Helmut Kubista

Evidence for structural and functional diversity among SDS-resistant SNARE complexes in neuroendocrine cells

The core complex, formed by the SNARE proteins synaptobrevin 2, syntaxin 1 and SNAP-25, is an important component of the synaptic fusion machinery and shows remarkable in vitro stability, as exemplified by its SDS-resistance. In western blots, antibodies against one of these SNARE proteins reveal the existence of not only an SDS-resistant ternary complex but also as many as five bands between 60 and >200 kDa. Structural conformation as well as possible functions of these various complexes remained elusive. In western blots of protein extracts from PC12 cell membranes, an antibody against SNAP-25 detected two heat-sensitive SDS-resistant bands with apparent molecular weights of 100 and 230 kDa. A syntaxin antibody recognized only the 230 kDa band and required heat-treatment of the blotting membrane to detect the 100 kDa band. Various antibodies against synaptobrevin failed to detect SNARE complexes in conventional western blots and detected either the 100 kDa band or the 230 kDa band on heat-treated blotting membranes. When PC12 cells were exposed to various extracellular K+-concentrations (to evoke depolarization-induced Ca2+ influx) or permeabilized in the presence of basal or elevated free Ca2+, levels of these SNARE complexes were altered differentially: moderate Ca2+ rises (≤1 μM) caused an increase, whereas Ca2+ elevations of more than 1 μM led to a decrease in the 230 kDa band. Under both conditions the 100 kDa band was either increased or remained unchanged. Our data show that various SDS-resistant complexes occur in living cells and indicate that they represent SNARE complexes with different structures and diverging functions. The distinct behavior of these complexes under release-promoting conditions indicates that these SNARE structures have different roles in exocytosis.

Attenuation of the P2Y receptor‐mediated control of neuronal Ca2+ channels in PC12 cells by antithrombotic drugs

In PC12 cells, adenine nucleotides inhibit voltage‐activated Ca2+ currents and adenylyl cyclase activity, and the latter effect was reported to involve P2Y12 receptors. To investigate whether these two effects are mediated by one P2Y receptor subtype, we used the antithrombotic agents 2‐methylthio‐AMP (2‐MeSAMP) and N6‐(2‐methyl‐thioethyl)‐2‐(3,3,3‐trifluoropropylthio)‐β,γ‐dichloromethylene‐ATP (AR‐C69931MX). ADP reduced A2A receptor‐dependent cyclic AMP synthesis with half maximal effects at 0.1–0.17 μM. In the presence of 30 μM 2‐MeSAMP or 100 nM AR‐C69931MX, concentration response curves were shifted to the right by factors of 39 and 30, indicative of pA2 values of 6.1 and 8.5, respectively. The inhibition of Ca2+ currents by ADP was attenuated by 10–1000 nM AR‐C69931MX and by 3–300 μM 2‐MeSAMP. ADP reinhibited Ca2+ currents after removal of 2‐MeSAMP within less than 15 s, but required 2 min to do so after removal of AR‐C69931MX. ADP inhibited Ca2+ currents with half maximal effects at 5–20 μM. AR‐C69931MX (10–100 nM) displaced concentration response curves to the right, and the resulting Schild plot showed a slope of 1.09 and an estimated pKB value of 8.7. Similarly, 10–100 μM 2‐MeSAMP also caused rightward shifts resulting in a Schild plot with a slope of 0.95 and an estimated pKB of 5.4. The inhibition of Ca2+ currents by 2‐methylthio‐ADP and ADPβS was also antagonized by AR‐C69931MX, which (at 30 nM) caused a rightward shift of the concentration response curve for ADPβS by a factor of 3.8, indicative of a pA2 value of 8.1. These results show that antithrombotic drugs antagonize the inhibition of neuronal Ca2+ channels by adenine nucleotides, which suggests that this effect is mediated by P2Y12 receptors.

Concomitant facilitation of GABAA receptors and KV7 channels by the non-opioid analgesic flupirtine

Flupirtine is a non‐opioid analgesic that has been in clinical use for more than 20 years. It is characterized as a selective neuronal potassium channel opener (SNEPCO). Nevertheless, its mechanisms of action remain controversial and are the purpose of this study.

CSTX-1, a toxin from the venom of the hunting spider Cupiennius salei, is a selective blocker of L-type calcium channels in mammalian neurons

The inhibitor cystine-knot motif identified in the structure of CSTX-1 from Cupiennius salei venom suggests that this toxin may act as a blocker of ion channels. Whole-cell patch-clamp experiments performed on cockroach neurons revealed that CSTX-1 produced a slow voltage-independent block of both mid/low- (M-LVA) and high-voltage-activated (HVA) insect Ca(v) channels. Since C. salei venom affects both insect as well as rodent species, we investigated whether Ca(v) channel currents of rat neurons are also inhibited by CSTX-1. CSTX-1 blocked rat neuronal L-type, but no other types of HVA Ca(v) channels, and failed to modulate LVA Ca(v) channel currents. Using neuroendocrine GH3 and GH4 cells, CSTX-1 produced a rapid voltage-independent block of L-type Ca(v) channel currents. The concentration-response curve was biphasic in GH4 neurons and the subnanomolar IC(50) values were at least 1000-fold lower than in GH3 cells. L-type Ca(v) channel currents of skeletal muscle myoballs and other voltage-gated ion currents of rat neurons, such as I(Na(v)) or I(K(v)) were not affected by CSTX-1. The high potency and selectivity of CSTX-1 for a subset of L-type channels in mammalian neurons may enable the toxin to be used as a molecular tool for the investigation of this family of Ca(v) channels.

P2Y1 receptors mediate an activation of neuronal calcium-dependent K+ channels

Molecularly defined P2Y receptor subtypes are known to regulate the functions of neurons through an inhibition of KV7 K+ and CaV2 Ca2+ channels and via an activation or inhibition of Kir3 channels. Here, we searched for additional neuronal ion channels as targets for P2Y receptors. Rat P2Y1 receptors were expressed in PC12 cells via an inducible expression system, and the effects of nucleotides on membrane currents and intracellular Ca2+ were investigated. At a membrane potential of −30 mV, ADP induced transient outward currents in a concentration‐dependent manner with half‐maximal effects at 4 μm. These currents had reversal potentials close to the K+ equilibrium potential and changed direction when extracellular Na+ was largely replaced by K+, but remained unaltered when extracellular Cl− was changed. Currents were abolished by P2Y1 antagonists and by blockade of phospholipase C. ADP also caused rises in intracellular Ca2+, and ADP‐evoked currents were abolished when inositol trisphosphate‐sensitive Ca2+ stores were depleted. Blockers of KCa2, but not those of KCa1.1 or KCa3.1, channels largely reduced ADP‐evoked currents. In hippocampal neurons, ADP also triggered outward currents at −30 mV which were attenuated by P2Y1 antagonists, depletion of Ca2+ stores, or a blocker of KCa2 channels. These results demonstrate that activation of neuronal P2Y1 receptors may gate Ca2+‐dependent K+ (KCa2) channels via phospholipase C‐dependent increases in intracellular Ca2+ and thereby define an additional class of neuronal ion channels as novel effectors for P2Y receptors. This mechanism may form the basis for the control of synaptic plasticity via P2Y1 receptors.

Enhanced currents through L-type calcium channels in cardiomyocytes disturb the electrophysiology of the dystrophic heart

Duchenne muscular dystrophy (DMD), induced by mutations in the gene encoding for the cytoskeletal protein dystrophin, is an inherited disease characterized by progressive muscle weakness. Besides the relatively well characterized skeletal muscle degenerative processes, DMD is also associated with cardiac complications. These include cardiomyopathy development and cardiac arrhythmias. The current understanding of the pathomechanisms in the heart is very limited, but recent research indicates that dysfunctional ion channels in dystrophic cardiomyocytes play a role. The aim of the present study was to characterize abnormalities in L-type calcium channel function in adult dystrophic ventricular cardiomyocytes. By using the whole cell patch-clamp technique, the properties of currents through calcium channels in ventricular cardiomyocytes isolated from the hearts of normal and dystrophic adult mice were compared. Besides the commonly used dystrophin-deficient mdx mouse model for human DMD, we also used mdx-utr mice, which are both dystrophin- and utrophin-deficient. We found that calcium channel currents were significantly increased, and channel inactivation was reduced in dystrophic cardiomyocytes. Both effects enhance the calcium influx during an action potential (AP). Whereas the AP in dystrophic mouse cardiomyocytes was nearly normal, implementation of the enhanced dystrophic calcium conductance in a computer model of a human ventricular cardiomyocyte considerably prolonged the AP. Finally, the described dystrophic calcium channel abnormalities entailed alterations in the electrocardiograms of dystrophic mice. We conclude that gain of function in cardiac L-type calcium channels may disturb the electrophysiology of the dystrophic heart and thereby cause arrhythmias.

Inhibition of transmitter release from rat sympathetic neurons via presynaptic M1 muscarinic acetylcholine receptors

Background and purpose:  M2, M3 and/or M4 muscarinic acetylcholine receptors have been reported to mediate presynaptic inhibition in sympathetic neurons. M1 receptors mediate an inhibition of Kv7, CaV1 and CaV2.2 channels. These effects cause increases and decreases in transmitter release, respectively, but presynaptic M1 receptors are generally considered facilitatory. Here, we searched for inhibitory presynaptic M1 receptors.

The anticonvulsant retigabine is a subtype selective modulator of GABAA receptors

Within its range of therapeutic plasma concentrations, the anticonvulsant retigabine (ezogabine) is believed to selectively act on Kv7 channels. Here, the contribution of specific γ‐aminobutyric acid (GABA)A receptor subtypes to the antiseizure effects of retigabine was investigated.

Dynamic interplay of excitatory and inhibitory coupling modes of neuronal L-type calcium channels

L-type voltage-gated calcium channels (LTCCs) have long been considered as crucial regulators of neuronal excitability. This role is thought to rely largely on coupling of LTCC-mediated Ca(2+) influx to Ca(2+)-dependent conductances, namely Ca(2+)-dependent K(+) (K(Ca)) channels and nonspecific cation (CAN) channels, which mediate afterhyperpolarizations (AHPs) and afterdepolarizations (ADPs), respectively. However, in which manner LTCCs, K(Ca) channels, and CAN channels co-operate remained scarcely known. In this study, we examined how activation of LTCCs affects neuronal depolarizations and analyzed the contribution of Ca(2+)-dependent potassium- and cation-conductances. With the use of hippocampal neurons in primary culture, pulsed current-injections were applied in the presence of tetrodotoxin (TTX) for stepwise depolarization and the availability of LTCCs was modulated by BAY K 8644 and isradipine. By varying pulse length and current strength, we found that weak depolarizing stimuli tend to be enhanced by LTCC activation, whereas in the course of stronger depolarizations LTCCs counteract excitation. Both effect modes appear to involve the same channels that mediate ADP and AHP, respectively. Indeed, ADPs were activated at lower stimulation levels than AHPs. In the absence of TTX, activation of LTCCs prolonged or shortened burst firing, depending on the initial burst duration, and invariably augmented brief unprovoked (such as excitatory postsynaptic potentials) and provoked electrical events. Hence, regulation of membrane excitability by LTCCs involves synchronous activity of both excitatory and inhibitory Ca(2+)-activated ion channels. The overall enhancing or dampening effect of LTCC stimulation on excitability does not only depend on the relative abundance of the respective coupling partner but also on the stimulus intensity.

Raised Activity of L-Type Calcium Channels Renders Neurons Prone to Form Paroxysmal Depolarization Shifts

Neuronal L-type voltage-gated calcium channels (LTCCs) are involved in several physiological functions, but increased activity of LTCCs has been linked to pathology. Due to the coupling of LTCC-mediated Ca2+ influx to Ca2+-dependent conductances, such as KCa or non-specific cation channels, LTCCs act as important regulators of neuronal excitability. Augmentation of after-hyperpolarizations may be one mechanism that shows how elevated LTCC activity can lead to neurological malfunctions. However, little is known about other impacts on electrical discharge activity. We used pharmacological up-regulation of LTCCs to address this issue on primary rat hippocampal neurons. Potentiation of LTCCs with Bay K8644 enhanced excitatory postsynaptic potentials to various degrees and eventually resulted in paroxysmal depolarization shifts (PDS). Under conditions of disturbed Ca2+ homeostasis, PDS were evoked frequently upon LTCC potentiation. Exposing the neurons to oxidative stress using hydrogen peroxide also induced LTCC-dependent PDS. Hence, raising LTCC activity had unidirectional effects on brief electrical signals and increased the likeliness of epileptiform events. However, long-lasting seizure-like activity induced by various pharmacological means was affected by Bay K8644 in a bimodal manner, with increases in one group of neurons and decreases in another group. In each group, isradipine exerted the opposite effect. This suggests that therapeutic reduction in LTCC activity may have little beneficial or even adverse effects on long-lasting abnormal discharge activities. However, our data identify enhanced activity of LTCCs as one precipitating cause of PDS. Because evidence is continuously accumulating that PDS represent important elements in neuropathogenesis, LTCCs may provide valuable targets for neuroprophylactic therapy.