Jürgen R. Schwarz

KCNQ1 potassium channels play a pivotal role in the physiology and pathophysiology of several human excitable and epithelial tissues. The latest cryo-electron microscopy (cryo-EM) structures provide unique insights into channel function and pharmacology, opening avenues for different therapeutic strategies against human diseases associated with KCNQ1 mutations. However, these structures also raise fundamental questions about the mechanisms of ion permeation. Cryo-EM structures thought to represent the open state of the channel feature a cavity region not wide enough for accommodation of hydrated K ⁺ . To understand how K ⁺ passes through the cavity constriction, we utilized microsecond-scale molecular dynamics (MD) simulations using the KCNQ1/KCNE3 cryo-EM structure, characterized mutants at the G345 residue situated at the narrowest point of the cavity, and recorded single channels. The findings indicate that ions become partially dehydrated at the constriction, which enables permeation. MD simulations demonstrate that the constriction can impede the flow of ions through the channel’s pore, a finding that is corroborated by mutational screening and single-channel recordings. Reduced channel conductance is the key mechanism underlying reported pathological KCNQ1 mutations at or near the constriction site.

Potassium channels stabilize the resting potential and neuronal excitability. Among them, erg (ether-à-go-go–related gene) K ⁺ channels represent a subfamily of voltage-gated channels, consisting of erg1, erg2, and erg3 subunits; however, their subunit-specific neuronal functions in vivo are barely understood. To find erg3- and erg1-mediated functions, we generated global Kcnh7 ( erg3 ) and conditional Kcnh2 ( erg1 ) knockout mice. We found that erg3 channels stabilize the resting potential and dampen spontaneous activity in cerebellar Purkinje cells (PCs) and hippocampal CA1 neurons, whereas erg1 channels have suprathreshold functions. Lack of erg3 subunits induced hyperexcitability with increased action potential firing in PCs, but not in CA1 neurons. Notably, erg3 depletion caused depressive-like behavior with reduced locomotor activity, strongly decreased digging behavior, and shorter latencies to fall off a rotating wheel, while learning and memory remained unchanged. Our data show that erg K ⁺ channels containing erg3 subunits mediate a neuronal subthreshold K ⁺ current that plays important roles in the regulation of locomotor behavior in vivo.

In myelinated nerve fibres, action potentials are generated at nodes of Ranvier. These structures are located at interruptions of the myelin sheath, forming narrow gaps with small rings of axolemma freely exposed to the extracellular space. The mammalian node contains a high density of Na⁺ channels and K⁺‐selective leakage channels. Voltage‐dependent Kv1 channels are only present in the juxta‐paranode. Recently, the leakage channels have been identified as K2P channels (TRAAK, TREK‐1). K2P channels are K⁺‐selective ‘background’ channels, characterized by outward rectification and their ability to be activated, e.g. by temperature, mechanical stretch or arachidonic acid. We are only beginning to elucidate the peculiar functions of nodal K2P channels. I will discuss two functions of the nodal K2P‐mediated conductance. First, at body temperature K2P channels have a high open probability, thereby inducing a resting potential of about −85 mV. This negative resting potential reduces steady‐state Na⁺ channel inactivation and ensures a large Na⁺ inward current upon a depolarizing stimulus. Second, the K2P conductance is involved in nodal action potential repolarization. The identification of nodal K2P channels is exciting since it shows that the nodal K⁺ conductance is not a fixed value but can be changed: it can be increased or decreased by a broad range of K2P modulators, thereby modulating, for example, the resting potential. The functional importance of nodal K2P channels will be exemplified by describing in more detail the function of the K2P conductance increase by raising the temperature from room temperature to 37°C. image

Mutations in the gene encoding the microtubule-severing protein spastin (spastic paraplegia 4 [SPG4]) cause hereditary spastic paraplegia (HSP), associated with neurodegeneration, spasticity, and motor impairment. Complicated forms (complicated HSP [cHSP]) further include cognitive deficits and dementia; however, the etiology and dysfunctional mechanisms of cHSP have remained unknown. Here, we report specific working and associative memory deficits upon spastin depletion in mice. Loss of spastin-mediated severing leads to reduced synapse numbers, accompanied by lower miniature excitatory postsynaptic current (mEPSC) frequencies. At the subcellular level, mutant neurons are characterized by longer microtubules with increased tubulin polyglutamylation levels. Notably, these conditions reduce kinesin-microtubule binding, impair the processivity of kinesin family protein (KIF) 5, and reduce the delivery of presynaptic vesicles and postsynaptic α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors. Rescue experiments confirm the specificity of these results by showing that wild-type spastin, but not the severing-deficient and disease-associated K388R mutant, normalizes the effects at the synaptic, microtubule, and transport levels. In addition, short hairpin RNA (shRNA)-mediated reduction of tubulin polyglutamylation on spastin knockout background normalizes KIF5 transport deficits and attenuates the loss of excitatory synapses. Our data provide a mechanism that connects spastin dysfunction with the regulation of kinesin-mediated cargo transport, synapse integrity, and cognition.

TRAAK is a membrane tension-activated K⁺ channel that has been associated through behavioral studies to mechanical nociception. We used specific monoclonal antibodies in mice to show that TRAAK is localized exclusively to nodes of Ranvier, the action potential propagating elements of myelinated nerve fibers. Approximately 80 percent of myelinated nerve fibers throughout the central and peripheral nervous system contain TRAAK in what is likely an all-nodes or no-nodes per axon fashion. TRAAK is not observed at the axon initial segment where action potentials are first generated. We used polyclonal antibodies, the TRAAK inhibitor RU2 and node clamp amplifiers to demonstrate the presence and functional properties of TRAAK in rat nerve fibers. TRAAK contributes to the ‘leak’ K⁺ current in mammalian nerve fiber conduction by hyperpolarizing the resting membrane potential, thereby increasing Na⁺ channel availability for action potential propagation. We speculate on why nodes of Ranvier contain a mechanosensitive K⁺ channel.

Mammalian ether‐à‐go‐go (EAG) channels are voltage‐gated K⁺ channels. They are encoded by the KCNH gene family and divided into three subfamilies, eag (Kv10), erg (eag‐related gene; Kv11) and elk (eag‐like; Kv12). All EAG channel subtypes are expressed in the brain where they effectively modulate neuronal excitability. This Topical Review describes the biophysical properties of each of the EAG channel subtypes, their function in neurons and the neurological diseases induced by EAG channel mutations. In contrast to the function of erg currents in the heart, where they contribute to repolarization of the cardiac action potential, erg currents in neurons are involved in the maintenance of the resting potential, setting of action potential threshold and frequency accommodation. They can even support high frequency firing by preventing a depolarization‐induced Na⁺ channel block. EAG channels are modulated differentially, e.g. eag channels by intracellular Ca²⁺, erg channels by extracellular K⁺ and GPCRs, and elk channels by changes in pH. So far, only currents mediated by erg channels have been recorded in neurons with the help of selective blockers. Neuronal eag and elk currents have not been isolated due to the lack of suitable channel blockers. However, findings in KO mice indicate a physiological role of eag1 currents in synaptic transmission and an involvement of elk2 currents in cognitive performance. Human eag1 and eag2 gain‐of‐function mutations underlie syndromes associated with epileptic seizures. image

We investigated the subthreshold properties of an erg (ether-à-go-go-related gene) K⁺current in Purkinje cells of neonatal mice. Action potentials recorded from Purkinje cells in cerebellar slices exhibited a decreased threshold potential and increased frequency of spontaneous and repetitive activity following application of the specific erg channel blocker E-4031. Accommodation was absent before and after drug application. The erg current of these Purkinje cells activated at membrane potentials near −60 mV and exhibited fast gating kinetics. The functional importance of fast gating subthreshold erg channels in Purkinje cells was corroborated by comparing the results of action potential clamp experiments with erg1a, erg1b, erg2, and erg3 currents heterologously expressed in HEK cells. Computer simulations based on a NEURON model of Purkinje cells only reproduced the effects of the native erg current when an erg channel conductance like that of erg3 was included. Experiments with subunit-sensitive toxins (BeKm-1, APETx1) indicated that erg channels in Purkinje cells are presumably mediated by heteromeric erg1/erg3 or modified erg1 channels. Following mGluR1 activation, the native erg current was reduced by ∼70%, brought about by reduction of the maximal erg current and a shift of the activation curve to more positive potentials. The Purkinje cell erg current contributed to the sustained current component of the biphasic mGluR1 response. Activation of mGluR1 by the agonist 3,4-dihydroxyphenylglycol increased Purkinje cell excitability, similar to that induced by E-4031. The results indicated that erg currents can be modulated and may contribute to the mGluR1-induced plasticity changes in Purkinje cells.

HERG (human ether-à-go-go-related gene) K⁺ currents fulfill important ionic functions in cardiac and other excitable cells. In addition, HERG channels influence cell growth and migration in various types of tumor cells. The mechanisms underlying these functions are still not resolved. Here, we investigated the role of HERG channels for cell growth in a cell line (SW2) derived from small cell lung cancer (SCLC), a malignant variant of lung cancer. The two HERG1 isoforms (HERG1a, HERG1b) as well as HERG2 and HERG3 are expressed in SW2 cells. Inhibition of HERG currents by acute or sustained application of E-4031, a specific ERG channel blocker, depolarized SW2 cells by 10–15 mV. This result indicated that HERG K⁺ conductance contributes considerably to the maintenance of the resting potential of about −45 mV. Blockage of HERG channels by E-4031 for up to 72 h did not affect cell proliferation. In contrast, siRNA-induced inhibition of HERG1 protein expression decreased cell proliferation by about 50%. Reduction of HERG1 protein expression was confirmed by Western blots. HERG current was almost absent in SW2 cells transfected with siRNA against HERG1. Qualitatively similar results were obtained in three other SCLC cell lines (OH1, OH3, H82), suggesting that the HERG1 channel protein is involved in SCLC cell growth, whereas the ion-conducting function of HERG1 seems not to be important for cell growth.

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 I_Ks. 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 I_Ks, 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 I_Ks. In isotonic KCl 50% of I_Ks 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 I_Ks were consistent with those of heterologously expressed homomeric KCNQ2 channels. The ability of XE991 to selectively block I_Ks was further exploited to study I_Ks 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 I_Ks: 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 I_Ks current is mediated by KCNQ channels, which in large fibres of rat sciatic nerve appear to be KCNQ2 homomers.