Fast Activation Kinetics Research Articles

Design of Protease Activated Optical Contrast Agents That Exploit a Latent Lysosomotropic Effect for Use in Fluorescence-Guided Surgery.

There is a need for new molecular-guided contrast agents to enhance surgical procedures such as tumor resection that require a high degree of precision. Cysteine cathepsins are highly up-regulated in a wide variety of cancers, both in tumor cells and in the tumor-supporting cells of the surrounding stroma. Therefore, tools that can be used to dynamically monitor their activity in vivo could be used as imaging contrast agents for intraoperative fluorescence image guided surgery (FGS). Although multiple classes of cathepsin-targeted substrate probes have been reported, most suffer from overall fast clearance from sites of protease activation, leading to reduced signal intensity and duration in vivo. Here we describe the design and synthesis of a series of near-infrared fluorogenic probes that exploit a latent cationic lysosomotropic effect (LLE) to promote cellular retention upon protease activation. These probes show tumor-specific retention, fast activation kinetics, and rapid systemic distribution. We demonstrate that they are suitable for detection of diverse cancer types including breast, colon and lung tumors. Most importantly, the agents are compatible with the existing, FDA approved, da Vinci surgical system for fluorescence guided tumor resection. Therefore, our data suggest that the probes reported here can be used with existing clinical instrumentation to detect tumors and potentially other types of inflammatory lesions to guide surgical decision making in real time.

A Novel Modulator of Kv3 Potassium Channels Regulates the Firing of Parvalbumin-Positive Cortical Interneurons.

Kv3.1 and Kv3.2 high voltage-activated potassium channels, which display fast activation and deactivation kinetics, are known to make a crucial contribution to the fast-spiking phenotype of certain neurons. Pharmacological experiments show that the blockade of native Kv3 currents with low concentrations of tetraethylammonium or 4-aminopyridine impairs the expression of this firing phenotype. In particular, Kv3 channels are highly expressed by fast-spiking, parvalbumin-positive interneurons in corticolimbic brain circuits, which modulate the synchronization of cortical circuits and the generation of brain rhythms. Here, we describe a novel small molecule, (5R)-5-ethyl-3-(6-{[4-methyl-3-(methyloxy)phenyl]oxy}-3-pyridinyl)-2,4-imidazolidinedione (AUT1), which modulates Kv3.1 and Kv3.2 channels in human recombinant and rodent native neurons. AUT1 increased whole currents mediated by human Kv3.1b and Kv3.2a channels, with a concomitant leftward shift in the voltage dependence of activation. A less potent effect was observed on hKv3.3 currents. In mouse somatosensory cortex slices in vitro, AUT1 rescued the fast-spiking phenotype of parvalbumin-positive-fast-spiking interneurons following an impairment of their firing capacity by blocking a proportion of Kv3 channels with a low concentration of tetraethylammonium. Notably, AUT1 had no effect on interneuron firing when applied alone. Together, these data confirm the role played by Kv3 channels in the regulation of the firing phenotype of somatosensory interneurons and suggest that AUT1 and other Kv3 modulators could represent a new and promising therapeutic approach to the treatment of disorders associated with dysfunction of inhibitory feedback in corticolimbic circuits, such as schizophrenia.

Nitric oxide influences potassium currents in inner hair cells isolated from guinea-pig cochlea.

Nitric oxide (NO) is a diffusible second messenger, which regulates neurotransmission, serving as the principal endothelium-derived relaxing factor. NO also acts as an ion channel modulator. Nitric oxide synthase (NOS) has been identified in the inner ear, although its physiological role remains unclear. In the present study, the effects of NO onto K currents in inner hair cells (IHCs) were investigated. IHCs were acutely isolated and K currents were recorded by conventional whole-cell voltage-clamp recordings. NO donors sodium nitroprusside (SNP) were applied directly to the cells. In 1mM SNP solutions, the amplitude of outward K currents (IK,f and IK,s) reversely decreased; however, fast activation kinetics was preserved. In the current-voltage relationship curves, the maximal slope conductances were 53.2nS and 44.2nS in control solutions and 1mM SNP solutions, respectively. At the membrane potential of +110mV, the amplitudes of outward currents were 9.2±2.9nA in control solutions and 7.3±2.7nA in 1mM SNP solutions, showing a significant difference. NO acts as a K channel modulator in IHCs. A fast K current suppression may account for the high-frequency hearing impairment by the prevention of fast repolarization.

Cold rolling under inert atmosphere: A powerful tool for Mg activation

In the present work, we report results from a new processing route for Mg: cold rolling under inert atmosphere, followed by a short-time HEBM step (CR + HEBM). This processing route is presented as a powerful tool to reduce the activation time for Mg hydrogenation samples with no need to introduce any additives. Mg samples were extensively processed by CR and CR + HEBM and their effects on the microstructure and hydrogen storage properties were studied. The Mg samples processed by CR + HEBM presented refined microstructure (grain sizes around 4–7 nm), strong texture and are certainly free of oxide layer formation. These conditions lead to relatively fast activation kinetics and enhanced kinetics behavior that has not been reported in the literature so far.

Origin of intrinsic irregular firing in cortical interneurons

Cortical spike trains are highly irregular both during ongoing, spontaneous activity and when driven at high firing rates. There is uncertainty about the source of this irregularity, ranging from intrinsic noise sources in neurons to collective effects in large-scale cortical networks. Cortical interneurons display highly irregular spike times (coefficient of variation of the interspike intervals >1) in response to dc-current injection in vitro. This is in marked contrast to cortical pyramidal cells, which spike highly irregularly in vivo, but regularly in vitro. We show with in vitro recordings and computational models that this is due to the fast activation kinetics of interneuronal K(+) currents. This explanation holds over a wide parameter range and with Gaussian white, power-law, and Ornstein-Uhlenbeck noise. The intrinsically irregular spiking of interneurons could contribute to the irregularity of the cortical network.

Novel Mutations Mapping to the Fourth Sodium Channel Domain of Nav1.7 Result in Variable Clinical Manifestations of Primary Erythromelalgia

We identified and clinically investigated two patients with primary erythromelalgia mutations (PEM), which are the first reported to map to the fourth domain of Nav1.7 (DIV). The identified mutations (A1746G and W1538R) were cloned and transfected to cell cultures followed by electrophysiological analysis in whole-cell configuration. The investigated patients presented with PEM, while age of onset was very different (3 vs. 61 years of age). Electrophysiological characterization revealed that the early onset A1746G mutation leads to a marked hyperpolarizing shift in voltage dependence of steady-state activation, larger window currents, faster activation kinetics (time-to-peak current) and recovery from steady-state inactivation compared to wild-type Nav1.7, indicating a pronounced gain-of-function. Furthermore, we found a hyperpolarizing shift in voltage dependence of slow inactivation, which is another feature commonly found in Nav1.7 mutations associated with PEM. In silico neuron simulation revealed reduced firing thresholds and increased repetitive firing, both indicating hyperexcitability. The late-onset W1538R mutation also revealed gain-of-function properties, although to a lesser extent. Our findings demonstrate that mutations encoding for DIV of Nav1.7 can not only be linked to congenital insensitivity to pain or paroxysmal extreme pain disorder but can also be causative of PEM, if voltage dependency of channel activation is affected. This supports the view that the degree of biophysical property changes caused by a mutation may have an impact on age of clinical manifestation of PEM. In summary, these findings extent the genotype–phenotype correlation profile for SCN9A and highlight a new region of Nav1.7 that is implicated in PEM.Electronic supplementary materialThe online version of this article (doi:10.1007/s12017-012-8216-8) contains supplementary material, which is available to authorized users.

Proton Channels in Normal and Malignant B Cells

HVCN1 is the only mammalian voltage-gated proton channel, highly expressed in B cell cancers such as lymphoma and chronic lymphocytic leukaemia (CLL). In normal B cells, we showed that HVCN1 sustains ROS production by the NADPH oxidase, required for optimal B Cell Receptor (BCR) signalling. In malignant B cells, HVCN1 appears to sustain tumour growth, since HVCN1 downregulation impairs cell survival. We are currently addressing whether HVCN1 supports cancer cells via maintaining ROS production and BCR signalling. During our initial investigation on HVCN1 in B cells, we identified a shorter translational isoform of 253 aa, translated from a second initiation site 20 aa downstream from the first ATG. HVCN1 Short is specifically expressed by malignant B cells: we have evidence that it has different electrophysiological properties, such as conducting larger currents with faster activation kinetics after PMA stimulation. In addition, while HVCN1 Long is internalised with the BCR upon stimulation, HVCN1 short is not, indicating it could mediate proton currents constitutively, thus further helping tumour cells maintain their aberrant pH and sustaining BCR signalling.Support: Bennett Fellowship from Leukaemia and Lymphoma Research (ref n: 12002) to MC. NSF MCB-0943362, NIH R01-GM087507 to TD.

Voltage-dependent Sodium Channels and Calcium-activated Potassium Channels in Human Odontoblasts In Vitro

IntroductionTransmembrane ionic signaling regulates many cellular processes in both physiological and pathologic settings. In this study, the biophysical properties of voltage-dependent Na+ channels in odontoblasts derived from human dental pulp (HOB cells) were investigated together with the effect of bradykinin on intracellular Ca2+ signaling and expression of Ca2+-activated K+ channels. MethodsIonic channel activity was characterized by using whole-cell patch-clamp recording and fura-2 fluorescence. ResultsMean resting membrane potential in the HOB cells was −38 mV. Depolarizing steps from a holding potential of −80 mV activated transient voltage-dependent inward currents with rapid activation/inactivation properties. At a holding potential of −50 mV, no inward current was recorded. Fast-activation kinetics exhibited dependence on membrane potential, whereas fast-inactivation kinetics did not. Steady-state inactivation was described by a Boltzmann function with a half-maximal inactivation potential of −70 mV, indicating that whereas the channels were completely inactivated at physiological resting membrane potential, they could be activated when the cells were hyperpolarized. Inward currents disappeared in Na+-free extracellular solution. Bradykinin activated intracellular Ca2+-releasing and influx pathways. When the HOB cells were clamped at a holding potential of −50 mV, outward currents were recorded at positive potentials, indicating sensitivity to inhibitors of intermediate-conductance Ca2+-activated K+ channels. ConclusionsHuman odontoblasts expressed voltage-dependent Na+ channels, bradykinin receptors, and Ca2+-activated K+ channels, which play an important role in driving cellular functions by channel-receptor signal interaction and membrane potential regulation.

Differential regulation of HCN channel isoform expression in thalamic neurons of epileptic and non-epileptic rat strains

Hyperpolarization-activated cyclic nucleotide-gated cation (HCN) channels represent the molecular substrate of the hyperpolarization-activated inward current (Ih). Although these channels act as pacemakers for the generation of rhythmic activity in the thalamocortical network during sleep and epilepsy, their developmental profile in the thalamus is not yet fully understood. Here we combined electrophysiological, immunohistochemical, and mathematical modeling techniques to examine HCN gene expression and Ih properties in thalamocortical relay (TC) neurons of the dorsal part of the lateral geniculate nucleus (dLGN) in an epileptic (WAG/Rij) compared to a non-epileptic (ACI) rat strain. Recordings of TC neurons between postnatal day (P) 7 and P90 in both rat strains revealed that Ih was characterized by higher current density, more hyperpolarized voltage dependence, faster activation kinetics, and reduced cAMP-sensitivity in epileptic animals. All four HCN channel isoforms (HCN1-4) were detected in dLGN, and quantitative analyses revealed a developmental increase of protein expression of HCN1, HCN2, and HCN4 but a decrease of HCN3. HCN1 was expressed at higher levels in WAG/Rij rats, a finding that was correlated with increased expression of the interacting proteins filamin A (FilA) and tetratricopeptide repeat-containing Rab8b-interacting protein (TRIP8b). Analysis of a simplified computer model of the thalamic network revealed that the alterations of Ih found in WAG/Rij rats compensate each other in a way that leaves Ih availability constant, an effect that ensures unaltered cellular burst activity and thalamic oscillations. These data indicate that during postnatal developmental the hyperpolarizing shift in voltage dependency (resulting in less current availability) is compensated by an increase in current density in WAG/Rij thereby possibly limiting the impact of Ih on epileptogenesis. Because HCN3 is expressed higher in young versus older animals, HCN3 likely does not contribute to alterations in Ih in older animals.

Abstract 4888: Development of a novel FAST cathepsin-activatable NIR agent for tumor imaging

Abstract Fluorescence molecular tomography (FMT) and near-infrared (NIR) imaging agents enable the quantification of kinetic changes in tumor biology in vivo, information useful for the detection, staging, and monitoring of chemotherapeutic efficacy. In particular, imaging agents that become fluorescent upon protease activation in vivo have emerged as valuable tools in oncology research due to the important role of proteases in tumor progression and invasion. Our objective was to develop a novel pan-cathepsin agent with the capability of fast activation kinetics and the potential for early imaging time points in monitoring tumor progression in vivo. To develop this agent, we compared it to an established and well characterized pan-cathepsin activatable imaging agent (ProSenseTM750, a high molecular weight, PEG-decorated NIR-labeled polylysine), which has a long circulation half-life of approximately 20h and offers a long and stable imaging window from 24 to 48h post-injection. ProSenseTM750 FAST is a newly developed 22 kDa molecule based on a novel scaffold consisting of a discrete pair of fluorophores separated by a cleavable sequence and attached to a small pharmacokinetic modifier. To compare selectivity profiles, the agents were assessed for activation by a variety of proteases, including cathepsins and matrix metalloproteases (MMPs). Based on their profiles, both agents show broad pan-cathepsin activation, cleaved by cathepsins B, S, L, and K, with little or no cleavage by MMPs. Consistent with its small architecture, ProSense750 FAST is efficiently activated, approximately 5X faster than the larger ProSense750, offering protease assay readouts as early as 1-2h. In mouse macrophages and 4T1 mouse breast adenocarcinoma cells, both agents were effectively up-taken and activated within the lysosomes for fluorescence microscopy assessment. Nearly complete inhibition of agent activation was achieved using the cell-permeable calpain/cysteine inhibitor E64d. In vivo, FMT imaging of ProSense750 FAST in a 4T-1 tumor model revealed clearly detectable tumor fluorescence as early as 2h after injection, reflecting its short pharmacokinetics (t1/2 ∼ 30 min) and quick in vitro activation profile, with quantification of peak signal around 6h and wash out by 72h. Animals can be optimally imaged at 6 or 24h + 1h, requiring scheduled injection/imaging times. The larger ProSense750 agent, in contrast, shows later optimal imaging (24-48h) but allows greater flexibility in imaging times for large animal cohorts (+ 3h). Careful assessment of organ biodistribution in 4T1 tumor-bearing mice showed different metabolic routes for the two agents, with ProSense750 cleared predominantly through the liver and ProSense750 FAST by renal clearance. These agents offer powerful tools for predicting and monitoring changes in tumor biology in preclinical models of disease. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 4888. doi:10.1158/1538-7445.AM2011-4888

Intramolecular Ca v1.1 Chimeras Reveal the Molecular Mechanism Determining the Characteristic Gating Behaviour of the Skeletal Muscle Calcium Channel

The Ca2+ channel CaV1.1 is the voltage sensor of skeletal muscle excitation-contraction coupling. The classical skeletal muscle CaV1.1 isoform has poor voltage sensitivity and conducts a small, slowly activating Ca2+ current. In contrast, a splice variant lacking exon 29 (α1S-ΔE29) (Tuluc et al.,2009) has an 8-fold higher current amplitude, fast activation-kinetics, and a 30mV left-shifted voltage-dependence of activation. Therefore, the extracellular loop in repeat IV (IVS3-IVS4) mainly coded by exon 29 is a critical determinant of the characteristic gating properties of CaV1.1. Here we used intramolecular chimeras between repeats I and IV to characterize the structural basis of the gating properties of CaV1.1. Inserting exon 29 (alone or in combination with IVS3) into the corresponding region of repeat I was ineffective. However, in combination with the voltage sensor (IVS4) it fully restored α1S-like amplitude and voltage-sensitivity to α1S-ΔE29. Interestingly, all three chimeras exhibit faster activation kinetics. Secondary structure predictions showed that the long IVS3-IVS4 loop contains a beta-sheet while the short loop forms a coil. Point mutations in exon 29 which abolish the beta-sheet fully mimic the effects of deleting exon 29 regarding the kinetic properties and increase the current amplitude by 3-fold and left-shift the voltage dependence by −15mV. Together with previous findings (Nakai et al., 1994) our data suggest that the S3-S4 loop of the first repeat determines activation kinetics, while the corresponding loop plus voltage sensor in the fourth repeat with its unique secondary structure dictate the voltage-dependence, amplitude, and kinetics of skeletal muscle Ca2+ currents. Grants: PT (MFI-2007-417), BEF (FWF-P20059-B05).

Deficient Regulation of Gbetagamma Effectors by Fluorescently Labeled Galpha i3 Subunits Reveals Distinct Aspects of Coupling to GIRK and Cav2.2 Channels

It has been proposed that Gαi subunits regulate, and form signaling complexes with, effectors primarily regulated by Gβγ, notably G protein activated K+ channel (GIRK) and N-type Ca2+ channel CaV2.2. To investigate Gαi signaling mechanisms, we tested three Gαi3 derivatives tagged by YFP and CFP (Gαi3xFP). Gαi3NT carried a myristoylation signal followed by xFP, preceding the Gα protein, similar to other GαixFPs reported as fully functional [An internally labeled Gαi3117 was previously used to study Gα-Gβγ interactions [Gibson SK & Gilman AG, PNAS 103: 212]. In a novel Gαi3CT, xFP was fused C-terminally to Gαi3. All Gαi3xFPs and wt-Gαi3 bound Gβγ and GIRK1. However, Gβγ did not enhance the Gαi3xFP-GIRK1 interaction, as observed for wt-Gαi3. Only Gαi3117 and Gαi3CT, but not Gαi3NT, controlled GIRK's basal activity. Muscarinic 2 receptor (m2R) activated the GIRK channel via pertussis toxin-insensitive wt-Gαi3, Gαi3117 and Gαi3NT, but not Gαi3CT. However, only Gαi3NT produced fast activation kinetics comparable to wt-Gαi3, whereas Gαi3117 greatly slowed the activation kinetics. As expected, activation of m2R inhibited CaV2.2 in a voltage-dependent manner but only via wt-Gαi3 and Gαi3NT, and not via Gαi3117 or Gαi3CT. xFP-Gβγ, but none of the xFP-labeled Gα subunits, showed FRET with xFP-GIRK1. Our results suggest that xFP labeling of Gα, even at positions widely regarded as producing functional Gα proteins, distort some or all of Gα-effector interactions and regulations. Incidentally, the differential regulation of basal and evoked GIRK activity by Gαi3NT and Gαi3117 reveal that control of GIRK's basal activity and the provision of “free” Gβγ for GIRK activation (following GPCR activation) are distinct functions of the Gα subunit. Neither mechanism is a prerequisite for the other. Supported by ISF (ND) and BSF (CWD, ND).

Functional Characterization of Inward Rectifier Potassium Ion Channel in Murine Fetal Ventricular Cardiomyocytes

Aims: Previous studies have shown the dramatic changes in electrical properties of murine fetal cardiomyocytes, while details on inward rectifier potassium current (IK1) are still seldom discussed. Thus we aimed to characterize the functional expression and functional role of IK1 in murine fetal ventricular cardiomyocytes. Methods: Whole cell patch clamp was applied to investigate the electrophysiological properties of IK1. Quantitative real-time PCR, western blotting and double-label immunofluorescence were further utilized to find out the molecular basis of IK1. Results: Compared to early developmental stage (EDS), IK1 at late developmental stage (LDS) displayed higher current density, stronger rectifier property and faster activation kinetics. It was paralleled with the downregulation of Kir2.3 and the upregulation of Kir2.1/Kir2.2. IK1 contributed to maintain the maximum diastolic potential (MDP), late repolarization phase (LRP) as well as the action potential duration (APD). However, the contribution to MDP and velocity of LRP did not change significantly with maturation. Conclusions: During fetal development, the switch of IK1 subtypes from Kir2.1/Kir2.3 to Kir2.1 resulted in the dramatic changes in IK1 electrophysiological properties.

Time and Voltage-Dependent Slowing in the Off Gating Currents from Kv3.1

Kv3.1 channels are characterized by fast activation and deactivation kinetics with a quite positive threshold of activation. In the central nervous system they are involved in the high-frequency firing pattern of neurons. Given some controversy on Kv3.1 channel gating, we performed an in-depth analysis of WT Kv3.1 gating currents elicited in mammalian Ltk- cells. The Q/V curves determined from integrating either QON or QOFF were comparable and were best fitted with a double Boltzmann distribution. The Q/V curves were shifted by approximately 15 mV towards more hyperpolarized potentials compared to the ionic G/V curve, indicating that a substantial charge was moved during closed-state transitions. The double component in the Q/V curve and a bi-exponential decay of IgON indicated that the charge was carried by at least two distinct transition steps, contrary to earlier reports for Kv3.2b, but more consistent with Shaker, Kv1.5 and Kv2.1 channels. Since the integrals of QON and QOFF were identical, there was no charge immobilization within a 125 ms depolarization. The time course of IgOFF was fast after short depolarizations, but displayed Shaker-like slowing with increasing step duration for depolarizations positive to the threshold for ionic current activation. Since 4-AP blocks the concerted opening step in Shaker, we used it to separate the two components of IgOFF. In the presence of 3 mM 4-AP only the fast IgOFF remained, indicating that the slowing is linked to a final concerted step in channel opening which appears to be a general gating feature of Kv channels.

Motor Protein Function in Skeletal Muscle—A Multiple Scale Approach to Contractility

We present an approach to skeletal muscle contractility and its regulation over different scales ranging from biomechanical studies in intact muscle fibers down to the motility and interaction of single motor protein molecules. At each scale, shortening velocities as a measure for weak cross-bridge cycling rates are extracted and compared. Experimental approaches include transmitted light microscopy, second harmonic generation imaging of contracting myofibrils, and fluorescence microscopy of single molecule motility. Each method yields image sequences that are analyzed with automated image processing algorithms to extract the contraction velocity. Using this approach, we show how to isolate the contribution of the motor proteins actin and myosin and their modulation by regulatory proteins from the concerted action of electro-mechanical activation on a more complex cellular scale. The advantage of this approach is that averaged contraction velocities can be determined on the different scales ranging from isolated motor proteins to sarcomere levels in myofibrils and myofibril arrays within the cellular architecture. Our results show that maximum shortening velocities during in situ electrical activation of sarcomere contraction in intact single muscle cells can substantially deviate from sliding velocities obtained in oriented in vitro motility assays of isolated motor proteins showing that biophysical contraction kinetics not simply translate linearly between contractility scales. To adequately resolve the very fast initial mechanical activation kinetics of shortening at each scale, it was necessary to implement high-speed imaging techniques. In the case of intact fibers and single molecule motility, we achieved a major increase in temporal resolution up to frame rates of 200-1000 fps using CMOS image sensor technology. The data we obtained at this unprecedented temporal resolution and the parameters extracted can be used to validate results obtained from computational models of motor protein interaction and skeletal muscle contractility in health and muscle disease. Our approach is feasible to explain the possible underlying mechanisms that contribute to different shortening velocities at different scales and complexities.

Biophysical Properties of 9 KCNQ1 Mutations Associated With Long-QT Syndrome

Inherited long-QT syndrome is characterized by prolonged QT interval on the ECG, syncope, and sudden death caused by ventricular arrhythmia. Causative mutations occur mostly in cardiac potassium and sodium channel subunit genes. Confidence in mutation pathogenicity is usually reached through family genotype-phenotype tracking, control population studies, molecular modeling, and phylogenetic alignments; however, biophysical testing offers a higher degree of validating evidence. By using in vitro electrophysiological testing of transfected mutant and wild-type long-QT syndrome constructs into Chinese hamster ovary cells, we investigated the biophysical properties of 9 KCNQ1 missense mutations (A46T, T265I, F269S, A302V, G316E, F339S, R360G, H455Y, and S546L) identified in a New Zealand-based long-QT syndrome screening program. We demonstrate through electrophysiology and molecular modeling that 7 of the missense mutations have profound pathological dominant-negative loss-of-function properties, confirming their likely disease-causing nature. This supports the use of these mutations in diagnostic family screening. Two mutations (A46T, T265I) show suggestive evidence of pathogenicity within the experimental limits of biophysical testing, indicating that these variants are disease-causing via delayed- or fast-activation kinetics. Further investigation of the A46T family has revealed an inconsistent cosegregation of the variant with the clinical phenotype. Electrophysiological characterization should be used to validate long-QT syndrome pathogenicity of novel missense channelopathies. When such results are inconclusive, great care should be taken with genetic counseling and screening of such families, and alternative disease-causing mechanisms should be considered.

Compartmentalized β Subunit Distribution Determines Characteristics and Ethanol Sensitivity of Somatic, Dendritic, and Terminal Large-Conductance Calcium-Activated Potassium Channels in the Rat Central Nervous System

Neurons are highly differentiated and polarized cells, whose various functions depend upon the compartmentalization of ion channels. The rat hypothalamic-neurohypophysial system (HNS), in which cell bodies and dendrites reside in the hypothalamus, physically separated from their nerve terminals in the neurohypophysis, provides a particularly powerful preparation in which to study the distribution and regional properties of ion channel proteins. Using electrophysiological and immunohistochemical techniques, we characterized the large-conductance calcium-activated potassium (BK) channel in each of the three primary compartments (soma, dendrite, and terminal) of HNS neurons. We found that dendritic BK channels, in common with somatic channels but in contrast to nerve terminal channels, are insensitive to iberiotoxin. Furthermore, analysis of dendritic BK channel gating kinetics indicates that they, like somatic channels, have fast activation kinetics, in contrast to the slow gating of terminal channels. Dendritic and somatic channels are also more sensitive to calcium and have a greater conductance than terminal channels. Finally, although terminal BK channels are highly potentiated by ethanol, somatic and dendritic channels are insensitive to the drug. The biophysical and pharmacological properties of somatic and dendritic versus nerve terminal channels are consistent with the characteristics of exogenously expressed alphabeta1 versus alphabeta4 channels, respectively. Therefore, one possible explanation for our findings is a selective distribution of auxiliary beta1 subunits to the somatic and dendritic compartments and beta4 to the terminal compartment. This hypothesis is supported immunohistochemically by the appearance of distinct punctate beta1 or beta4 channel clusters in the membrane of somatic and dendritic or nerve terminal compartments, respectively.

Biological Pacemaker Engineered by Nonviral Gene Transfer in a Mouse Model of Complete Atrioventricular Block

We hypothesized that a nonviral gene delivery of the hyperpolarization-activated HCN2 channel combined with the beta(2)-adrenergic receptor (ADRB2) would generate a functional pacemaker in a mouse model of complete atrioventricular block (CAVB) induced by radiofrequency ablation of the His bundle. Plasmids encoding HCN2 and ADRB2 mixed with tetronic 304, a poloxamine block copolymer, were injected in the left ventricular free wall (HCN2-ADRB2 mice). Sham mice received a noncoding plasmid. CAVB was induced 5 days later. Ventricular escape rhythms in HCN2-ADRB2 mice were significantly faster than in sham mice at day 15 after ablation and later. In HCN2-ADRB2 mice, QRS complexes were larger than in sham mice and characterized by abnormal axes. Immunostaining of GFP-HCN2 fusion protein showed an expression of HCN2 channel in left ventricular myocardium for at least 45 days after injection. In the mouse, CAVB induces progressive hypertrophy and heart failure leading to 50% mortality after 110 days. HCN2-ADRB2 mice survived 3 weeks longer than sham mice. Finally, beta-adrenergic input increased ventricular escape rhythms significantly more in HCN2-ADRB2 mice than in sham mice. In conclusion, nonviral gene transfer can produce a functional cardiac biological pacemaker regulated by sympathetic input, which improves life expectancy in a mouse model of CAVB.

Spatiotemporal Analysis of Differential Akt Regulation in Plasma Membrane Microdomains

As a central kinase in the phosphatidylinositol 3-kinase pathway, Akt has been the subject of extensive research; yet, spatiotemporal regulation of Akt in different membrane microdomains remains largely unknown. To examine dynamic Akt activity in membrane microdomains in living cells, we developed a specific and sensitive fluorescence resonance energy transfer-based Akt activity reporter, AktAR, through systematic testing of different substrates and fluorescent proteins. Targeted AktAR reported higher Akt activity with faster activation kinetics within lipid rafts compared with nonraft regions of plasma membrane. Disruption of rafts attenuated platelet-derived growth factor (PDGF)-stimulated Akt activity in rafts without affecting that in nonraft regions. However, in insulin-like growth factor-1 (IGF)-1 stimulation, Akt signaling in nonraft regions is dependent on that in raft regions. As a result, cholesterol depletion diminishes Akt activity in both regions. Thus, Akt activities are differentially regulated in different membrane microdomains, and the overall activity of this oncogenic pathway is dependent on raft function. Given the increased abundance of lipid rafts in some cancer cells, the distinct Akt-activating characteristics of PDGF and IGF-1, in terms of both effectiveness and raft dependence, demonstrate the capabilities of different growth factor signaling pathways to transduce differential oncogenic signals across plasma membrane.

Abstract 357: Novel Gain of Function SCN5A Mutation Causes Familial Atrial Fibrillation

Background : Few genetic etiologies are known for familial or sporadic cases of atrial fibrillation, which is the most common arrhythmia in older adults. We report a mother and son, both with atrial fibrillation, who were found to have a novel mutation in the sodium channel gene, SCN5A. Case History: The proband, a 50 year old male, first developed paroxysmal atrial fibrillation following orthopedic surgery. At age 57, he presented with increasing fatigue and shortness of breath and was found to have chronic atrial fibrillaion. Cardiac testing found no evidence of ischemia, normal left ventricular function and mild left atrial dilation. His mother had paroxysmal atrial fibrillation, diagnosed at age 63, and a history of well-controlled hypertension and palpitations. Her atrial fibrillation was initially controlled by sotalol, but worsening symptoms led to a change to amiodarone at age 83. Her echo showed mild biatrial enlargement. ECGs for the proband and his mother have never demonstrated conduction system disease, long QT, or a Brugada pattern. Methods and Results: Genomic DNA isolated from blood lymphocytes was screened using direct sequencing techniques. Genes previously reported to be associated with atrial fibrillation, KCNA5, GJA5 and KCNQ1, were screened and no mutations were found. In the sodium channel gene, SCN5A, a novel missense mutation was identified (c. 1493K>R) in both the proband and his mother, but not in an unaffected sibling or in the proband’s 2 asymptomatic offspring. The mutation is in a highly conserved lysine residue in the voltage-gated sodium channel gene family. Functional studies recorded macroscopic sodium currents from tsA201 cells expressing both the wild-type (Nav1.5/WT) and mutant channel (Nav1.5/K1493R). The mutant channel exhibited fast activation and inactivation kinetics and an increase in current density. No effect on steady-state activation and inactivation were observed, however, current deactivation was faster in mutant Nav1.5/K1493R as compared to the WT channels. Conclusions: This is the first report of an inherited SCN5A mutation in familial atrial fibrillation. Functional studies suggest a ’gain-of-function’ effect of the identified Nav1.5/K1493R mutation.