Shift In Voltage Dependence Of Activation Research Articles

The Potential Antidepressant Compound Org 34167 Modulates HCN Channels Via a Novel Mode of Action.

Org 34167 is a small molecule hyperpolarization-activated cyclic nucleotide-gated (HCN) channel modulator that has been trialed in humans for its potential antidepressant activity. The precise action of Org 34167 is not fully understood. Here we use two-electrode voltage clamp recordings and an allosteric model to explore the interaction of Org 34167 with human HCN1 channels. The impact of Org 34167 on channel function included a hyperpolarizing shift in activation voltage dependence and a slowing of activation kinetics. Furthermore, a reduction in the maximum open probability at extreme hyperpolarization argued for an additional voltage-independent mechanism. Org 34167 had a similar impact on a truncated HCN1 channel lacking the C-terminal nucleotide binding domain, thus ruling out an interaction with this domain. Fitting a gating model, derived from a 10-state allosteric scheme, predicted that Org 34167 strongly reduced the equilibrium constant for the voltage-independent pore domain to favor a closed pore, as well as reducing the voltage sensing domain-pore domain coupling and shifting the zero voltage equilibrium constant of the voltage sensing domain to favor the inactive state. SIGNIFICANCE STATEMENT: The brain penetrant small molecule Org 34167 has been reported to have an antidepressant action by targeting HCN channels; however, its mode of action is unknown. We used heterologously expressed human HCN1 channels to show that Org 34167 inhibits channel activity by modulating kinetic parameters associated with the channel pore domain, voltage sensing domain, and interdomain coupling.

Antidepressant-like activity of a brain penetrant HCN channel inhibitor in mice.

Changes in Hyperpolarization-Activated Cyclic Nucleotide-Gated (HCN) channel function have been linked to depressive-like traits, making them potential drug targets. However, there is currently no peer-reviewed data supporting the use of a small molecule modulator of HCN channels in depression treatment. Org 34167, a benzisoxazole derivative, has been patented for the treatment of depression and progressed to Phase I trials. In the current study, we analysed the biophysical effects of Org 34167 on HCN channels in stably transfected human embryonic kidney 293 (HEK293) cells and mouse layer V neurons using patch-clamp electrophysiology, and we utilised three high-throughput screens for depressive-like behaviour to assess the activity of Org 34167 in mice. The impact of Org 34167 on locomotion and coordination were measured by performing rotarod and ledged beam tests. Org 34167 is a broad-spectrum inhibitor of HCN channels, slowing activation and causing a hyperpolarising shift in voltage-dependence of activation. It also reduced I h-mediated sag in mouse neurons. Org 34167 (0.5mg/kg) reduced marble burying and increased the time spent mobile in the Porsolt swim and tail suspension tests in both male and female BALB/c mice, suggesting reduced depressive-like behaviour. Although no adverse effects were seen at 0.5mg/kg, an increase in dose to 1mg/kg resulted in visible tremors and impaired locomotion and coordination. These data support the premise that HCN channels are valid targets for anti-depressive drugs albeit with a narrow therapeutic index. Drugs with higher HCN subtype selectivity are needed to establish if a wider therapeutic window can be obtained.

Interactions of HCN4 with LRMP and IRAG

The hyperpolarization-activated cyclic-nucleotide sensitive channel, HCN4, is critical for excitability of cardiac pacemaker cells. We recently identified two isoform-specific regulators of HCN4, lymphoid restricted membrane protein (LRMP, IRAG2) and inositol triphosphate receptor-associated guanylate-cyclase substrate (IRAG). LRMP and IRAG are ER-transmembrane proteins with cytoplasmic N-termini and coiled-coil domains. Interestingly these homologous proteins exhibit opposing effects on HCN4, with LRMP causing a loss-of-function by inhibiting the cAMP-dependent depolarizing shift in voltage-dependent activation, and IRAG causing a gain-of-function by inducing a depolarizing shift in voltage-dependence in the absence of cAMP.

Alternative Splicing at N Terminus and Domain I Modulates CaV1.2 Inactivation and Surface Expression

The CaV1.2 L-type calcium channel is a key conduit for Ca2+ influx to initiate excitation-contraction coupling for contraction of the heart and vasoconstriction of the arteries and for altering membrane excitability in neurons. Its α1C pore-forming subunit is known to undergo extensive alternative splicing to produce many CaV1.2 isoforms that differ in their electrophysiological and pharmacological properties. Here, we examined the structure-function relationship of human CaV1.2 with respect to the inclusion or exclusion of mutually exclusive exons of the N-terminus exons 1/1a and IS6 segment exons 8/8a. These exons showed tissue selectivity in their expression patterns: heart variant 1a/8a, one smooth-muscle variant 1/8, and a brain isoform 1/8a. Overall, the 1/8a, when coexpressed with CaVβ2a, displayed a significant and distinct shift in voltage-dependent activation and inactivation and inactivation kinetics as compared to the other three splice variants. Further analysis showed a clear additive effect of the hyperpolarization shift in V1/2inact of CaV1.2 channels containing exon 1 in combination with 8a. However, this additive effect was less distinct for V1/2act. However, the measured effects were β-subunit-dependent when comparing CaVβ2a with CaVβ3 coexpression. Notably, calcium-dependent inactivation mediated by local Ca2+-sensing via the N-lobe of calmodulin was significantly enhanced in exon-1-containing CaV1.2 as compared to exon-1a-containing CaV1.2 channels. At the cellular level, the current densities of the 1/8a or 1/8 variants were significantly larger than the 1a/8a and 1a/8 variants when coexpressed either with CaVβ2a or CaVβ3 subunit. This finding correlated well with a higher channel surface expression for the exon 1-CaV1.2 isoform that we quantified by protein surface-expression levels or by gating currents. Our data also provided a deeper molecular understanding of the altered biophysical properties of alternatively spliced human CaV1.2 channels by directly comparing unitary single-channel events with macroscopic whole-cell currents.

Gating mechanism of Kv11.1 (hERG) K+ channels without covalent connection between voltage sensor and pore domains

Kv11.1 (hERG, KCNH2) is a voltage-gated potassium channel crucial in setting the cardiac rhythm and the electrical behaviour of several non-cardiac cell types. Voltage-dependent gating of Kv11.1 can be reconstructed from non-covalently linked voltage sensing and pore modules (split channels), challenging classical views of voltage-dependent channel activation based on a S4–S5 linker acting as a rigid mechanical lever to open the gate. Progressive displacement of the split position from the end to the beginning of the S4–S5 linker induces an increasing negative shift in activation voltage dependence, a reduced zg value and a more negative ΔG0 for current activation, an almost complete abolition of the activation time course sigmoid shape and a slowing of the voltage-dependent deactivation. Channels disconnected at the S4–S5 linker near the S4 helix show a destabilization of the closed state(s). Furthermore, the isochronal ion current mode shift magnitude is clearly reduced in the different splits. Interestingly, the progressive modifications of voltage dependence activation gating by changing the split position are accompanied by a shift in the voltage-dependent availability to a methanethiosulfonate reagent of a Cys introduced at the upper S4 helix. Our data demonstrate for the first time that alterations in the covalent connection between the voltage sensor and the pore domains impact on the structural reorganizations of the voltage sensor domain. Also, they support the hypothesis that the S4–S5 linker integrates signals coming from other cytoplasmic domains that constitute either an important component or a crucial regulator of the gating machinery in Kv11.1 and other KCNH channels.

Nonlinear effects of hyperpolarizing shifts in activation of mutant Nav1.7 channels on resting membrane potential.

The Nav1.7 sodium channel is preferentially expressed within dorsal root ganglion (DRG) and sympathetic ganglion neurons. Gain-of-function mutations that cause the painful disorder inherited erythromelalgia (IEM) shift channel activation in a hyperpolarizing direction. When expressed within DRG neurons, these mutations produce a depolarization of resting membrane potential (RMP). The biophysical basis for the depolarized RMP has to date not been established. To explore the effect on RMP of the shift in activation associated with a prototypical IEM mutation (L858H), we used dynamic-clamp models that represent graded shifts that fractionate the effect of the mutation on activation voltage dependence. Dynamic-clamp recording from DRG neurons using a before-and-after protocol for each cell made it possible, even in the presence of cell-to-cell variation in starting RMP, to assess the effects of these graded mutant models. Our results demonstrate a nonlinear, progressively larger effect on RMP as the shift in activation voltage dependence becomes more hyperpolarized. The observed differences in RMP were predicted by the "late" current of each mutant model. Since the depolarization of RMP imposed by IEM mutant channels is known, in itself, to produce hyperexcitability of DRG neurons, the development of pharmacological agents that normalize or partially normalize activation voltage dependence of IEM mutant channels merits further study.NEW & NOTEWORTHY Inherited erythromelalgia (IEM), the first human pain disorder linked to a sodium channel, is widely regarded as a genetic model of neuropathic pain. IEM is produced by Nav1.7 mutations that hyperpolarize activation. These mutations produce a depolarization of resting membrane potential (RMP) in dorsal root ganglion neurons. Using dynamic clamp to explore the effect on RMP of the shift in activation, we demonstrate a nonlinear effect on RMP as the shift in activation voltage dependence becomes more hyperpolarized.

LRRK2 Regulates Voltage-Gated Calcium Channel Function.

Voltage-gated Ca2+ (CaV) channels enable Ca2+ influx in response to membrane depolarization. CaV2.1 channels are localized to the presynaptic membrane of many types of neurons where they are involved in triggering neurotransmitter release. Several signaling proteins have been identified as important CaV2.1 regulators including protein kinases, G-proteins and Ca2+ binding proteins. Recently, we discovered that leucine rich repeat kinase 2 (LRRK2), a protein associated with inherited Parkinson’s disease, interacts with specific synaptic proteins and influences synaptic transmission. Since synaptic proteins functionally interact with CaV2.1 channels and synaptic transmission is triggered by Ca2+ entry via CaV2.1, we investigated whether LRRK2 could impact CaV2.1 channel function. CaV2.1 channel properties were measured using whole cell patch clamp electrophysiology in HEK293 cells transfected with CaV2.1 subunits and various LRRK2 constructs. Our results demonstrate that both wild type (wt) LRRK2 and the G2019S LRRK2 mutant caused a significant increase in whole cell Ca2+ current density compared to cells expressing only the CaV2.1 channel complex. In addition, LRRK2 expression caused a significant hyperpolarizing shift in voltage-dependent activation while having no significant effect on inactivation properties. These functional changes in CaV2.1 activity are likely due to a direct action of LRRK2 as we detected a physical interaction between LRRK2 and the β3 CaV channel subunit via coimmunoprecipitation. Furthermore, effects on CaV2.1 channel function are dependent on LRRK2 kinase activity as these could be reversed via treatment with a LRRK2 inhibitor. Interestingly, LRRK2 also augmented endogenous voltage-gated Ca2+ channel function in PC12 cells suggesting other CaV channels could also be regulated by LRRK2. Overall, our findings support a novel physiological role for LRRK2 in regulating CaV2.1 function that could have implications for how mutations in LRRK2 contribute to Parkinson’s disease pathophysiology.

Probing the Contribution of Nav1.7 and Nav1.8 to Cold Tolerance in Hibernators

Voltage-gated sodium (Nav) channels play an important role in the transduction of sensory signals. Nav1.7 and Nav1.8 are primarily expressed in peripheral sensory neurons, particularly small diameter nociceptors and temperature sensors. Mutations that alter the functional properties of these channels cause severe pain disorders or pain insensitivity in humans.Small depolarizations of the neuronal membrane caused by sensory receptor channels activate Nav channels, which further depolarize the sensory neuron and generate action potentials. Here, we test this general paradigm with regard to cold sensitivity using a model organism that is tolerant to cold. Thirteen-lined ground squirrels (Ictidomys tridecemlineatus ) are able to tolerate very low temperatures during hibernation, but also show resistance to cold in the active state. Electrophysiological recordings of dissociated sensory neurons from active squirrels reveal that both Nav1.7 and Nav1.8 activity is suppressed at cold temperatures. The suppression is manifested as a shift in activation voltage-dependence and a reduction of current density. We propose that these changes regulate neuronal firing in response to cold and thus contribute to a mechanism for cold tolerance. Our results suggest a molecular model of cold resistance at the level of primary afferents.

Gabapentinoids Suppress Early Afterdepolarizations by Uncoupling α2δ-1 Subunits from CAV1.2 Channels: Implications for CAV Channel Gating-Modifiers as a New Class of Antiarrhythmics

Under certain pathological conditions, the repolarization phase of the cardiac action potential (AP) is interrupted by membrane potential oscillations called early afterdepolarizations (EADs), well-recognized triggers of deadly cardiac arrhythmias. The reactivation of L-type calcium current (ICa,L) plays a prominent role in EAD formation. In recent studies using the dynamic clamp technique, we have shown that a small depolarizing shift (<5mV) of the CaV1.2 steady-state activation potently suppresses EAD occurrence. Based on this findings, we hypothesized that pharmacological interventions that induce a depolarizing shift of macroscopic ICa,L activation (gating modifiers) can suppress EADs and their arrhythmogenic consequences. Gabapentinoids (gabapentin and pregabalin), drugs that selectively bind to α2δ-1 CaV subunits and are commonly used to treat epilepsy and neuropathic pain, are good candidates as Cav gating modifiers. We found that gabapentin induced a time-dependent depolarizing shift of the voltage-dependent activation of CaV1.2 channels expressed in Xenopus oocytes (α1C β3 α2δ-1; 1mM gabapentin, 2h incubation). Since α2δ-1 facilitates CaV1.2 voltage-dependent activation, these results suggest a functional uncoupling of α2δ-1 from a fraction of channels. Importantly, the maximal macroscopic conductance was unaltered, implying that CaV1.2 channel trafficking was not perturbed, in contrast to the effects shown in other CaV isoforms. We then studied gabapentinoids effect on EAD susceptibility in freshly-dissociated rabbit ventricular myocytes under EAD-promoting conditions (600μM H2O2). Strikingly, 1 mM pregabalin suppressed EAD occurrence and restored AP duration as predicted based on the depolarizing shift in voltage-dependent activation. Taken together, these results support the view that gabapentinoids, and more generally CaV1.2 gating modifiers, show promise in suppressing EAD-mediated arrhythmias and could constitute a novel class of antiarrhythmics.

Protein kinase C bidirectionally modulates Ih and hyperpolarization-activated cyclic nucleotide-gated (HCN) channel surface expression in hippocampal pyramidal neurons.

Hyperpolarization-activated cyclic nucleotide-gated (HCN) channels, particularly that of the HCN1 isoform, are enriched in the distal dendrites of hippocampal CA1 pyramidal neurons; these channels have physiological functions with respect to decreasing neuronal excitability. In the present study, we aimed to investigate phosphorylation as a mechanism controlling Ih amplitude and HCN1 surface expression in hippocampal principal neurons under normal physiological conditions. Tyrosine phosphorylation decreased Ih amplitude at maximal activation (maximal Ih ), without altering HCN1 surface expression, in two classes of hippocampal principal neurons. Inhibition of serine/threonine protein phosphatases 1 and 2A decreased maximal Ih and HCN1 surface expression in hippocampal principal neurons. Protein kinase C (PKC) activation irreversibly diminished Ih and HCN1 surface expression, whereas PKC inhibition augmented Ih and HCN1 surface expression. PKC activation increased HCN1 channel phosphorylation. These results demonstrate the novel finding of a phosphorylation mechanism, dependent on PKC activity, which bidirectionally modulates Ih amplitude and HCN1channel surface expression in hippocampal principal neurons under normal physiological conditions. Hyperpolarization-activated, cyclic nucleotide-gated (HCN) ion channels attenuate excitability in hippocampal pyramidal neurons. Loss of HCN channel-mediated current (Ih ), particularly that mediated by the HCN1 isoform, occurs with the development of epilepsy. Previously, we showed that, following pilocarpine-induced status epilepticus, there are two independent changes in HCN function in dendrites: decreased Ih amplitude associated with a loss of HCN1 surface expression and a hyperpolarizing shift in voltage-dependence of activation (gating). The hyperpolarizing shift in gating was attributed to decreased phosphorylation as a result of a loss of p38 mitogen-activated protein kinase activity and increased calcineurin activity; however, the mechanisms controlling Ih amplitude and HCN1 surface expression under epileptic or normal physiological conditions are poorly understood. We aimed to investigate phosphorylation as a mechanism regulating Ih amplitude and HCN1 surface expression (i.e. as is the case for HCN gating) in hippocampal principal neurons under normal physiological conditions. We discovered that inhibition of either tyrosine phosphatases or the serine/threonine protein phosphatases 1 and 2A decreased Ih at maximal activation in hippocampal CA1 pyramidal dendrites and pyramidal-like principal neuron somata from naïve rats. Furthermore, we found that inhibition of PP1/PP2A decreased HCN1 surface expression, whereas tyrosine phosphatase inhibition did not. Protein kinase C (PKC) activation reduced Ih amplitude and HCN1 surface expression, whereas PKC inhibition produced the opposite effect. Inhibition of protein phosphatases 1 and 2A and activation of PKC increased the serine phosphorylation state of the HCN1 protein. The effect of PKC activation on Ih was irreversible. These results indicate that PKC bidirectionally modulates Ih amplitude and HCN1 surface expression in hippocampal principal neurons.

Serum Factor Alters T-Type Cav3.2 Gating Kinetics and Current Density

T-type Calcium channels play a critical role in regulating neuronal excitability and modulating sensory transmission. The Cav3.2 channel isoform is highly expressed in peripheral nociceptors as well as in the pain-processing regions of the dorsal horn of the spinal cord. Augmenting Cav3.2 currents has been shown to induce hyperexcitability in nociceptive neurons in vitro and hyperalgesia in vivo. These studies strongly suggest that potentiation of the Cav3.2 channel results in abnormal nociceptive transmission, which could contribute to a variety of clinical pain syndromes. Therefore, it is important to identify endogenously produced molecular species that modulate Cav3.2 currents. Using the patch-clamp technique and stably transfected human embryonic kidney cells (HEK-293) expressing the Cav3.2 channel, we have begun to characterize a factor found in fetal bovine serum (FBS) that profoundly affects Cav3.2 channel gating kinetics. Specifically, when compared to baseline recombinant currents, 1% serum produces maximal increases in current magnitude (350%; p<.001), conductance (150%, p<.001), rate of macroscopic inactivation (47.1%; p<.001) and deactivation (74.3%; p<.001). Furthermore, 1% serum induces a hyperpolarizing shift in voltage-dependence of activation (V50) (−4.77mV; p <.001) with minimal effect on voltage-dependence of inactivation. In contrast, we found that recombinant CaV3.1 currents were completely insensitive. Similar to recombinant Cav3.2 currents, T-currents from rat dorsal root ganglia (DRG) cells exhibited comparable changes, with 1% serum producing maximal increases in current magnitude (210%; p<.05), conductance (183%; p<.05), and rate of inactivation (71.4%; p<.001), in addition to inducing a hyperpolarizing shift in V50 (−9.402mV; p<.01). Future studies will focus on identifying this serum factor in order to evaluate its potential role in nociceptive signal modulation and cellular excitability. Supported in part by NIH grant R21DA034448 (SMT).

Electrophysiological Functions of Intracellular Amyloid β in Specific for Cultured Human Neurones and its Impairment Properties

Prevailing role of intracellular amyloid <TEX>${\beta}$</TEX> (<TEX>$iA{\beta}$</TEX>) in Alzheimer's disease (AD) initiation and progression attracts more and more attention in recent years. To address whether <TEX>$iA{\beta}$</TEX> induces early alterations of electrophysiological properties in cultured human primary neurons, we delivered <TEX>$iA{\beta}$</TEX> with adenovirus and measured the electrophysiological properties of infected neurons with whole-cell recordings. Our results show that <TEX>$iA{\beta}$</TEX> induces an increase in neuronal resting membrane potentials, a decrease in <TEX>$K^+$</TEX> currents and a hyperpolarizing shift in voltage-dependent activation of <TEX>$K^+$</TEX> currents. These results suggest the electrophysiological impairments induced by <TEX>$iA{\beta}$</TEX> may be responsible for its neuronal toxicity.

Characterization of KCNQ1 atrial fibrillation mutations reveals distinct dependence on KCNE1

The IKs potassium channel, critical to control of heart electrical activity, requires assembly of α (KCNQ1) and β (KCNE1) subunits. Inherited mutations in either IKs channel subunit are associated with cardiac arrhythmia syndromes. Two mutations (S140G and V141M) that cause familial atrial fibrillation (AF) are located on adjacent residues in the first membrane-spanning domain of KCNQ1, S1. These mutations impair the deactivation process, causing channels to appear constitutively open. Previous studies suggest that both mutant phenotypes require the presence of KCNE1. Here we found that despite the proximity of these two mutations in the primary protein structure, they display different functional dependence in the presence of KCNE1. In the absence of KCNE1, the S140G mutation, but not V141M, confers a pronounced slowing of channel deactivation and a hyperpolarizing shift in voltage-dependent activation. When coexpressed with KCNE1, both mutants deactivate significantly slower than wild-type KCNQ1/KCNE1 channels. The differential dependence on KCNE1 can be correlated with the physical proximity between these positions and KCNE1 as shown by disulfide cross-linking studies: V141C forms disulfide bonds with cysteine-substituted KCNE1 residues, whereas S140C does not. These results further our understanding of the structural relationship between KCNE1 and KCNQ1 subunits in the IKs channel, and provide mechanisms for understanding the effects on channel deactivation underlying these two atrial fibrillation mutations.

CaBP1 Regulates Voltage-dependent Inactivation and Activation of CaV1.2 (L-type) Calcium Channels

CaBP1 is a Ca(2+)-binding protein that regulates the gating of voltage-gated (Ca(V)) Ca(2+) channels. In the Ca(V)1.2 channel α(1)-subunit (α(1C)), CaBP1 interacts with cytosolic N- and C-terminal domains and blunts Ca(2+)-dependent inactivation. To clarify the role of the α(1C) N-terminal domain in CaBP1 regulation, we compared the effects of CaBP1 on two alternatively spliced variants of α(1C) containing a long or short N-terminal domain. In both isoforms, CaBP1 inhibited Ca(2+)-dependent inactivation but also caused a depolarizing shift in voltage-dependent activation and enhanced voltage-dependent inactivation (VDI). In binding assays, CaBP1 interacted with the distal third of the N-terminal domain in a Ca(2+)-independent manner. This segment is distinct from the previously identified calmodulin-binding site in the N terminus. However, deletion of a segment in the proximal N-terminal domain of both α(1C) isoforms, which spared the CaBP1-binding site, inhibited the effect of CaBP1 on VDI. This result suggests a modular organization of the α(1C) N-terminal domain, with separate determinants for CaBP1 binding and transduction of the effect on VDI. Our findings expand the diversity and mechanisms of Ca(V) channel regulation by CaBP1 and define a novel modulatory function for the initial segment of the N terminus of α(1C).

The Cardiac Conduction System

The human heart beats 2.5 billion times during a normal lifespan, a feat accomplished by cells of the cardiac conduction system (CCS). The functional components of the CCS can be broadly divided into the impulse-generating nodes and the impulse-propagating His-Purkinje system. Human diseases of the conduction system have been identified that alter impulse generation, impulse propagation, or both. CCS dysfunction is primarily due to acquired conditions such as myocardial ischemia/infarct, age-related degeneration, procedural complications, and drug toxicity. Inherited forms of CCS disease are rare, but each new mutation provides invaluable insight into the molecular mechanisms governing CCS development and function. Applying a multidisciplinary approach, which includes human genetic screening, biophysical analysis, and transgenic mouse technology, has yielded a broad array of gene families involved in maintaining normal CCS physiology (Figure 1). In this review, we discuss gene families that have been implicated in human CCS diseases of rhythm, conduction block, accessory conduction, and development (Table). We also investigate evolving therapeutic strategies that may serve as adjuvant or replacement therapy to current implantable pacemakers. Figure 1. Cardiac conduction system cell. Genes identified in human cardiac conduction system disease are highlighted. View this table: Table. Genetic Basis of Conduction System Disease The human sinoatrial node (SAN) is a crescent-shaped, intramural structure with its head located subepicardially at the junction of the right atrium and the superior vena cava and its tail extending 10 to 20 mm along the crista terminalis.26 The SAN has complex 3-dimensional tissue architecture with central and peripheral components made up of distinct ion channel and gap junction expression profiles.27 Central and peripheral cells have different action potential characteristics and conduction properties (Figure 2).27 Experimental and computational models have demonstrated that SAN heterogeneity is necessary to maintain normal automaticity and impulse conduction.28,–,30 Figure 2. Electrophysiological heterogeneity of the …

Homeostatic regulation of M-current modulates synaptic integration in secretomotor, but not vasomotor, sympathetic neurons in the bullfrog

We compared how vasomotor C neurons and secretomotor B neurons integrated identical patterns of virtual synaptic activity using dynamic clamp, perforated-patch recordings from dissociated bullfrog sympathetic ganglion cells. The synaptic template modelled one strong nicotinic synapse and nine weak synapses, each firing randomly at 5 Hz, with strength normalized to each cell. B neurons initially fired at 12 Hz, but this declined within seconds, decreasing 27% after 40 s and recovering slowly as evidenced by the threshold synaptic conductance for firing (tau(recovery) = 136 + or - 23 s). C neurons gave an identical initial response that remained steady, declining only 6% after 40 s. The difference resulted from an activity-dependent 379 + or - 65% increase in M-current (I(M)) in B cells (tau(recovery) = 153 + or - 22 s), which was absent in C cells. In addition, action potential afterhyperpolarizations were 2-fold longer in B cells, but this did not produce the differential response to synaptic stimulation. Activity-dependent increases in I(M) were sensitive to 100 microm Cd(2+) and 2.5 microm oxotremorine M (oxo-M), a muscarinic agonist, and fully blocked by zero Ca(2+), 10 microm oxo-M and 2.5 microm oxo-M plus 50 microm wortmannin, a PIP(2) synthesis inhibitor. A leftward shift in voltage-dependent activation could not fully account for the I(M) increase. Firing at 0.5 Hz was sufficient to modulate I(M). Opposing influences of activity and muscarinic excitation thus produce homeostatic I(M) regulation, to stabilize excitability and postsynaptic output in secretomotor sympathetic neurons. Absence of this regulation in vasomotor neurons suggests a different integrative function, where synaptic gain increases in proportion to presynaptic activity.

The amiodarone derivative KB130015 activates hERG1 potassium channels via a novel mechanism

Human ether à go-go related gene (hERG1) potassium channels underlie the repolarizing I Kr current in the heart. Since they are targets of various drugs with cardiac side effects we tested whether the amiodarone derivative 2-methyl-3-(3,5-diiodo-4-carboxymethoxybenzyl)benzofuran (KB130015) blocks hERG1 channels like its parent compound. Using patch-clamp and two-electrode voltage-clamp techniques we found that KB130015 blocks native and recombinant hERG1 channels at high voltages, but it activates them at low voltages. The activating effect has an apparent EC 50 value of 12 µM and is brought about by an about 4-fold acceleration of activation kinetics and a shift in voltage-dependent activation by − 16 mV. Channel activation was not use-dependent and was independent of inactivation gating. KB130015 presumably binds to the hERG1 pore from the cytosolic side and functionally competes with hERG1 block by amiodarone, E4031 (N-[4-[[1-[2-(6-methyl-2-pyridinyl)ethyl]-4-piperidinyl] carbonyl] phenyl] methanesulfonamide dihydrochloride), and sertindole. Vice versa, amiodarone attenuates hERG1 activation by KB130015. Based on synergic channel activation by mallotoxin and KB130015 we conclude that the hERG1 pore contains at least two sites for activators that are functionally coupled among each other and to the cavity-blocker site. KB130015 and amiodarone may serve as lead structures for the identification of hERG1 pore-interacting drugs favoring channel activation vs. block.

Distinctive Inactivation Defects of Differing Mutant Calcium Channels Underlying Timothy Syndrome

Timothy Syndrome (TS) is a multisystem disorder characterized by autism, immune deficiencies, and cardiac arrhythmias. Intriguingly, the underlying defect comes down to a single point mutation (either G402S or G406R) in the IS6 region of CaV1.2 channels. These channels are critical conduits of Ca2+ entry into the heart, smooth muscle and brain. As such, these channels employ two forms of feedback regulation−voltage-dependent inactivation (VDI) and Ca2+/calmodulin-dependent inactivation (CDI). In TS, these regulatory mechanisms are disrupted, resulting in inappropriate Ca2+ feedback. Given that the pattern of multisystem pathology differs for the two types of mutant channels, we here undertook in-depth biophysical analysis of the altered inactivation in each of these constructs. As reported, both mutants exhibited strongly attenuated VDI. Rather surprisingly, however, both constructs also demonstrated a clear reduction of CDI, in contrast to a previous study reporting selective weakening of VDI (PNAS105:11987). Further analysis revealed that the CDI deficits in the two mutants may arise from very different mechanisms. For G406R, voltage-dependent activation is strongly shifted to more negative potentials, while estimated maximal open probability (PO/max) at saturating depolarization was only slightly altered. According to an allosteric mechanism of CDI (Biophys J96:222a), this favoring of channel activation would reduce CDI, because opening would be enhanced even within inactivated channels (i.e., the current decrease seen upon channel inactivation would be lessened). By contrast, the G402S mutation caused a marked depolarizing shift in voltage-dependent activation, with largely unchanged PO/max. This outcome would sharply diminish channel opening at physiological voltages, yielding attenuated CDI via decreased entry into inactivated states. Recognizing these divergent mechanisms of CDI disruption may shed light on the differing disease phenotypes elaborated by the two mutations, and ultimately prove beneficial in tailoring treatments for each TS population.

Intracellular amyloid induces impairments on electrophysiological properties of cultured human neurons

The role of intracellular amyloid β (iAβ) in Alzheimer's disease (AD) initiation and progression attracts more and more attention in recent years. To address whether iAβ induces early alterations of electrophysiological properties in cultured human primary neurons, we delivered iAβ with adeno-virus and measured the electrophysiological properties of infected neurons with whole-cell recordings. Our results show that iAβ induces an increase in neuronal resting membrane potentials, a decrease in K+ currents and a hyperpolarizing shift in voltage-dependent activation of K+ currents. These results suggest the electrophysiological impairments induced by iAβ may be responsible for its neuronal toxicity.

Association with the Auxiliary Subunit PEX5R/Trip8b Controls Responsiveness of HCN Channels to cAMP and Adrenergic Stimulation

Hyperpolarization-activated cyclic nucleotide-gated (HCN) channels are key modulators of neuronal activity by providing the depolarizing cation current I(h) involved in rhythmogenesis, dendritic integration, and synaptic transmission. These tasks critically depend on the availability of HCN channels, which is dynamically regulated by intracellular cAMP; the range of this regulation, however, largely differs among neurons in the mammalian brain. Using affinity purification and high-resolution mass spectrometry, we identify the PEX5R/Trip8b protein as the beta subunit of HCN channels in the mammalian brain. Coassembly of PEX5R/Trip8b affects HCN channel gating in a subtype-dependent and mode-specific way: activation of HCN2 and HCN4 by cAMP is largely impaired, while gating by phosphoinositides and basal voltage-dependence remain unaffected. De novo expression of PEX5R/Trip8b in cardiomyocytes abolishes beta-adrenergic stimulation of HCN channels. These results demonstrate that PEX5R/Trip8b is an intrinsic auxiliary subunit of brain HCN channels and establish HCN-PEX5R/Trip8b coassembly as a mechanism to control the channels' responsiveness to cyclic nucleotide signaling.