DTX-K caused the largest increases, latency and jitter in Kcna1(-/-) cells and in 3 nM DTX-K-treated cells were similar to each other but increased compared with +/+. The near-phenocopy of the Kcna1(-/-) cells with 3 nM DTX-K shows that acute blockade of a subset of the Kv1.1-containing channels is functionally similar to the chronic elimination of all Kv1.1 subunits. During rapid stimulation (100-500 Hz), Ikl reduction increased jitter in response to both large and small inputs. These data show that Ikl is critical for maintaining AP temporal precision at physiologically relevant firing rates.     

Manipulation of the potassium channel Kv1.1 and its effect on neuronal excitability in rat sensory neurons

Potassium channels play a critical role in regulating many aspects of action potential (AP) firing. To establish the contribution of the voltage-dependent potassium channel Kv1.1 in regulating excitability, we used the selective blocker dendrotoxin-K (DTX-K) and small interfering RNA (siRNA) targeted to Kv1.1 to determine their effects on AP firing in small-diameter capsaicin-sensitive sensory neurons. A 5-min exposure to 10 nM DTX-K suppressed the total potassium current (I(K)) measured at +40 mV by about 33%. DTX-K produced a twofold increase in the number of APs evoked by a ramp of depolarizing current. Associated with increased firing was a decrease in firing threshold and rheobase. DTX-K did not alter the resting membrane potential or the AP duration. A 48-h treatment with siRNA targeted to Kv1.1 reduced the expression of this channel protein by about 60% as measured in Western blots. After treatment with siRNA, I(K) was no longer sensitive to DTX-K, indicating a loss of functional protein. Similarly, after siRNA treatment exposure to DTX-K had no effect on the number of evoked APs, firing threshold, or rheobase. However, after siRNA treatment, the firing threshold had values similar to those obtained after acute exposure to DTX-K, suggesting that the loss of Kv1.1 plays a critical role in setting this parameter of excitability. These results demonstrate that Kv1.1 plays an important role in limiting AP firing and that siRNA may be a useful approach to establish the role of specific ion channels in the absence of selective antagonists.     

Potassium channels Kv1.1, Kv1.2 and Kv1.6 influence excitability of rat visceral sensory neurons

Voltage-gated potassium channels, Kv1.1, Kv1.2 and Kv1.6, were identified as PCR products from mRNA prepared from nodose ganglia. Immunocytochemical studies demonstrated expression of the proteins in all neurons from ganglia of neonatal animals (postnatal days 0-3) and in 85-90 % of the neurons from older animals (postnatal days 21-60). In voltage clamp studies, α-dendrotoxin (α-DTX), a toxin with high specificity for these members of the Kv1 family, was used to examine their contribution to K⁺ currents of the sensory neurons. α-DTX blocked current in both A- and C-type neurons. The current had characteristics of a delayed rectifier with activation positive to −50 mV and little inactivation during 250 ms pulses. In current-clamp experiments α-DTX, used to eliminate the current, had no effect on resting membrane potential and only small effects on the amplitude and duration of the action potential of A- and C-type neurons. However, there were prominent effects on excitability. α-DTX lowered the threshold for initiation of discharge in response to depolarizing current steps, reduced spike after-hyperpolarization and increased the frequency/pattern of discharge of A- and C-type neurons at membrane potentials above threshold. Model simulations were consistent with these experimental results and demonstrated how the other major K⁺ currents function in response to the loss of the α-DTX-sensitive current to effect these changes in action potential wave shape and discharge.     

Presynaptic Rat Kv1.2 Channels Suppress Synaptic Terminal Hyperexcitability Following Action Potential Invasion

Voltage-gated K⁺ channels activating close to resting membrane potentials are widely expressed and differentially located in axons, presynaptic terminals and cell bodies. There is extensive evidence for localisation of Kv1 subunits at many central synaptic terminals but few clues to their presynaptic function. We have used the calyx of Held to investigate the role of presynaptic Kv1 channels in the rat by selectively blocking Kv1.1 and Kv1.2 containing channels with dendrotoxin-K (DTX-K) and tityustoxin-Kα (TsTX-Kα) respectively. We show that Kv1.2 homomers are responsible for two-thirds of presynaptic low threshold current, whilst Kv1.1/Kv1.2 heteromers contribute the remaining current. These channels are located in the transition zone between the axon and synaptic terminal, contrasting with the high threshold K⁺ channel subunit Kv3.1 which is located on the synaptic terminal itself. Kv1 homomers were absent from bushy cell somata (from which the calyx axons arise); instead somatic low threshold channels consisted of heteromers containing Kv1.1, Kv1.2 and Kv1.6 subunits. Current-clamp recording from the calyx showed that each presynaptic action potential (AP) was followed by a depolarising after-potential (DAP) lasting around 50 ms. Kv1.1/Kv1.2 heteromers had little influence on terminal excitability, since DTX-K did not alter AP firing. However TsTX-Kα increased DAP amplitude, bringing the terminal closer to threshold for generating an additional AP. Paired pre- and postsynaptic recordings confirmed that this aberrant AP evoked an excitatory postsynaptic current (EPSC). We conclude that Kv1.2 channels have a general presynaptic function in suppressing terminal hyperexcitability during the depolarising after-potential.     

Properties and molecular basis of the mouse urinary bladder voltage-gated K+ current

Potassium channels play an important role in controlling the excitability of urinary bladder smooth muscle (UBSM). Here we describe the biophysical, pharmacological and molecular properties of the mouse UBSM voltage-gated K⁺ current ( I _{K ( V)}). The I _{K ( V)} activated, deactivated and inactivated slowly with time constants of 29.9 ms at +30 mV, 131 ms at −40 mV and 3.4 s at +20 mV. The midpoints of steady-state activation and inactivation curves were 1.1 mV and −61.4 mV, respectively. These properties suggest that I _{K ( V)} plays a role in regulating the resting membrane potential and contributes to the repolarization and after-hyperpolarization phases of action potentials. The I _{K ( V)} was blocked by tetraethylammonium ions with an IC₅₀ of 5.2 m m and was unaffected by 1 m m 4-aminopyridine. RT-PCR for voltage-gated K⁺ channel (K_V) subunits revealed the expression of Kv2.1, Kv5.1, Kv6.1, Kv6.2 and Kv6.3 in isolated UBSM myocytes. A comparison of the biophysical properties of UBSM I _{K ( V)} with those reported for Kv2.1 and Kv5.1 and/or Kv6 heteromultimeric channels demonstrated a marked similarity. We propose that heteromultimeric channel complexes composed of Kv2.1 and Kv5.1 and/or Kv6 subunits form the molecular basis of the mouse UBSM I _{K ( V)}.     

Altered expression of potassium channel subunit mRNA and alpha-dendrotoxin sensitivity of potassium currents in rat dorsal root ganglion neurons after axotomy

Previous studies have raised the possibility that a decrease in voltage-gated K+ currents may contribute to hyperexcitability of injured dorsal root ganglion (DRG) neurons and the emergence of neuropathic pain. We examined the effects of axotomy on mRNA levels for various Kv1 family subunits and voltage-gated K+ currents in L4-L5 DRG neurons from sham-operated and sciatic nerve-transected rats. RNase protection assay revealed that Kv1.1 and Kv 1.2 mRNAs are highly abundant while Kv1.3, Kv1.4, Kv1.5 and Kv1.6 mRNAs were detected at lower levels in L4-L5 DRGs from sham and intact rats. Axotomy significantly decreased Kv1.1, Kv1.2, Kv1.3 and Kv1.4 mRNA levels by approximately 35%, approximately 60%, approximately 40% and approximately 80%, respectively, but did not significantly change Kv1.5 or Kv1.6 mRNA levels. Patch clamp recordings revealed two types of K+ currents in small-sized L4-L5 DRG neurons: sustained delayed rectifier currents elicited from a -40 mV holding potential and slowly inactivating A-type currents that was additionally activated from a -120 mV holding potential. Axotomy decreased both types of K+ currents by 50-60% in injured DRG neurons. In addition, axotomy increased the alpha-dendrotoxin sensitivity of the delayed rectifier, but not slow A-type K+ currents in injured DRG neurons. These results suggest that Kv1.1 and Kv1.2 subunits are major components of voltage-gated K+ channels in L4-L5 DRG neurons and that the decreased expression of Kv1-family subunits significantly contributes to the reduction and altered kinetics of Kv current in axotomized neurons.     

Initial segment Kv2.2 channels mediate a slow delayed rectifier and maintain high frequency action potential firing in medial nucleus of the trapezoid body neurons

The medial nucleus of the trapezoid body (MNTB) is specialized for high frequency firing by expression of Kv3 channels, which minimize action potential (AP) duration, and Kv1 channels, which suppress multiple AP firing, during each calyceal giant EPSC. However, the outward K⁺ current in MNTB neurons is dominated by another unidentified delayed rectifier. It has slow kinetics and a peak conductance of ∼37 nS; it is half-activated at −9.2 ± 2.1 mV and half-inactivated at −35.9 ± 1.5 mV. It is blocked by several non-specific potassium channel antagonists including quinine (100 μ m ) and high concentrations of extracellular tetraethylammonium (TEA; IC₅₀= 11.8 m m ), but no specific antagonists were found. These characteristics are similar to recombinant Kv2-mediated currents. Quantitative RT-PCR showed that Kv2.2 mRNA was much more prevalent than Kv2.1 in the MNTB. A Kv2.2 antibody showed specific staining and Western blots confirmed that it recognized a protein ∼110 kDa which was absent in brainstem tissue from a Kv2.2 knockout mouse. Confocal imaging showed that Kv2.2 was highly expressed in axon initial segments of MNTB neurons. In the absence of a specific antagonist, Hodgkin–Huxley modelling of voltage-gated conductances showed that Kv2.2 has a minor role during single APs (due to its slow activation) but assists recovery of voltage-gated sodium channels (Nav) from inactivation by hyperpolarizing interspike potentials during repetitive AP firing. Current-clamp recordings during high frequency firing and characterization of Nav inactivation confirmed this hypothesis. We conclude that Kv2.2-containing channels have a distinctive initial segment location and crucial function in maintaining AP amplitude by regulating the interspike potential during high frequency firing.     

Localization and function of the Kv3.1b subunit in the rat medulla oblongata: focus on the nucleus tractus solitarii

The voltage-gated potassium channel subunit Kv3.1 confers fast firing characteristics to neurones. Kv3.1b subunit immunoreactivity (Kv3.1b-IR) was widespread throughout the medulla oblongata, with labelled neurones in the gracile, cuneate and spinal trigeminal nuclei. In the nucleus of the solitary tract (NTS), Kv3.1b-IR neurones were predominantly located close to the tractus solitarius (TS) and could be GABAergic or glutamatergic. Ultrastructurally, Kv3.1b-IR was detected in NTS terminals, some of which were vagal afferents. Whole-cell current-clamp recordings from neurones near the TS revealed electrophysiological characteristics consistent with the presence of Kv3.1b subunits: short duration action potentials (4.2 ± 1.4 ms) and high firing frequencies (68.9 ± 5.3 Hz), both sensitive to application of TEA (0.5 m m ) and 4-aminopyridine (4-AP; 30 μ m ). Intracellular dialysis of an anti-Kv3.1b antibody mimicked and occluded the effects of TEA and 4-AP in NTS and dorsal column nuclei neurones, but not in dorsal vagal nucleus or cerebellar Purkinje cells (which express other Kv3 subunits, but not Kv3.1b). Voltage-clamp recordings from outside-out patches from NTS neurones revealed an outward K⁺ current with the basic characteristics of that carried by Kv3 channels. In NTS neurones, electrical stimulation of the TS evoked EPSPs and IPSPs, and TEA and 4-AP increased the average amplitude and decreased the paired pulse ratio, consistent with a presynaptic site of action. Synaptic inputs evoked by stimulation of a region lacking Kv3.1b-IR neurones were not affected, correlating the presence of Kv3.1b in the TS with the pharmacological effects.     

Kv3-like potassium channels are required for sustained high-frequency firing in basal ganglia output neurons

The GABA projection neurons in the substantial nigra pars reticulata (SNr) are key output neurons of the basal ganglia motor control circuit. These neurons fire sustained high-frequency, short-duration spikes that provide a tonic inhibition to their targets and are critical to movement control. We hypothesized that a robust voltage-activated K(+) conductance that activates quickly and resists inactivation is essential to the remarkable fast-spiking capability in these neurons. Semi-quantitative RT-PCR (qRT-PCR) analysis on laser capture-microdissected nigral neurons indicated that mRNAs for Kv3.1 and Kv3.4, two key subunits for forming high activation threshold, fast-activating, slow-inactivating, 1 mM tetraethylammonium (TEA)-sensitive, fast delayed rectifier (I(DR-fast)) type Kv channels, are more abundant in fast-spiking SNr GABA neurons than in slow-spiking nigral dopamine neurons. Nucleated patch clamp recordings showed that SNr GABA neurons have a strong Kv3-like I(DR-fast) current sensitive to 1 mM TEA that activates quickly at depolarized membrane potentials and is resistant to inactivation. I(DR-fast) is smaller in nigral dopamine neurons. Pharmacological blockade of I(DR-fast) by 1 mM TEA impaired the high-frequency firing capability in SNr GABA neurons. Taken together, these results indicate that Kv3-like channels mediating fast-activating, inactivation-resistant I(DR-fast) current are critical to the sustained high-frequency firing in SNr GABA projection neurons and hence movement control.     

A-type voltage-gated K+ currents influence firing properties of isolectin B4-positive but not isolectin B4-negative primary sensory neurons

Voltage-gated K+ channels (Kv) in primary sensory neurons are important for regulation of neuronal excitability. The dorsal root ganglion (DRG) neurons are heterogeneous, and the types of native Kv currents in different groups of nociceptive DRG neurons are not fully known. In this study, we determined the difference in the A-type Kv current and its influence on the firing properties between isolectin B4 (IB4)-positive and -negative DRG neurons. Whole cell voltage- and current-clamp recordings were performed on acutely dissociated small DRG neurons of rats. The total Kv current density was significantly higher in IB+-positive than that in IB(4)-negative neurons. Also, 4-aminopyridine (4-AP) produced a significantly greater reduction in Kv currents in IB4-positive than in IB4-negative neurons. In contrast, IB4-negative neurons exhibited a larger proportion of tetraethylammonium-sensitive Kv currents. Furthermore, IB4-positive neurons showed a longer latency of firing and required a significantly larger amount of current injection to evoke action potentials. 4-AP significantly decreased the latency of firing and increased the firing frequency in IB4-positive but not in IB4-negative neurons. Additionally, IB4-positive neurons are immunoreactive to Kv1.4 but not to Kv1.1 and Kv1.2 subunits. Collectively, this study provides new information that 4-AP-sensitive A-type Kv currents are mainly present in IB4-positive DRG neurons and preferentially dampen the initiation of action potentials of this subpopulation of nociceptors. The difference in the density of A-type Kv currents contributes to the distinct electrophysiological properties of IB4-positive and -negative DRG neurons.     

Ether-à-go-go-related gene K+ channels contribute to threshold excitability of mouse auditory brainstem neurons

The ionic basis of excitability requires identification and characterisation of expressed channels and their specific roles in native neurons. We have exploited principal neurons of the medial nucleus of the trapezoid body (MNTB) as a model system for examining voltage-gated K⁺ channels, because of their known function and simple morphology. Here we show that channels of the ether-à-go-go -related gene family (ERG, Kv11; encoded by kcnh ) complement Kv1 channels in regulating neuronal excitability around threshold voltages. Using whole-cell patch clamp from brainstem slices, the selective ERG antagonist E-4031 reduced action potential (AP) threshold and increased firing on depolarisation. In P12 mice, under voltage-clamp with elevated [K⁺]_o (20 m m ), a slowly deactivating current was blocked by E-4031 or terfenadine ( V _0.5,act=−58.4 ± 0.9 mV, V _0.5,inact=−76.1 ± 3.6 mV). Deactivation followed a double exponential time course (τ_slow= 113.8 ± 6.9 ms, τ_fast= 33.2 ± 3.8 ms at −110 mV, τ_fast 46% peak amplitude). In P25 mice, deactivation was best fitted by a single exponential (τ_fast= 46.8 ± 5.8 ms at −110 mV). Quantitative RT-PCR showed that ERG1 and ERG3 were the predominant mRNAs and immunohistochemistry showed expression as somatic plasma membrane puncta on principal neurons. We conclude that ERG currents complement Kv1 currents in limiting AP firing at around threshold; ERG may have a particular role during periods of high activity when [K⁺]_o is elevated. These ERG currents suggest a potential link between auditory hyperexcitability and acoustic startle triggering of cardiac events in familial LQT2.     

Involvement of kv1 potassium channels in spreading acidification and depression in the cerebellar cortex

Spreading acidification and depression (SAD) is a form of propagated activity in the cerebellar cortex characterized by acidification and a transient depression in excitability. This study investigated the role of Kv1 potassium channels in SAD using neutral red, flavoprotein autofluorescence, and voltage-sensitive dye optical imaging in the mouse cerebellar cortex, in vivo. The probability of evoking SAD was greatly increased by blocking Kv1.1 as well as Kv1.2 potassium channels by their specific blockers dendrotoxin K (DTX-K) and tityustoxin (TsTX), respectively. DTX-K not only greatly lowered the threshold for evoking SAD but also resulted in multiple cycles of spread and spontaneous SAD. The occurrence of spontaneous SAD originating from spontaneous parallel fiber-like beams of activity suggests that blocking Kv1 channels increased parallel fiber excitability. This was confirmed by the generation of parallel fiber-like beams with the microinjection of glutamate into the upper molecular layer in the presence of DTX-K. The dramatic effects of DTX-K suggest a possible connection between SAD and episodic ataxia type 1 (EA1), a Kv1.1 potassium channelopathy. The threshold for evoking SAD was significantly lowered in the Kv1.1 heterozygous knockout mouse compared with wild-type littermates. Carbamazepine and acetazolamide, both effective in the treatment of EA1, significantly decreased the likelihood of evoking SAD. Blocking GABAergic neurotransmission did not alter the effectiveness of DTX-K. The cyclin D2 null mouse, which lacks cerebellar stellate cells, also exhibited SAD. Therefore blocking Kv1 potassium channels establishes the conditions needed to generate SAD. Furthermore, the results are consistent with the hypothesis that SAD may underlie the transient attacks of ataxia characterizing EA1.     

Characterization of the heteromeric potassium channel formed by kv2.1 and the retinal subunit kv8.2 in Xenopus oocytes

Kv8.2 (KCNV2) subunits do not form homotetrameric potassium channels, although they coassemble with Kv2.1 to constitute functional heteromers. High expression of Kv8.2 was reported in the human retina and its mutations were linked to the visual disorder "cone dystrophy with supernormal rod electroretinogram." We detected abundant Kv8.2 expression in the photoreceptor layer of mouse retina, where Kv2.1 is also known to be present. When the two subunits were coexpressed in Xenopus oocytes in equal amounts, Kv8.2 abolished the current of Kv2.1. If the proportion of Kv8.2 was reduced then the current of heteromeric channels emerged. Kv8.2 shifted the steady-state activation of Kv2.1 to more negative potentials, without affecting the voltage dependence of inactivation. This gave rise to a window current within the -40 to -10 mV membrane potential range. Ba2+ inhibited the heteromeric channel and shifted its activation to more positive potentials. These electrophysiological and pharmacological properties resemble those of the voltage-gated K+ current (named I Kx) described in amphibian retinal rods. Furthermore, oocytes expressing Kv2.1/Kv8.2 developed transient hyperpolarizing overshoots in current-clamp experiments, whereas those expressing only Kv2.1 failed to do so. Similar overshoots are characteristic responses of photoreceptors to light flashes. We demonstrated that Kv8.2 G476D, analogous to a disease-causing human mutation, eliminated Kv2.1 current, if the subunits were coexpressed equally. However, Kv8.2 G476D did not form functional heteromers under any conditions. Therefore we suggest that the custom-tailored current of Kv2.1/Kv8.2 functionally contributes to photoreception, and this is the reason that mutations of Kv8.2 lead to a genetic visual disorder.     

Two types of voltage-gated K(+) currents in dissociated heart ventricular muscle cells of the snail Lymnaea stagnalis

We have used a combination of current-clamp and voltage-clamp techniques to characterize the electrophysiological properties of enzymatically dissociated Lymnaea heart ventricle cells. Dissociated ventricular muscle cells had average resting membrane potentials of -55 +/- 5 mV. When hyperpolarized to potentials between -70 and -63 mV, ventricle cells were capable of firing repetitive action potentials (8.5 +/- 1.2 spikes/min) that failed to overshoot 0 mV. The action potentials were either simple spikes or more complex spike/plateau events. The latter were always accompanied by strong contractions of the muscle cell. The waveform of the action potentials were shown to be dependent on the presence of extracellular Ca(2+) and K(+) ions. With the use of the single-electrode voltage-clamp technique, two types of voltage-gated K(+) currents were identified that could be separated by differences in their voltage sensitivity and time-dependent kinetics. The first current activated between -50 and -40 mV. It was relatively fast to activate (time-to-peak; 13.7 +/- 0.7 ms at +40 mV) and inactivated by 53.3 +/- 4.9% during a maintained 200-ms depolarization. It was fully available for activation below -80 mV and was completely inactivated by holding potentials more positive than -40 mV. It was completely blocked by 5 mM 4-aminopyridine (4-AP) and by concentrations of tetraethylammonium chloride (TEA) >10 mM. These properties characterize this current as a member of the A-type family of voltage-dependent K(+) currents. The second voltage-gated K(+) current activated at more depolarized potentials (-30 to -20 mV). It activated slower than the A-type current (time-to-peak; 74.1 +/- 3.9 ms at +40 mV) and showed little inactivation (6.2 +/- 2.1%) during a maintained 200-ms depolarization. The current was fully available for activation below -80 mV with a proportion of the current still available for activation at potentials as positive as 0 mV. The current was completely blocked by 1-3 mM TEA. These properties characterize this current as a member of the delayed rectifier family of voltage-dependent K(+) currents. The slow activation rates and relatively depolarized activation thresholds of the two K(+) currents are suggestive that their main role is to contribute to the repolarization phase of the action potential.     

Modification of rat brain Kv1.4 channel gating by association with accessory Kvβ1.1 and β2.1 subunits

 We have examined the effects of co-expression of Kvβ1.1 and Kvβ2.1 subunits on the gating of rat brain Kv1.4 channels, expressed in Xenopus oocytes. Expression of Kv1.4 subunits alone produced a rapidly inactivating ”A” type current, which activated at potentials beyond –60 mV in a solution containing high levels of rubidium. Current activation curves obtained from tail current measurements were fitted with a Boltzmann function, with V _1/2 = –47 mV and k = 10 mV. Neither the Kvβ1.1 nor Kvβ2.1 subunits altered the voltage dependence of activation. Both subunits accelerated the activation time constant of Kv1.4, without affecting its voltage dependence. Surprisingly, the Kvβ2.1 subunit, which lacks an N-terminal inactivation domain, was almost as effective as the Kvβ1.1 subunit in speeding up Kv1.4. Steady-state inactivation of Kv1.4 was unchanged upon co-expression with either Kvβ1.1 or Kvβ2.1 subunits. Kv1.4 recovered from inactivation with two time constants; apart from an ≈ 50% lengthening of the slow time constant with a high Kvβ2.1 injection ratio, neither time constant was altered by either the Kvβ1.1 or Kvβ2.1 subunits, suggesting little interaction with recovery from C-type inactivation. Clearly, β subunits have the potential to modify the gating of Kv1.4 channels in the brain more subtly than has been suggested previously. Received: 17 March 1997 / Accepted: 30 June 1997     

Kv3.4 subunits enhance the repolarizing efficiency of Kv3.1 channels in fast-spiking neurons

Neurons with the capacity to discharge at high rates--'fast-spiking' (FS) neurons--are critical participants in central motor and sensory circuits. It is widely accepted that K+ channels with Kv3.1 or Kv3.2 subunits underlie fast, delayed-rectifier (DR) currents that endow neurons with this FS ability. Expression of these subunits in heterologous systems, however, yields channels that open at more depolarized potentials than do native Kv3 family channels, suggesting that they differ. One possibility is that native channels incorporate a subunit that modifies gating. Molecular, electrophysiological and pharmacological studies reported here suggest that a splice variant of the Kv3.4 subunit coassembles with Kv3.1 subunits in rat brain FS neurons. Coassembly enhances the spike repolarizing efficiency of the channels, thereby reducing spike duration and enabling higher repetitive spike rates. These results suggest that manipulation of K3.4 subunit expression could be a useful means of controlling the dynamic range of FS neurons.     

IGF-1 up-regulates K+ channels via PI3-kinase, PDK1 and SGK1

Involvement of voltage-gated (Kv) potassium channels in IGF-1-induced proliferation of HEK293 cells was studied by patch-clamp, RT-PCR and FACS analysis. IGF-1 up-regulated outwardly rectifying whole-cell K+ current starting after 1 h of incubation and reaching a maximum after 4-6 h. The IGF-1-stimulated current was voltage-gated with an activation threshold of -30 mV to -40 mV, a half-maximal activation at +5.3+/-1.8 mV, and time constants for activation and inactivation of 4.5+/-0.4 ms and 43.5+/-5.6 ms ( n=10), respectively. The current was inhibited by TEA, margatoxin, agitoxin-2 and stichodactyla toxin. PCR amplification of different Kv subunits from HEK293 cDNA demonstrated the expression of Kv1.1, Kv1.2, Kv1.3, Kv1.4, Kv3.1 and Kv3.4 mRNA. Quantitative RT-PCR showed up-regulation of Kv1.1, 1.2 and 1.3 mRNA by IGF-1. The effect of IGF-1 on K+ current was blocked by inhibitors of phosphatidylinositol 3-kinase (PI3-kinase), wortmannin and LY294002, and mimicked by overexpression of human 3-phosphoinositide-dependent protein kinase-1 (hPDK1) or serum- and glucocorticoid-dependent kinase-1 (hSGK1), both sequential downstream targets of PI3-kinase. IGF-1-induced proliferation of HEK293 cells was inhibited by both K+ channel blockers and inhibitors of PI3-kinase. In conclusion, IGF-1 through PI3-kinase, PDK1 and SGK1 up-regulates Kv channels, an effect required for the proliferative action of the growth factor.