Jürgen R. Schwarz

KCNQ channels mediate IKs, a slow K+ current regulating excitability in the rat node of Ranvier

Mutations that reduce the function of KCNQ2 channels cause neuronal hyperexcitability, manifested as epileptic seizures and myokymia. These channels are present in nodes of Ranvier in rat brain and nerve and have been proposed to mediate the slow nodal potassium current IKs. We have used immunocytochemistry, electrophysiology and pharmacology to test this hypothesis and to determine the contribution of KCNQ channels to nerve excitability in the rat. When myelinated nerve fibres of the sciatic nerve were examined by immunofluorescence microscopy using antibodies against KCNQ2 and KCNQ3, all nodes showed strong immunoreactivity for KCNQ2. The nodes of about half the small and intermediate sized fibres showed labelling for both KCNQ2 and KCNQ3, but nodes of large fibres were labelled by KCNQ2 antibodies only. In voltage‐clamp experiments using large myelinated fibres, the selective KCNQ channel blockers XE991 (IC50= 2.2 μm) and linopirdine (IC50= 5.5 μm) completely inhibited IKs, as did TEA (IC50= 0.22 mm). The KCNQ channel opener retigabine (10 μm) shifted the activation curve to more negative membrane potentials by −24 mV, thereby increasing IKs. In isotonic KCl 50% of IKs was activated at −62 mV. The activation curve shifted to more positive potentials as [K+]o was reduced, so that the pharmacological and biophysical properties of IKs were consistent with those of heterologously expressed homomeric KCNQ2 channels. The ability of XE991 to selectively block IKs was further exploited to study IKs function in vivo. In anaesthetized rats, the excitability of tail motor axons was indicated by the stimulus current required to elicit a 40% of maximal compound muscle action potential. XE991 (2.5 mg kg−1i.p.) eliminated all nerve excitability functions previously attributed to IKs: accommodation to 100 ms subthreshold depolarizing currents, the post‐depolarization undershoot in excitability, and the late subexcitability after a single impulse or short trains of impulses. Due to reduced spike‐frequency adaptation after XE991 treatment, 100 ms suprathreshold current injections generated long trains of action potentials. We conclude that the nodal IKs current is mediated by KCNQ channels, which in large fibres of rat sciatic nerve appear to be KCNQ2 homomers.

Ancillary Subunits Associated With Voltage-Dependent K+ Channels

Since the first discovery of Kvbeta-subunits more than 15 years ago, many more ancillary Kv channel subunits were characterized, for example, KChIPs, KCNEs, and BKbeta-subunits. The ancillary subunits are often integral parts of native Kv channels, which, therefore, are mostly multiprotein complexes composed of voltage-sensing and pore-forming Kvalpha-subunits and of ancillary or beta-subunits. Apparently, Kv channels need the ancillary subunits to fulfill their many different cell physiological roles. This is reflected by the large structural diversity observed with ancillary subunit structures. They range from proteins with transmembrane segments and extracellular domains to purely cytoplasmic proteins. Ancillary subunits modulate Kv channel gating but can also have a great impact on channel assembly, on channel trafficking to and from the cellular surface, and on targeting Kv channels to different cellular compartments. The importance of the role of accessory subunits is further emphasized by the number of mutations that are associated in both humans and animals with diseases like hypertension, epilepsy, arrhythmogenesis, periodic paralysis, and hypothyroidism. Interestingly, several ancillary subunits have in vitro enzymatic activity; for example, Kvbeta-subunits are oxidoreductases, or modulate enzymatic activity, i.e., KChIP3 modulates presenilin activity. Thus different modes of beta-subunit association and of functional impact on Kv channels can be delineated, making it difficult to extract common principles underlying Kvalpha- and beta-subunit interactions. We critically review present knowledge on the physiological role of ancillary Kv channel subunits and their effects on Kv channel properties.

Na currents and action potentials in rat myelinated nerve fibres at 20 and 37° C

Abstract(1) Action potentials and membrane currents were recorded in single myelinated rat nerve fibres at 20 and 37° C. Three experiments were also performed in single cat nerve fibres. (2) K currents were blocked by internal CsCl and external TEA. The steady state and kinetic parameters of Na activation and inactivation were determined at both temperatures. (3) When the temperature was raised from 20 to 37° C, steady state Na activation,m∞(V), and inactivation,h∞(V), did not change significantly. (4) The time constant of Na activation, τm, was determined within the potential range of −40 to 125 mV at 20° C andV=40–60 mV at 37° C. The temperature coefficient, Q10, of τm was 2.2. (5) The decay in the Na current was described by two exponentials at both temperatures. The amplitude of the slow phase was 1–10%. The time constant of the fast phase of Na inactivation, τh1, was determined at both temperatures within the potential range of −50 mV to 125 mV. The Q10 of τh1 was 2.9 and did not depend on potential. (6) The Na equilibrium potential was 152 mV at 20° C and 144 mV at 37° C. The leakage conductance was 24 nS at 20° C and 43 nS at 37° C. These differences were interpreted as signs of fibre deterioration at higher temperature. (7) The results from the current and voltage clamp experiments performed in the cat nerve were essentially the same as those in the rat nerve fibres. (8) The action potentials computed on the basis of the voltage clamp results at 20° C were similar to the ones actually measured. This was also true for those action potentials predicted for 37° C on the basis of the 20° C data, thegL andVNa values measured at 37° C, and the Q10 values of the time constants. (9) Steady state values and kinetic parameters of K permeability were adopted from the literature. As in the experiments the influence ofPK on the shape of the predicted action potential was almost negligible at both temperatures.

Phenytoin and Carbamazepine: Potential‐ and Frequency‐Dependent Block of Na Currents in Mammalian Myelinated Nerve Fibers

Summary: Voltage clamp experiments were performed in single myelinated nerve fibers of the rat and the effects of phenytoin (PHD and carbamazepine (CBZ) on the ionic membrane currents were studied. PHT and CBZ are almost selective blockers of Na channels. The main part of this inhibition is a potential‐dependent block which can be removed by hyperpolarization. The dose‐response curve of PHT was described as a first‐order reaction with Kd= 37 μM; 100 μM CBZ was equally as effective 100 μM PHT. Both drugs shift the steady‐state Na inactiva‐tion curve (h1(V)) to more negative membrane potentials and decrease its steepness. PHT and CBZ have equimolar effects on the shift and decrease in steepness of h1(V). All of the drug‐induced effects depend on drug concentration. Both drugs induce a slowing of recovery from Na inactivation. PHT has a stronger slowing effect than CBZ. From this ensues a frequency‐dependent block, which is more pronounced in the presence of PHT than of CBZ. The effects of both drugs can be interpreted in the framework of the modulated receptor hypothesis.

Activation of T Cell Calcium Influx by the Second Messenger ADP-ribose

Stimulation of Jurkat T cells by high concentrations of concanavalin A (ConA) induced an elevation of the endogenous adenosine diphosphoribose (ADPR) concentration and an inward current significantly different from the Ca2+ release-activated Ca2+ current (ICRAC). Electrophysiological characterization and activation of a similar current by infusion of ADPR indicated that the ConA-induced current is carried by TRPM2. Expression of TRPM2 in the plasma membrane of Jurkat T cells was demonstrated by reverse transcription-PCR, Western blot, and immunofluorescence. Inhibition of ADPR formation reduced ConA-mediated, but not store-operated, Ca2+ entry and prevented ConA-induced cell death of Jurkat cells. Moreover, gene silencing of TRPM2 abolished the ADPR- and ConA-mediated inward current. Thus, ADPR is a novel second messenger significantly involved in ConA-mediated cell death in T cells.

Aromatase Inhibition Abolishes LTP Generation in Female But Not in Male Mice

Inhibitors of aromatase, the final enzyme of estradiol synthesis, are suspected of inducing memory deficits in women. In previous experiments, we found hippocampal spine synapse loss in female mice that had been treated with letrozole, a potent aromatase inhibitor. In this study, we therefore focused on the effects of letrozole on long-term potentiation (LTP), which is an electrophysiological parameter of memory and is known to induce spines, and on phosphorylation of cofilin, which stabilizes the spine cytoskeleton and is required for LTP in mice. In acute slices of letrozole-treated female mice with reduced estradiol serum concentrations, impairment of LTP started as early as after 6 h of treatment and progressed further, together with dephosphorylation of cofilin in the same slices. Theta-burst stimulation failed to induce LTP after 1 week of treatment. Impairment of LTP was followed by spine and spine synapse loss. The effects were confirmed in vitro by using hippocampal slice cultures of female mice. The sequence of effects in response to letrozole were similar in ovariectomized female and male mice, with, however, differences as to the degree of downregulation. Our data strongly suggest that impairment of LTP, followed by loss of mushroom spines and spine synapses in females, may have implications for memory deficits in women treated with letrozole.

Modulation of rat erg1, erg2, erg3 and HERG K+ currents by thyrotropin-releasing hormone in anterior pituitary cells via the native signal cascade.

1 The mechanism of thyrotropin‐releasing hormone (TRH)‐induced ether‐à‐go‐go‐related gene (erg) K+ current modulation was investigated with the perforated‐patch whole‐cell technique in clonal somatomammotroph GH3/B6 cells. These cells express a small endogenous erg current known to be reduced by TRH. GH3/B6 cells were injected with cDNA coding for rat erg1, erg2, erg3 and HERG K+ channels. The corresponding erg currents were isolated with the help of the specific erg channel blockers E‐4031 and dofetilide and their biophysical properties were determined. 2 TRH (1 μm) was able to significantly reduce the different erg currents. The voltage dependence of activation was shifted by 15 mV (erg1), 10 mV (erg2) and 6 mV (erg3) to more positive potentials without strongly affecting erg inactivation. TRH reduced the maximal available erg current amplitude by 12 % (erg1), 13 % (erg2) and 39 % (erg3) and accelerated the time course of erg1 and erg2 channel deactivation, whereas erg3 deactivation kinetics were not significantly altered. The effects of TRH on HERG currents did not differ from those on its rat homologue erg1. In addition, coinjection of rat MiRP1 with HERG cDNA did not influence the TRH‐induced modulation of HERG channels. 3 Rat erg1 currents recorded in the cell‐attached configuration were reduced by application of TRH to the extra‐patch membrane in the majority of the experiments, confirming the involvement of a diffusible second messenger. 4 Application of the phorbol ester phorbol 12‐myristate 13‐acetate (PMA; 1 μm) shifted the voltage dependence of erg1 activation in the depolarizing direction, but it did not reduce the maximal current amplitude. The voltage shift could not be explained by a selective effect on protein kinase C (PKC) since the PKC inhibitor bisindolylmaleimide I did not block the effects of TRH and PMA on erg1. In addition, cholecystokinin, known to activate the phosphoinositol pathway similarly to TRH, did not significantly affect the erg1 current. 5 Various agents interfering with different known TRH‐elicited cellular responses were not able to completely mimic or inhibit the TRH effects on erg1. Tested substances included modulators of the cAMP‐protein kinase A pathway, arachidonic acid, inhibitors of tyrosine kinase and mitogen‐activated protein kinase, sodium nitroprusside and cytochalasin D. 6 The results demonstrate that all three members of the erg channel subfamily are modulated by TRH in GH3/B6 cells. In agreement with previous studies on the TRH‐induced modulation of the endogenous erg current in prolactin‐secreting anterior pituitary cells, the TRH effects on overexpressed erg1 channels are not mediated by any of the tested signalling pathways.

The erg‐like potassium current in rat lactotrophs

1 The ether‐à‐go‐go‐related gene (erg)‐like K+ current in rat lactotrophs from primary culture was characterized and compared with that in clonal rat pituitary cells (GH3/B6). The class III antiarrhythmic E‐4031 known to block specifically erg K+ channels was used to isolate the erg‐like current as the E‐4031‐sensitive current. The experiments were performed in 150 mM K+ external solution using the patch‐clamp technique. 2 The erg‐like K+ current elicited with hyperpolarizing pulses negative to ‐100 mV consisted of a fast and a pronounced slowly deactivating current component. The contribution of the slow component to the total current amplitude was potential dependent and varied from cell to cell. At ‐100 mV it ranged from 50 to 85 % and at ‐140 mV from 21 to 45 %. 3 The potential‐dependent channel availability curves determined with 2 s prepulses were fitted with the sum of two Boltzmann functions. The function related to the slowly deactivating component of the erg‐like current was shifted by more than 40 mV to more negative membrane potentials compared with that of the fast component. 4 In contrast to that of native lactotrophs studied under identical conditions, the erg‐like K+ current of GH3/B6 cells was characterized by a predominant fast deactivating current component, with similar kinetic and steady‐state properties to the fast deactivating current component of native lactotrophs. 5 Thyrotrophin‐releasing hormone reduced the erg‐like current in native lactotrophs via an intracellular signal cascade which seemed to involve a pathway independent from protein kinase A and protein kinase C. 6 RT‐PCR studies on cytoplasm from single lactotrophs revealed the presence of mRNA of the rat homologue of the human ether‐à‐go‐go‐related gene HERG (r‐erg1) as well as mRNA of the two other cloned r‐erg cDNAs (r‐erg2 and r‐erg3) in different combinations. In GH3/B6 cells, only the transcripts of r‐erg1 and r‐erg2 were found.

Functions of erg K+ channels in excitable cells

Ether‐à‐go‐go‐related gene (erg) channels are voltage‐dependent K+ channels mediating inward‐rectifying K+ currents because of their peculiar gating kinetics. These characteristics are essential for repolarization of the cardiac action potential. Inherited and acquired malfunctioning of erg channels may lead to the long QT‐syndrome. However, erg currents have also been recorded in many other excitable cells, like smooth muscle fibres of the gastrointestinal tract, neuroblastoma cells or neuroendocrine cells. In these cells erg currents contribute to the maintenance of the resting potential. Changes in the resting potential are related to cell‐specific functions like increase in hormone secretion, frequency adaptation or increase in contractility.

Potassium inactivation in single myelinated nerve fibres of Xenopus laevis.

Summary1.Voltage clamp measurements were performed on single myelinated nerve fibres of the frog Xenopus laevis.2.During long-lasting depolarizations the potassium current decayed in a fast phase with a time constant of about 0.6 sec and a following slow phase with a time constant between 3.6 (V=0) and 20 sec (V=100 mV).3.The decay of the potassium current was the result of an inactivation of the potassium permeability and not of a shift of the potassium equilibrium potential as shown by experiments in isotonic KCl solution.4.At a hyperpolarization of −20 mV the potassium inactivation was fully removed. It remained incomplete even at large depolarizations. The steady-state inactivation curve was S-shaped but not symmetrical.5.The experimental results could be described by extending the Hodgkin-Huxley equations introducing two terms of potassium inactivation.