Prolonged Depolarization Research Articles

Unique Type of Sodium Channel Inhibition by Riluzole

Riluzole is a persistent-selective sodium channel inhibitor (SCI), which has a therapeutic potential to treat several neurological and psychiatric disorders. SCIs form a large and diverse group, which includes widely different modes of action. Individual SCIs may be very potent in one protocol, but ineffective in another. We have developed a method for testing the “personality” of SCIs, i.e., we assess their potency under different voltage protocols. The pattern of their relative potencies gives a characteristic fingerprint which shows correlation with specific chemical properties of molecules, and may also predict their therapeutic profile. We have identified distinct modes of action for specific groups of SCIs. Riluzole was found to have a unique “personality” type; therefore, in this study we performed a detailed analysis of its mode of action. The classic SCIs lidocaine and carbamazepine as well as the persistent-selective ranolazine were used as reference compounds. We observed a paradoxical inverse use-dependence and an apparent transient facilitation on sodium channels in the presence of riluzole (but not of other drugs) when currents were evoked by short depolarizations: in such protocols, riluzole appeared to be ineffective. On the other hand, in protocols with prolonged moderate depolarizations the drug was remarkably potent; suggesting that it strongly enhanced closed state inactivation. Recovery from fast inactivation was significantly impeded by riluzole, while recovery from slow inactivated state was - remarkably - even slightly accelerated. As a possible mechanism we propose that riluzole has an exceptionally fast binding kinetics, a high affinity for pre-open closed- and fast inactivated states, while a low affinity for slow inactivated state.

Molecular Determinants of Slow Inactivation in Voltage-Gated Potassium Channels

Potassium channels respond to prolonged depolarizations with structural rearrangements that result in inactivated, nonconducting channels through a process termed slow inactivation. Significant efforts to understand this process have been made using structural, theoretical and experimental approaches yet the molecular details of slow inactivation in eukaryotic voltage-gated potassium (Kv) channels remain poorly understood. Data gleaned from prokaryotic (KcsA) and eukaryotic (Kv1.2/2.1) channels have implicated two adjacent and highly conserved aromatic side chains near the selectivity filter as critical determinants for slow inactivation. In particular, the indole nitrogens of both side chains, Trp434 and Trp435 in Shaker potassium channels, have been proposed to contribute to a hydrogen bond network that modulates slow inactivation, yet the direct demonstration of this notion is missing in Kv channels. Here we incorporate unnatural derivatives of Trp to directly demonstrate that the indole nitrogen of Trp434 is a crucial component of the slow inactivation process through two complementary substitutions. By subtly increasing the acidity of the indole nitrogen through fluorination (and therefore strengthening its hydrogen bond donor ability), the rate of slow inactivation was substantially decreased (4-fold slower than WT). Conversely, the novel unnatural amino acid side chain Ind, which lacks the indole nitrogen but is otherwise isosteric to Trp, increased the rate of inactivation more than 10-fold when Ind-containing channels were co-expressed with WT channels, as Ind-containing channels alone did not produce ionic current. In contrast to Trp434, the indole nitrogen of Trp435 does not contribute to slow inactivation as even relatively nonconservative mutations to Phe or Tyr at this site do not affect slow inactivation. Taken together, these results directly demonstrate the functional importance of the H-bonding ability of Trp434 in open pore stability in Kv channels.

The role of glutamatergic inputs onto parvalbumin-positive interneurons: relevance for schizophrenia

Cognitive impairment, a core feature of schizophrenia, has been suggested to arise from a disturbance of gamma oscillations that is due to decreased neurotransmission from the parvalbumin (PV) subtype of interneurons. Indeed, PV interneurons have uniquely fast membrane and synaptic properties that are crucially important for network functions such as feedforward inhibition or gamma oscillations. The causes leading to impairment of PV neurotransmission in schizophrenia are still under investigation. Interestingly, NMDA receptors (NMDARs) antagonism results in schizophrenia-like symptoms in healthy adults. Additionally, systemic NMDAR antagonist administration increases prefrontal cortex pyramidal cell firing, apparently by producing disinhibition, and repeated exposure to NMDA antagonists leads to changes in the GABAergic markers that mimic the impairments found in schizophrenia. Based on these findings, PV neuron deficits in schizophrenia have been proposed to be secondary to (NMDAR) hypofunction at glutamatergic synapses onto these cells. However, NMDARs generate long-lasting postsynaptic currents that result in prolonged depolarization of the postsynaptic cells, a property inconsistent with the role of PV cells in network dynamics. Here, we review evidence leading to the conclusion that cortical disinhibition and GABAergic impairment produced by NMDAR antagonists are unlikely to be mediated via NMDARs at glutamatergic synapses onto mature cortical PV neurons.

Alternative Splicing Regulates Kv3.1 Polarized Targeting to Adjust Maximal Spiking Frequency

Synaptic inputs received at dendrites are converted into digital outputs encoded by action potentials generated at the axon initial segment in most neurons. Here, we report that alternative splicing regulates polarized targeting of Kv3.1 voltage-gated potassium (Kv) channels to adjust the input-output relationship. The spiking frequency of cultured hippocampal neurons correlated with the level of endogenous Kv3 channels. Expression of axonal Kv3.1b, the longer form of Kv3.1 splice variants, effectively converted slow-spiking young neurons to fast-spiking ones; this was not the case for Kv1.2 or Kv4.2 channel constructs. Despite having identical biophysical properties as Kv3.1b, dendritic Kv3.1a was significantly less effective at increasing the maximal firing frequency. This suggests a possible role of channel targeting in regulating spiking frequency. Mutagenesis studies suggest the electrostatic repulsion between the Kv3.1b N/C termini, created by its C-terminal splice domain, unmasks the Kv3.1b axonal targeting motif. Kv3.1b axonal targeting increased the maximal spiking frequency in response to prolonged depolarization. This finding was further supported by the results of local application of channel blockers and computer simulations. Taken together, our studies have demonstrated that alternative splicing controls neuronal firing rates by regulating the polarized targeting of Kv3.1 channels.

The afterhyperpolarizing potential following a train of action potentials is suppressed in an acute epilepsy model in the rat Cornu Ammonis 1 area

In hippocampal Cornu Ammonis 1 (CA1) neurons, a prolonged depolarization evokes a train of action potentials followed by a prominent afterhyperpolarizing potential (AHP), which critically dampens neuronal excitability. Because it is not known whether epileptiform activity alters the AHP and whether any alteration of the AHP is independent of inhibition, we acutely induced epileptiform activity by bath application of the GABA A receptor blocker gabazine (5 μM) in the rat hippocampal slice preparation and studied its impact on the AHP using intracellular recordings. Following 10 min of gabazine wash-in, slices started to develop spontaneous epileptiform discharges. This disinhibition was accompanied by a significant shift of the resting membrane potential of CA1 neurons to more depolarized values. Prolonged depolarizations (600 ms) elicited a train of action potentials, the number of which was not different between baseline and gabazine treatment. However, the AHP following the train of action potentials was significantly reduced after 20 min of gabazine treatment. When the induction of epileptiform activity was prevented by co-application of 6-cyano-7-nitroquinoxaline-2,3-dione disodium (CNQX, 10 μM) and d-(−)-2-amino-5-phosphonopentanoic acid ( d-AP5, 50 μM) to block α-amino-3-hydroxy-5-methylisoxazolepropionate (AMPA) and N-methyl- d-aspartate (NMDA) receptors, respectively, the AHP was preserved despite of GABA A receptor inhibition suggesting that the epileptiform activity was required to suppress the AHP. Moreover, the AHP was also preserved when the slices were treated with the protein kinase blockers H-9 (100 μM) and H-89 (1 μM). These results demonstrate that the AHP following a train of action potentials is rapidly suppressed by acutely induced epileptiform activity due to a phosphorylation process—presumably involving protein kinase A.

Prolonged Membrane Depolarization Enhances Midbrain Dopamine Neuron Differentiation via Epigenetic Histone Modifications

Understanding midbrain dopamine (DA) neuron differentiation is of importance, because of physiological and clinical implications of this neuronal subtype. We show that prolonged membrane depolarization induced by KCl treatment promotes DA neuron differentiation from neural precursor cells (NPCs) derived from embryonic ventral midbrain (VM). Interestingly, the depolarization-induced increase of DA neuron yields was not abolished by L-type calcium channel blockers, along with no depolarization-mediated change of intracellular calcium level in the VM-derived NPCs (VM-NPCs), suggesting that the depolarization effect is due to a calcium-independent mechanism. Experiments with labeled DA neuron progenitors indicate that membrane depolarization acts at the differentiation fate determination stage and promotes the expression of DA phenotype genes (tyrosine hydroxylase [TH] and DA transporter [DAT]). Recruitment of Nurr1, a transcription factor crucial for midbrain DA neuron development, to the promoter of TH gene was enhanced by depolarization, along with increases of histone 3 acetylation (H3Ac) and trimethylation of histone3 on lysine 4 (H3K4m3), and decreases of H3K9m3 and H3K27m3 in the consensus Nurr1 binding regions of TH promoter. Depolarization stimuli on differentiating VM-NPCs also induced dissociation of methyl CpG binding protein 2 and related repressor complex molecules (repressor element-1 silencing transcription factor corepressor and histone deacetylase 1) from the CpG sites of TH and DAT promoters. Based on these findings, we suggest that membrane depolarization promotes DA neuron differentiation by opening chromatin structures surrounding DA phenotype genes and inhibiting the binding of corepressors, thus allowing transcriptional activators such as Nurr1 to access DA neuron differentiation gene promoter regions.

Atrial electromechanical function

longitudinal with the mitral annulus moving back towards the rear of the atrium after the P-wave and during the PR interval and returning to its resting position at the time of the first heart sound, marking end-diastole. It is worth mentioning that not only does this pattern of function mirror that of the left ventricle, although in the opposite direction, but the atrium also does not function in isolation of the ventricle. Myocardial development studies have shown close relationship between age-related changes in the function of the atrium and the ventricle which themselves are closely related. As left ventricular lengthening velocities reduce with age, those of left atrial shortening reciprocally increase in order to secure normal stroke volume entering the left ventricle. 3 Conventional measurements of the left atrial size rely on longitudinal and transverse diameters as well as its area measurements. Segmental left atrial myocardial function is easily assessed from the longitudinal motion amplitude and velocity of the lateral, septal, and posterior segments as shown by the mitral annulus excursion during atrial systole. This is easily achieved using tissue Doppler or speckle tracking techniques. The amplitude of left atrial longitudinal motion is also assessed by the same techniques or by conventional M-mode. Similar to the left ventricle, the main determinant of left atrial contractile function is its intrinsic myocardial function, which has its well-established maturation course, between foetal life and adolescence. 4 As the atrium gets large in size due to various pathologies, with the commonest secondary to left ventricular and valve diseases, its overall contractile function falls and eventually pump failure and atrial fibrillation. Even in patients with paroxysmal atrial fibrillation and only small increase in atrial size we have shown significant reduction in segmental left atrial function particularly in areas adjacent to the pulmonary veins. 5 Critical investigation of atrial function shows that such pathophysiological course is not that simple. We and others have previously shown that atrial flutter and fibrillation are commonly preceded by progressive prolongation of P-wave duration which itself proved a good predictor for the occurrence of atrial arrhythmias. 6,7 This is explained on the basis of progressive atrial enlargement and myocardial stretching, which increase its surface area and hence the prolonged depolarization time. Such behaviour is similar to that occurring in dilated left ventricles with prolonged depolarization time and broad QRS duration. Another important component of left atrial function is its electromechanical delay, similar to that seen in the left ventricle. This can easily be studied by the same Doppler echocardiographic techniques mentioned above and left atrial motion measured with reference to the onset of its electrical depolarization, the P-wave. Studies have used the onset of segmental left atrial shortening (excursion) or peak shortening velocity as two possible landmarks for electromechanical delay. Normal values for the two measurements have been determined and well documented. Normally, the three left atrial segments shorten in a synchronous fashion with no more than few milliseconds time difference between them, again a finding similar to that seen in the left

Plasma membrane insertion of TRPC5 channels contributes to the cholinergic plateau potential in hippocampal CA1 pyramidal neurons

In cultured hippocampal neurons, transient receptor potential 5 (TRPC5) channels are translocated and inserted into plasma membranes of hippocampal neurons to generate nonselective cation (NSC) currents. We investigated whether TRPC5 channel translocation also contributes to the generation of NSC currents underlying the afterdepolarizations and plateau potentials (PPs) in hippocampal pyramidal cells that are induced by muscarinic receptor activation. Using a biotinylation assay to quantify the change in surface membrane proteins in acute hippocampal slices, we found that muscarinic stimulation significantly enhanced the levels of TRPC5 protein on the membrane surface but not those of TRPC1 or TRPC4 channels. We then investigated the pharmacological sensitivity of the cation current observed during muscarinic stimulation to determine if a component could be due to TRPC5 channels. The TRPC channel antagonists 2-APB and SKF96365 strongly depressed the generation of PPs, the underlying tail currents (I(tail)) and the associated dendritic Ca(2+) influx induced by muscarinic receptor activation in pyramidal neurons. High intracellular concentrations of ATP, which specifically inhibit TRPC5 channels, depressed I(tail). In addition, pretreatment with the calmodulin (CaM) inhibitor W-7, which depresses recombinant TRPC5 currents, inhibited both the cation current (I(tail)) and the surface insertion of TRPC5 channels. Finally, the phosphatidylinositide 3-kinase (PI(3)K) inhibitor wortmannin, which blocks translocation of TRPC5 channels in cell culture, also inhibited both the I(tail) and the surface insertion of TRPC5 channels. Therefore, we conclude that insertion of TRPC5 channels contributes to the generation of the prolonged afterdepolarizations following muscarinic stimulation. This altered plasma membrane expression of TRPC5 channels in pyramidal neurons may play an important role in the generation of prolonged neuronal depolarization and bursting during the epileptiform seizure discharges of epilepsy.

Chronically Epileptic Human and Rat Neocortex Display a Similar Resistance Against Spreading Depolarization In Vitro

Experimental and clinical evidence suggests that prolonged spreading depolarizations (SDs) are a promising target for therapeutic intervention in stroke because they recruit tissue at risk into necrosis by protracted intracellular calcium surge and massive glutamate release. Unfortunately, unlike SDs in healthy tissue, they are resistant to drugs such as N-methyl-d-aspartate-receptor antagonists. This drug resistance of SD in low perfusion areas may be due to the gradual rise of extracellular potassium before SD onset. Brain slices from patients undergoing surgery for intractable epilepsy allow for screening of drugs, targeting pharmacoresistant SDs under elevated potassium in human tissue. However, network changes associated with epilepsy may interfere with tissue susceptibility to SD. This could distort the results of pharmacological tests. We investigated the threshold for SD, induced by a gradual rise of potassium, in neocortex slices of patients with intractable epilepsy and of chronically epileptic rats as well as age-matched and younger control rats using combined extracellular potassium/field recordings and intrinsic optical imaging. Both age and epilepsy significantly increased the potassium threshold, which was similarly high in epileptic rat and human slices (23.6±2.4 mmol/L versus 22.3±2.8 mmol/L). Our results suggest that chronic epilepsy confers resistance against SD. This should be considered when human tissue is used for screening of neuroprotective drugs. The finding of similar potassium thresholds for SD in epileptic human and rat neocortex challenges previous speculations that the resistance of the human brain against SD is markedly higher than that of the rodent brain.

“Comorbidity” between epilepsy and headache/migraine: the other side of the same coin!

Dear Editor, We read with interest the paper by Toldo and co-workers addressing the important issue of comorbidity between epilepsy and headache/migraine [1]. In fact, although these are both chronic disorders with episodic attacks and their association has been long recognized, the common molecular mechanisms remain so far elusive [2–4]. Indeed, recent data suggest shared genetic substrates and phenotypic-genotypic correlations with mutations in some ion transporters genes, including CACNA1A, ATP1A2, SCN1A [3, 4]. In their latest study, Toldo et al. evaluated the distribution of five polymorphisms of SCN1A, the gene encoding the a-subunit of the neuronal voltage-gated sodium channel Nav1.1, in children and adolescents with headache and epilepsy compared to controls. They concluded that SCN1A is not involved in the pathogenesis of comorbidity between headache/migraine and epilepsy [1]. Despite this negative study, neuronal hyperexcitability and increased susceptibility to cortical spreading depression remain important molecular mechanisms in the pathophysiology of this association. In the coming decade it is possible that International efforts to collect large well-phenotyped samples and the current technical possibilities of massive genotyping will shed light on the genetic mechanisms involved in with headache/migraine and epilepsy. On the other hand, the role of additional, non genetic factors influencing the excitation threshold, e.g., mitochondrial dysfunction, disturbance in neurotransmitters metabolism, or inflammatory factors-alone or in combination-can not be excluded a priori [3, 4]. Indeed, any of these triggering factors, irrespective of their nature (genetically determined or not), could potentially lead to a paroxysmal and transient cortical excitability change leading to prolonged neuronal depolarization (seizure) or spreading depression (headache/migraine) [2, 3]. Among the potential practical implications arising from these observations, there is the urgent need for a revision of either International Classifications of Epilepsy and Headache disorders [2, 3, 5]. Finally, insight into the molecular mechanisms involved in the association between headache/migraine and epilepsy is crucial to identify drug targets for improving patients’ treatment.

Swelling-Activated Potassium Channel in Porcine Pigmented Ciliary Epithelial Cells

Ion channels in the ciliary epithelium play critical roles in the formation of aqueous humor in the eye. The present study identified a novel, swelling-activated K(+) current in freshly dissociated porcine pigmented ciliary epithelial cells. Ciliary epithelial cells were freshly dissociated from porcine eyes. Whole-cell currents were recorded by the patch-clamp technique in pigmented and nonpigmented ciliary epithelial cell (PCE-NPCE) pairs or single PCE cells. The 0-current potential was -49 ± 13 mV in PCE-NPCE cell pairs (n = 97) and -52 ± 12 mV in single PCE cells (n = 30). Whole-cell currents in these cells were dominated by an outwardly rectifying K(+) current activated by potentials more positive than -90 mV, which never inactivated during prolonged depolarization. The K(+) current was significantly augmented by hypotonic cell perfusion. External Ba(2+) was a blocker of this K(+) conductance (IC(50) of 0.38 mM), but the conductance was insensitive to external TEA(+). Linopirdine, a specific inhibitor of KCNQ channels, effectively blocked the K(+) current in these PCE cells. Porcine PCE cells express a swelling-activated K(+) channel, which may be a member of the KCNQ/Kv7 channel family. This K(+) channel is active near resting potentials and could contribute to the regulation of cell volume and water transport via the ciliary epithelia.

Parkin Interacts with Ambra1 to Induce Mitophagy

Mutations in the gene encoding Parkin are a major cause of recessive Parkinson's disease. Recent work has shown that Parkin translocates from the cytosol to depolarized mitochondria and induces their autophagic removal (mitophagy). However, the molecular mechanisms underlying Parkin-mediated mitophagy are poorly understood. Here, we investigated whether Parkin interacts with autophagy-regulating proteins. We purified Parkin and associated proteins from HEK293 cells using tandem affinity purification and identified the Parkin interactors using mass spectrometry. We identified the autophagy-promoting protein Ambra1 (activating molecule in Beclin1-regulated autophagy) as a Parkin interactor. Ambra1 activates autophagy in the CNS by stimulating the activity of the class III phosphatidylinositol 3-kinase (PI3K) complex that is essential for the formation of new phagophores. We found Ambra1, like Parkin, to be widely expressed in adult mouse brain, including midbrain dopaminergic neurons. Endogenous Parkin and Ambra1 coimmunoprecipitated from HEK293 cells, SH-SY5Y cells, and adult mouse brain. We found no evidence for ubiquitination of Ambra1 by Parkin. The interaction of endogenous Parkin and Ambra1 strongly increased during prolonged mitochondrial depolarization. Ambra1 was not required for Parkin translocation to depolarized mitochondria but was critically important for subsequent mitochondrial clearance. In particular, Ambra1 was recruited to perinuclear clusters of depolarized mitochondria and activated class III PI3K in their immediate vicinity. These data identify interaction of Parkin with Ambra1 as a key mechanism for induction of the final clearance step of Parkin-mediated mitophagy.

Differential Control of Presynaptic CaMKII Activation and Translocation to Active Zones

The release of neurotransmitters, neurotrophins, and neuropeptides is modulated by Ca(2+) mobilization from the endoplasmic reticulum (ER) and activation of Ca(2+)/calmodulin-dependent protein kinase II (CaMKII). Furthermore, when neuronal cultures are subjected to prolonged depolarization, presynaptic CaMKII redistributes from the cytoplasm to accumulate near active zones (AZs), a process that is reminiscent of CaMKII translocation to the postsynaptic side of the synapse. However, it is not known how presynaptic CaMKII activation and translocation depend on neuronal activity and ER Ca(2+) release. Here these issues are addressed in Drosophila motoneuron terminals by imaging a fluorescent reporter of CaMKII activity and subcellular distribution. We report that neuronal excitation acts with ER Ca(2+) stores to induce CaMKII activation and translocation to a subset of AZs. Surprisingly, activation is slow, reflecting T286 autophosphorylation and the function of presynaptic ER ryanodine receptors (RyRs) and inositol trisphosphate receptors (IP3Rs). Furthermore, translocation is not simply proportional to CaMKII activity, as T286 autophosphorylation promotes activation, but does not affect translocation. In contrast, RNA interference-induced knockdown of the AZ scaffold protein Bruchpilot disrupts CaMKII translocation without affecting activation. Finally, RyRs comparably stimulate both activation and translocation, but IP3Rs preferentially promote translocation. Thus, Ca(2+) provided by different presynaptic ER Ca(2+) release channels is not equivalent. These results suggest that presynaptic CaMKII activation depends on autophosphorylation and global Ca(2+) in the terminal, while translocation to AZs requires Ca(2+) microdomains generated by IP3Rs.

Mode shift of the voltage sensors in Shaker K+ channels is caused by energetic coupling to the pore domain

The voltage sensors of voltage-gated ion channels undergo a conformational change upon depolarization of the membrane that leads to pore opening. This conformational change can be measured as gating currents and is thought to be transferred to the pore domain via an annealing of the covalent link between voltage sensor and pore (S4-S5 linker) and the C terminus of the pore domain (S6). Upon prolonged depolarizations, the voltage dependence of the charge movement shifts to more hyperpolarized potentials. This mode shift had been linked to C-type inactivation but has recently been suggested to be caused by a relaxation of the voltage sensor itself. In this study, we identified two ShakerIR mutations in the S4-S5 linker (I384N) and S6 (F484G) that, when mutated, completely uncouple voltage sensor movement from pore opening. Using these mutants, we show that the pore transfers energy onto the voltage sensor and that uncoupling the pore from the voltage sensor leads the voltage sensors to be activated at more negative potentials. This uncoupling also eliminates the mode shift occurring during prolonged depolarizations, indicating that the pore influences entry into the mode shift. Using voltage-clamp fluorometry, we identified that the slow conformational change of the S4 previously correlated with the mode shift disappears when uncoupling the pore. The effects can be explained by a mechanical load that is imposed upon the voltage sensors by the pore domain and allosterically modulates its conformation. Mode shift is caused by the stabilization of the open state but leads to a conformational change in the voltage sensor.

Spreading depolarizations have prolonged direct current shifts and are associated with poor outcome in brain trauma

Cortical spreading depolarizations occur spontaneously after ischaemic, haemorrhagic and traumatic brain injury. Their effects vary spatially and temporally as graded phenomena, from infarction to complete recovery, and are reflected in the duration of depolarization measured by the negative direct current shift of electrocorticographic recordings. In the focal ischaemic penumbra, peri-infarct depolarizations have prolonged direct current shifts and cause progressive recruitment of the penumbra into the core infarct. In traumatic brain injury, the effects of spreading depolarizations are unknown, although prolonged events have not been observed in animal models. To determine whether detrimental penumbral-type depolarizations occur in human brain trauma, we analysed electrocorticographic recordings obtained by subdural electrode-strip monitoring during intensive care. Of 53 patients studied, 10 exhibited spreading depolarizations in an electrophysiologic penumbra (i.e. isoelectric cortex with no spontaneous activity). All 10 patients (100%) with isoelectric spreading depolarizations had poor outcomes, defined as death, vegetative state, or severe disability at 6 months. In contrast, poor outcomes were observed in 60% of patients (12/20) who had spreading depolarizations with depression of spontaneous activity and only 26% of patients (6/23) who had no depolarizations (χ2, P<0.001). Spontaneous electrocorticographic activity and direct current shifts of depolarizations were further examined in nine patients. Direct current shift durations (n=295) were distributed with a significant positive skew (range 0:51-16:19 min:s), evidencing a normally distributed group of short events and a sub-group of prolonged events. Prolonged direct current shifts were more commonly associated with isoelectric depolarizations (median 2 min 36 s), whereas shorter depolarizations occurred with depression of spontaneous activity (median 2 min 10 s; P<0.001). In the latter group, direct current shift durations correlated with electrocorticographic depression periods, and were longer when preceded by periodic epileptiform discharges than by continuous delta (0.5-4.0 Hz) or higher frequency activity. Prolonged direct current shifts (>3 min) also occurred mainly within temporal clusters of events. Our results show for the first time that spreading depolarizations are associated with worse clinical outcome after traumatic brain injury. Furthermore, based on animal models of brain injury, the prolonged durations of depolarizations raise the possibility that these events may contribute to maturation of cortical lesions. Prolonged depolarizations, measured by negative direct current shifts, were associated with (i) isoelectricity or periodic epileptiform discharges; (ii) prolonged depression of spontaneous activity and (iii) occurrence in temporal clusters. Depolarizations with these characteristics are likely to reflect a worse prognosis.

TRPA1 and Sympathetic Activation Contribute to Increased Risk of Triggered Cardiac Arrhythmias in Hypertensive Rats Exposed to Diesel Exhaust

Background: Diesel exhaust (DE), which is emitted from on- and off-road sources, is a complex mixture of toxic gaseous and particulate components that leads to triggered adverse cardiovascular effects such as arrhythmias.Objective: We hypothesized that increased risk of triggered arrhythmias 1 day after DE exposure is mediated by airway sensory nerves bearing transient receptor potential (TRP) channels [e.g., transient receptor potential cation channel, member A1 (TRPA1)] that, when activated by noxious chemicals, can cause a centrally mediated autonomic imbalance and heightened risk of arrhythmia.Methods: Spontaneously hypertensive rats implanted with radiotelemeters were whole-body exposed to either 500 μg/m3 (high) or 150 μg/m3 (low) whole DE (wDE) or filtered DE (fDE), or to filtered air (controls), for 4 hr. Arrhythmogenesis was assessed 24 hr later by continuous intravenous infusion of aconitine, an arrhythmogenic drug, while heart rate (HR) and electrocardiogram (ECG) were monitored.Results: Rats exposed to wDE or fDE had slightly higher HRs and increased low-frequency:high-frequency ratios (sympathetic modulation) than did controls; ECG showed prolonged ventricular depolarization and shortened repolarization periods. Rats exposed to wDE developed arrhythmia at lower doses of aconitine than did controls; the dose was even lower in rats exposed to fDE. Pretreatment of low wDE–exposed rats with a TRPA1 antagonist or sympathetic blockade prevented the heightened sensitivity to arrhythmia.Conclusions: These findings suggest that a single exposure to DE increases the sensitivity of the heart to triggered arrhythmias. The gaseous components appear to play an important role in the proarrhythmic response, which may be mediated by activation of TRPA1, and subsequent sympathetic modulation. As such, toxic inhalants may partly exhibit their toxicity by lowering the threshold for secondary triggers, complicating assessment of their risk.

Gate Closure Strictly Follows Voltage-Sensor Movements in K V Channels

Kv channels are voltage-dependent potassium pores that shape the action potential duration and are critical for cell excitability. Detection of membrane potential (V) is done by a charged (Q) voltage sensor domain (VSD) whose reorientations generate a transient gating current (IQ). Prolonged depolarization of Shaker Kv channels pushes the VSD into the relaxed state, characterized by a slowing in IQOff. Kv channels also have two gates (in series) that seal off K+ permeation: the S6 bundle crossing (BC), directly tied to the VSD, and the selectivity filter (SF). Direct comparison of K+-conduction in Shaker, reflecting the status of the BC gate, with IQ shows a strong correlation between both. As IQOff slowed down with prolonged depolarizations, BC gate closure displayed a similar 2-fold slowing when the duration of a +20mV pre-pulse was increased from 0.2 to 10 seconds. Simultaneous monitoring of the VSD movement (fluorescence recordings) and channel gate closure (ionic recordings) in the TMRM-labeled Shaker mutant M356C showed that the slowing in IQOff and gate closure occurs simultaneously. This indicates that the gate is strictly controlled by the movements of the VSD and most importantly that the BC gate remains open even when the VSD relaxes. Consequently, K+ conduction continues as long as the SF gate does not close (inactivation). Interestingly, in Kv3.1 - a channel that regulates high frequency firing in-vivo - the opposite behavior was observed: prolonged depolarization speeded up both IQOff and gate closure. Thus, the effect of VSD relaxation differs between different subtypes of Kv channels suggesting that relaxation affects the excitability of cells differently depending on their depolarization history, either reducing excitability in cells expressing Shaker or increasing it in case of Kv3.1. (Support: NIH-GM030376, FWO-G025608)

Gate Closure in Kv1.5 Channels is not Dependent on the Status of the Selectivity-Filter

Voltage-gated potassium (Kv) channels are tetramers of 6-transmembrane domain (S1-S6) α-subunits. The activation gate that seals off the ion conducting pore is under control of the voltage-sensing domain and locates at the bundle crossing of the S6 segments. After channel opening most Kv channels display slow inactivation, a process that involves rearrangements of the selectivity filter (SF) resulting in a non-conducting channel although the S6-gate is open. Recent evidence argued for a strong coupling between inactivation and activation: after gate opening the S6 segment undergoes structural rearrangements that would be transmitted up to the level of the SF destabilizing its conformation. Substituting in Kv1.5 residue T480 that locates at the bottom of the SF by an alanine generated mutant channels which instead of inactivating displayed a second open state, characterised by slowly increasing current. Several molecular dynamics (MD) simulations showed a MD trajectory that displays a hydrophobic collapse of the central cavity with S6 dynamics decreasing the pore radius of the S6-gate. The simulations further showed that the SF did constrict in WT channels but not in the mutant. Ionic current measurements of the mutant T480A channels showed that after prolonged depolarizations - pushing the channels into the second conducting state - the activation gate could close and reopen with the channel remaining in the second conducting state. To convert the channels back to their original conducting state longer repolarizing times were needed. Thus, using the T480A pore mutant we could directly determine from ionic currents that gate closure in Kv1.5 channels does not depend on the status of the SF which, as suggested by Deutsch et al. implies the existence of a channel state with both a closed gate and inactivated selectivity filter. (Support: FWO-G025608)

Mode Shift of the Voltage Sensors in Shaker K + Channels is Caused by Energetic Coupling to the Pore Domain

The voltage sensors (S1-S4) of Kv channels are known to undergo a conformational change when triggered by membrane depolarisation that subsequently leads to opening of the central pore (S5-S6) and ion conduction. The electromechanical coupling between these domains is mediated by two well conserved regions: the C-terminus of the S6 segment and the S4-S5 linker. Upon change in the membrane potential, the structural rearrangements induced by the charged S4 segment can be measured as gating currents. During prolonged depolarization, voltage-gated ion channels present a behaviour called the “mode-shift”. This shift causes the QV to change towards negative potentials and essentially affects the energy required by the system to bring all sensors back to their resting state. To understand the structural cause of this process, we investigated the coupling between the pore and voltage sensor and its influence on the development of the mode shift. In our approach we used the cut-open voltage clamp fluorometry method to simultaneously measure gating or ionic currents and conformational changes of the S4 segment. We identified mutations that fully uncouple voltage sensors from the pore domain thus eliminating the mutual influence of these two domains on one another. The results show that, when eliminating the coupling between these domains, the voltage sensors require less energy to move. At the same time the mode shift occurring during prolonged depolarization is abolished. By instead preventing open state stabilization, voltage sensors are kept in the opposite mode. We also found that the mode shift is accompanied by a conformational change in the S4 segment. We propose that the pore influences the voltage sensor by the presence of a ‘’mechanical load’’ and allosterically induces a conformational change in the S4.

Genetic Algorithm Based Computer Simulation Reveals a New Property of Kv1.3 Inactivation

Kv1.3, a voltage-gated potassium channel found in human T cells, enters a non-conducting state during prolonged depolarization by the slow P/C type inactivation, whose exact mechanism is yet to be determined. We have previously shown that acidic extracellular pH (pHe) slows the inactivation process in the presence of low extracellular potassium concentration ([K+]e), whereas speeds it in the presence of high [K+]e. Our aim was to generate a gating scheme, based on a Markov model, which is able to explain the experimental results as a continuous function of pHe and [K+]e. We developed a new computer program based on the Gillespie algorithm that is able to generate simulated records from given gating schemes and compare the resulting curve to the original recording, i.e. it provides an indicator for the goodness of a given scheme. Optimizing of the fitted parameters was achieved using a genetic algorithm. The algorithm was implemented in parallel and run in multi-core and multi-processor environments. As the program enabled us to optimize the gating scheme for more than one measurement record at a time, we could conclude that our simple model having two open microstates and one inactivation pathway from both of them was unable to explain the effects of pHe and [K+]e. Introduction of a new binary parameter and therefore the duplication of inactivation pathways was needed to fully explain the experimental results. We therefore assume that not only the binding of a single potassium ion influences the speed of inactivation, but the conducting pore can have two conformations with different conduction and inactivation properties depending on the [K+]e.