Resting Membrane Voltage Research Articles

WIN55,212-2, a Dual Modulator of Cannabinoid Receptors and G Protein-Coupled Inward Rectifier Potassium Channels.

The coupling of cannabinoid receptors, CB1 and CB2, to G protein-coupled inward rectifier potassium channels, GIRK1 and GIRK2, modulates neuronal excitability in the human brain. The present study established and validated the functional expression in a Xenopus laevis oocyte expression system of CB1 and CB2 receptors, interacting with heteromeric GIRK1/2 channels and a regulator of G protein signaling, RGS4. This ex vivo system enables the discovery of a wide range of ligands interacting orthosterically or allosterically with CB1 and/or CB2 receptors. WIN55,212-2, a non-selective agonist of CB1 and CB2, was used to explore the CB1- or CB2-GIRK1/2-RGS4 signaling cascade. We show that WIN55,212-2 activates CB1 and CB2 at low concentrations whereas at higher concentrations it exerts a direct block of GIRK1/2. This illustrates a dual modulatory function, a feature not described before, which helps to explain the adverse effects induced by WIN55,212-2 in vivo. When comparing the effects with other typical cannabinoids such as Δ9-THC, CBD, CP55,940, and rimonabant, only WIN55,212-2 can significantly block GIRK1/2. Interestingly, the inward rectifier potassium channel, IRK1, a non-G protein-coupled potassium channel important for setting the resting membrane voltage and highly similar to GIRK1 and GIRK2, is not sensitive to WIN55,212-2, Δ9-THC, CBD, CP55,940, or rimonabant. From this, it is concluded that WIN55,212-2 selectively blocks GIRK1/2.

Quantum Electrochemical Equilibrium: Quantum Version of the Goldman–Hodgkin–Katz Equation

The resting membrane voltage of excitable cells such as neurons and muscle cells is determined by the electrochemical equilibrium of potassium and sodium ions. This voltage is calculated by using the Goldman–Hodgkin–Katz equation. However, from the quantum perspective, ions with significant quantum tunneling through closed channels can interfere with the electrochemical equilibrium and affect the value of the membrane voltage. Hence, in this case the equilibrium becomes quantum electrochemical. Therefore, the model of quantum tunneling of ions is used in this study to modify the Goldman–Hodgkin–Katz equation in such a way to calculate the resting membrane voltage at the point of equilibrium. According to the present calculations, it is found that lithium—with its lower mass—shows a significant depolarizing shift in membrane voltage. In addition to this, when the free gating energy of the closed channels decreases, even sodium and potassium ions depolarize the resting membrane voltage via quantum tunneling. This study proposes the concept of quantum electrochemical equilibrium, at which the electrical potential gradient, the concentration gradient and the quantum gradient (due to quantum tunneling) are balanced. Additionally, this concept may be used to solve many issues and problems in which the quantum behavior becomes more influential.

Accuracy considerations for capacitance estimation by voltage steps in cardiomyocytes

Electrophysiologists routinely use simple voltage steps to evaluate cell membrane capacitance derived from corresponding current responses. Frequently, the resting membrane voltage Vrest is employed as holding potential for the subsequent command voltage step and more or less accurate methods are utilised to analyse the transient current. Another choice as holding potential is the peak of the “quasi steady-state” current to voltage relationship, Vpeak. The aim of this study is the systematic evaluation of capacitance estimation accuracy from voltage step experiments depending on the choice of holding potential and analysis method. In this paper, a simulation approach is employed to analyse the current response of a model patch-clamp circuit. Four commonly accepted methods are implemented, utilizing different aspects of the transient current (charge, membrane time constant, and influence of the series resistance) in various combinations and with various degrees of refinement. This simulation study indicates an acceptable accuracy of the elaborated methods for capacitance estimation at holding potentials Vrest and Vpeak over a broad range of capacitance as well as series resistance values.Simple integration of the current transient provides sufficient accuracy at holding potentials, which effectively minimizes changes in resistive membrane current flow during command voltage steps (particularly around Vpeak). However, biphasic command protocols performed at Vpeak activate voltage dependent sodium channels, thereby possibly leading to the threshold voltage for an action potential. Compared to Vrest, all methods utilizing monophasic step protocols, gain additional accuracy, when applied at Vpeak as holding potential.

Endocannabinoid degradation inhibitors ameliorate neuronal and synaptic alterations following traumatic brain injury.

Our previous work showed that lateral fluid percussion injury to the sensorimotor cortex (SMC) of anesthetized rats increased neuronal synaptic hyperexcitability in layer 5 (L5) neurons in ex vivo brain slices 10 days postinjury. Furthermore, endocannabinoid (EC) degradation inhibition via intraperitoneal JZL184 injection 30 min postinjury attenuated synaptic hyperexcitability. This study tested the hypothesis that traumatic brain injury (TBI) induces synaptic and intrinsic neuronal alterations of L5 SMC pyramidal neurons and that these alterations are significantly attenuated by in vivo post-TBI treatment with EC degradation inhibitors. We tested the effects of systemically administered EC degradation enzyme inhibitors (JZL184, MJN110, URB597, or JZL195) with differential selectivity for fatty acid amide hydrolase (FAAH) and monoacylglycerol lipase (MAGL) on electrophysiological parameters in SMC neurons of TBI- and sham-treated rats 10 days post-TBI. We recorded intrinsic neuronal properties, including resting membrane voltage, input resistance, spike threshold, spiking responses to current input, voltage "sag" (rebound response to hyperpolarization-activated inward current), and burst firing. We also measured the frequency and amplitude of spontaneous excitatory postsynaptic currents. We then used the aggregate parameter sets (intrinsic + synaptic properties) to apply a machine learning classification algorithm to quantitatively compare neural population responses from each experimental group. Collectively, our electrophysiological and computational results indicate that sham neurons are the most distinguishable from TBI neurons. Administration of EC degradation inhibitors post-TBI exerted varying degrees of rescue, approximating the neuronal phenotype of sham neurons, with neurons from TBI/JZL195 (a dual MAGL/FAAH inhibitor) being most similar to neurons from sham rats.NEW & NOTEWORTHY This study elucidates neuronal properties altered by traumatic brain injury (TBI) in layer 5 of sensorimotor cortex, which may be implicated in post-TBI circuit dysfunction. We compared effects of systemic administration of four different endocannabinoid degradation inhibitors within a clinically relevant window postinjury. Electrophysiological measures and using a machine learning classification algorithm collectively suggest that pharmacological inhibitors targeting both monoacylglycerol lipase and fatty acid amide hydrolase (e.g., JZL195) may be most efficacious in attenuating TBI-induced neuronal dysfunction at site of injury.

Kv7 potassium channels as signal transduction intermediates in the control of microvascular tone.

Potassium channels are recognized as important regulators of cellular functions in most, if not all cell types. These cellular proteins assemble to form gated pores in the plasma membrane, which serve to regulate the flow of potassium ions (K+ ) from the cytosol to the extracellular space. In VSMCs, the open state of potassium channels enables the efflux of K+ and thereby establishes a negative resting voltage across the plasma membrane that inhibits the opening of VSCCs. Under these conditions, cytosolic Ca2+ concentrations are relatively low and Ca2+ -dependent contraction is inhibited. Recent research has identified Kv7 family potassium channels as important contributors to resting membrane voltage in VSMCs, with much of the research focusing on the effects of drugs that specifically activate or block these channels to produce corresponding effects on VSMC contraction and vascular tone. Increasingly, evidence is emerging that these channels are not just good drug targets-they are also essential intermediates in vascular signal transduction, mediating vasoconstrictor or vasodilator responses to a variety of physiological stimuli. This review will summarize recent research findings that support a crucial function of Kv7 channels in both positive (vasoconstrictive) and negative (vasorelaxant) regulation of microvascular tone.

Inward rectifying potassium currents resolved into components: modeling of complex drug actions.

Inward rectifier potassium currents (I Kir,x) belong to prominent ionic currents affecting both resting membrane voltage and action potential repolarization in cardiomyocytes. In existing integrative models of electrical activity of cardiac cells, they have been described as single current components. The proposed quantitative model complies with findings indicating that these channels are formed by various homomeric or heteromeric assemblies of channel subunits with specific functional properties. Each I Kir,x may be expressed as a total of independent currents via individual populations of identical channels, i.e., channels formed by the same combination of their subunits. Solution of the model equations simulated well recently observed unique manifestations of dual ethanol effect in rat ventricular and atrial cells. The model reflects reported occurrence of at least two binding sites for ethanol within I Kir,x channels related to slow allosteric conformation changes governing channel conductance and inducing current activation or inhibition. Our new model may considerably improve the existing models of cardiac cells by including the model equations proposed here in the particular case of the voltage-independent drug-channel interaction. Such improved integrative models may provide more precise and, thus, more physiologically relevant results.

Role of voltage-gated calcium channels in the regulation of aldosterone production from zona glomerulosa cells of the adrenal cortex.

Zona glomerulosa cells (ZG) of the adrenal gland constantly integrate fluctuating ionic, hormonal and paracrine signals to control the synthesis and secretion of aldosterone. These signals modulate Ca2+ levels, which provide the critical second messenger to drive steroid hormone production. Angiotensin II is a hormone known to modulate the activity of voltage-dependent L- and T-type Ca2+ channels that are expressed on the plasma membrane of ZG cells in many species. Because the ZG cell maintains a resting membrane voltage of approximately -85mV and has been considered electrically silent, low voltage-activated T-type Ca2+ channels are assumed to provide the primary Ca2+ signal that drives aldosterone production. However, this view has recently been challenged by human genetic studies identifying somatic gain-of-function mutations in L-type CaV 1.3 channels in aldosterone-producing adenomas of patients with primary hyperaldosteronism. We provide a review of these assumptions and challenges, and update our understanding of the state of the ZG cell in a layer in which native cellular associations are preserved. This updated view of Ca2+ signalling in ZG cells provides a unifying mechanism that explains how transiently activating CaV 3.2 channels can generate a significant and recurring Ca2+ signal, and how CaV 1.3 channels may contribute to the Ca2+ signal that drives aldosterone production.

Channels and Pumps in Aldosterone-Producing Adenomas

Primary aldosteronism is the most common cause of secondary hypertension, with a prevalence of about 5% of the general hypertensive population and 10% of patients referred to hypertension specialty clinics. Aldosterone-producing adenomas (APAs) account for approximately 30–50% of cases of primary aldosteronism, and idiopathic hyperaldosteronism accounts for most of the rest of the cases. Aldosterone synthesis is regulated primarily by the renin-angiotensin system, potassium, ACTH, and to a lesser degree other peptides (1). Isolated adrenal zona glomerulosa and fasciculata cells from different species maintain a negative membrane resting potential determined by regulating membrane permeability to potassium (2, 3). Zona glomerulosa cells with hyperpolarized resting membrane voltages are steroidogenically inactive (4). Stimulation with angiotensin II, ACTH, or potassium results in membrane depolarization, opening of plasma membrane calcium channels, and mobilization of intracellular calcium, which increases activation of the calcium-calmodulin pathway, ultimately resulting in an increase in steroid biosynthesis and secretion (1). Several potassium channels have been described in rodent, bovine, and human adrenal cells, including inward rectifying and leak potassium currents that provide background potassium currents (reviewed in Ref. 3).

Pharmacological Properties of Homomeric Kv7.4, Kv7.5 and Heteromeric Kv7.4/7.5 Channels: Effects of ICA069673 and Ml213

Kv7.4 and Kv7.5 voltage-activated potassium channels are proposed to contribute to the maintenance of resting membrane voltage in smooth muscle cells. We had previously provided evidence that Kv7.4 and Kv7.5 form predominantly heteromeric channels in vascular smooth muscle cells. Among new activators of Kv7 channels, ICA069673 and ML213 were reported to discriminate between Kv7.4 and Kv7.5 isoforms. Here we compare effects of ICA069673 and ML213 on homomeric human Kv7.4, Kv7.5 and heteromeric Kv7.4/7.5 channels exogenously expressed in A7r5 vascular smooth muscle cells. We found that in contradiction to previously published reports, ML213 does not discriminate among Kv7.4 and Kv7.5 isoforms. ML213 significantly increased the maximum conductance of Kv7.4, Kv7.5 and heteromeric Kv7.4/7.5 channels. In each case, the increase in maximal conductance was accompanied by a negative shift of the activation curves by 23 mV, 47 mV and 32 mV; EC50: 1.5 μM, 3.4 μM and 3.2 μM for Kv7.4, Kv7.5 and heteromeric Kv7.4/7.5 channels, respectively. Current deactivation rates decreased in a voltage dependent manner in the presence of the ML213 (10 μM) for all three channel combinations. In contrast to ML213, ICA069673 was unable to enhance currents through Kv7.5 homomeric channels. However, both Kv7.4 and heteromeric Kv7.4/7.5 channels responded to ICA069672 with a negative shift of activation curves by 57 mV (EC50 = 21 μM) and 66 mV (EC50 = 50 μM) respectively without increase in maximal conductance. Current deactivation rates decreased in the presence of ICA069673 with an opposite voltage dependence in comparison with deactivation rates in the presence of ML213. Based on these results, neither ML213 nor ICA069673 are able to fully discriminate between homomeric Kv7.4, Kv7.5 and heteromeric Kv7.4/7.5 channels exogenously expressed in A7r5 vascular smooth muscle cells.

AR Highlights

Xenopus laevis, commonly known as the African clawed frog, is a useful model in developmental biology. The tail of the Xenopus tadpole completely regenerates by 7–14 days after amputation. This regeneration is similar to tissue renewal in mammals. A ‘‘refractory period’’ occurs during development during which tail regenerative ability is lost in tadpoles. Because of this refractory period, the Xenopus tadpole tail is an ideal model for studying changes in regenerative ability in mammals. Tail regeneration in Xenopus tadpoles requires bioelectrical signaling, which involves slow changes in ion content and resting membrane voltage. For optimal treatment to provide bioelectrical signals for successful regeneration, techniques need to be developed for precise temporal control of resting membrane voltage. In this article, the authors discuss a technique that assists with control of resting membrane voltage in vivo. They demonstrate that this method controls regenerative responses in the tadpole tail. The authors propose a new hypothesis for how ion content is converted into alterations in cell behavior and transcription. The authors hypothesize that the sodium/butyratre transporter SLC5A8 is a mechanism by which some systems may link epigenetic chromatin modifications with bioelectric events (changes in ion content). Determining the mechanisms by which ion flow modulates gene expression will likely provide further understanding on cell biology and evolution of morphogenetic pathways, and contribute to development of treatments that use bioelectricity. Mammalian tissue repair involves a complex, dynamic interplay among cells located at the site of injury and white blood cells recruited to the site. These interactions lead to various processes, including inflammation and tissue differentiation, necessary for repair. In contrast, limb regeneration in urodeles (e.g., salamanders and newts) involves a short period of inflammation, but this is followed by blastema formation that results in a new organ that is fully functional. The authors focus on limb regeneration in Xenopus laevis, which as well as being a useful embryological model, also plays an important role in developmental studies of the immune system and for investigation of regenerative mechanisms. They first review studies on gene expression in amputated Xenopus limbs at various developmental stages involving regeneration and non-regeneration. These studies show that inflammation and regenerative capacity are inversely correlated. The authors also review non-mammalian immune system features involved in the role of inflammation in the balance between regeneration and fibrosis. The authors show results testing the hypothesis that factors controlling the inflammatory response to injury affect regeneration. They compare the effects of antiinflammatory agents and a proinflammatory adjuvant. The data reviewed by the authors suggest that studies on ontogenic immunological changes that occur in Xenopus laevis are useful for determining how inflammation affects blastema formation. An important question is the extent to which differentiating blastema cells resemble mammalian mesenchymal stem cells. Many lizards have the remarkable ability of tail and spinal cord regeneration. Although tail regeneration in lizards has been well studied, further characterization of the anatomy of the regenerated tale is required. The authors examined the anatomy of the original and regenerated tail in the green anole Anolis carolinensis. They chose this species as a model because the process of tail shedding and regeneration is well understood, as well as the fact that the genome has been sequenced, making it a good model for understanding genetic regulation for tail regeneration. The authors investigated the musculature, chondrology, and osteology of the original and regenerated tail in A. carolinensis. The authors found differences in musculature between the original and regenerated tail, suggesting major functional differences between the two types of tails. Notably, the regenerated tail is thought to be less able to perform fine movements. The authors' findings of detailed anatomy of the tail in A. carolinensis could be useful for future genetic and developmental studies on tail regeneration in lizards, and potentially to regeneration in mammals. Ellen C. Jensen*

Protein Kinase C-Dependent Modulation of Heterologously Expressed Homomeric Kv7.4, Kv7.5 and Heteromeric Kv7.4/7.5; Differential Sensitivities to Low Concentrations of Vasopressin and PMA in A7R5 Vascular Smooth Muscle Cells

Kv7.4 and Kv7.5 voltage-activated potassium channels are proposed to contribute to the maintenance of resting membrane voltage in smooth muscle cells. We had previously provided evidence that Kv7.4 and Kv7.5 form predominantly heteromeric channels when natively or exogenously expressed in vascular smooth muscle cells. Endogenous Kv7 currents in smooth muscle cells are suppressed upon activation of Gq coupled receptors. It remained to be elucidated if both Kv7.4 and Kv7.5 respond similarly to low concentrations of vasopressin (AVP), an agonist of the V1a Gq-coupled receptor. using patch-clamp techniques, we measured currents through human Kv7.4 and Kv7.5 channels expressed individually or together in A7r5 rat aortic smooth muscle cells and compared their sensitivity to AVP (100 pM and 500 pM) and to the protein kinase C (PKC) activator phorbol 12-myristate 13-acetate (PMA, 1nM). AVP (100 pM) and PMA suppressed currents through Kv7.4, Kv7.5 and Kv7.5/7.5. Currents were reduced with different voltage dependencies and potencies in the rank order: Kv7.5 > Kv7.4/7.5 > Kv7.4. Both AVP and PMA increased the steepness of Kv7.5 voltage-dependent activation and dramatically decreased Gmax. In contrast, AVP and PMA induced a rightward shift of the Kv7.4 activation curve with only a slight reduction in maximal conductance (Gmax). The modulation of Kv7.4/7.5 activation by AVP and PMA had intermediate biophysical characteristics that were distinct from the modulation of either of the homomeric configurations. These findings reinforce the significance of PKC-dependent regulation of the Kv7 channels and suggest a differential regulation of Kv7.4 and Kv7.5 channel subunits by PKC-dependent phosphorylation.

Enhanced Sarcoplasmic Reticulum Ca 2+ Leak and Increased Na + -Ca 2+ Exchanger Function Underlie Delayed Afterdepolarizations in Patients With Chronic Atrial Fibrillation

Delayed afterdepolarizations (DADs) carried by Na(+)-Ca(2+)-exchange current (I(NCX)) in response to sarcoplasmic reticulum (SR) Ca(2+) leak can promote atrial fibrillation (AF). The mechanisms leading to delayed afterdepolarizations in AF patients have not been defined. Protein levels (Western blot), membrane currents and action potentials (patch clamp), and [Ca(2+)](i) (Fluo-3) were measured in right atrial samples from 76 sinus rhythm (control) and 72 chronic AF (cAF) patients. Diastolic [Ca(2+)](i) and SR Ca(2+) content (integrated I(NCX) during caffeine-induced Ca(2+) transient) were unchanged, whereas diastolic SR Ca(2+) leak, estimated by blocking ryanodine receptors (RyR2) with tetracaine, was ≈50% higher in cAF versus control. Single-channel recordings from atrial RyR2 reconstituted into lipid bilayers revealed enhanced open probability in cAF samples, providing a molecular basis for increased SR Ca(2+) leak. Calmodulin expression (60%), Ca(2+)/calmodulin-dependent protein kinase-II (CaMKII) autophosphorylation at Thr287 (87%), and RyR2 phosphorylation at Ser2808 (protein kinase A/CaMKII site, 236%) and Ser2814 (CaMKII site, 77%) were increased in cAF. The selective CaMKII blocker KN-93 decreased SR Ca(2+) leak, the frequency of spontaneous Ca(2+) release events, and RyR2 open probability in cAF, whereas protein kinase A inhibition with H-89 was ineffective. Knock-in mice with constitutively phosphorylated RyR2 at Ser2814 showed a higher incidence of Ca(2+) sparks and increased susceptibility to pacing-induced AF compared with controls. The relationship between [Ca(2+)](i) and I(NCX) density revealed I(NCX) upregulation in cAF. Spontaneous Ca(2+) release events accompanied by inward I(NCX) currents and delayed afterdepolarizations/triggered activity occurred more often and the sensitivity of resting membrane voltage to elevated [Ca(2+)](i) (diastolic [Ca(2+)](i)-voltage coupling gain) was higher in cAF compared with control. Enhanced SR Ca(2+) leak through CaMKII-hyperphosphorylated RyR2, in combination with larger I(NCX) for a given SR Ca(2+) release and increased diastolic [Ca(2+)](i)-voltage coupling gain, causes AF-promoting atrial delayed afterdepolarizations/triggered activity in cAF patients.

Validation of XE991 use as a Selective Irreversible Blocker of Native Kv7 Channels in Smooth Muscle Myocytes

A concern was recently raised about the selectivity of XE991 and its use as a Kv7 channel blocker in single cell electrophysiology and in vitro functional assays. In the present study, we evaluated the effects of XE991 on different components of the endogenous delayed rectifier potassium currents recorded in freshly isolated guinea pig airway myocytes using the perforated-patch clamp technique and a 5 s voltage step protocol. Application of XE991 (10 μM) reversibly inhibited a transient component of the total current by 27% at voltages positive to −4 mV, whereas it irreversibly inhibited 71% of sustained non-inactivating currents at membrane voltages positive to −30 mV. Comparison of the activation properties of both components (reversibly inhibited transient component and irreversibly inhibited sustained component) revealed voltages of the half activation (V0.5) of +6 mV for the transient component and −31 mV for the irreversibly inhibited sustained current component. These values are in good agreement with the V0.5 values reported for cloned Kv1/Kv2 family channels and cloned Kv7 family channels, respectively. Endogenous Kv7 currents measured in smooth muscle cells of different origins also have V0.5 values ∼−30 mV and resting membrane voltages are typically −60 to −45 mV. Our findings suggest that only the sustained component of the total delayed rectifier potassium current that is irreversibly blocked by XE991 can be attributed to Kv7 current. In functional in vitro models, such as pressurized arteries or lung slices, irreversible contraction of the smooth muscle cells by XE991, which is associated with membrane depolarization and activation of voltage gated calcium channels, can be attributed to the inhibition of Kv7 channels.

Reply to Chadha et al.

Reply to Chadha et al.

Crystal Structure of the Eukaryotic Strong Inward-Rectifier K + Channel Kir2.2 at 3.1 Å Resolution

Inward-rectifier potassium (K+) channels conduct K+ ions most efficiently in one direction, into the cell. Kir2 channels control the resting membrane voltage in many electrically excitable cells, and heritable mutations cause periodic paralysis and cardiac arrhythmia. We present the crystal structure of Kir2.2 from chicken, which, excluding the unstructured amino and carboxyl termini, is 90% identical to human Kir2.2. Crystals containing rubidium (Rb+), strontium (Sr2+), and europium (Eu3+) reveal binding sites along the ion conduction pathway that are both conductive and inhibitory. The sites correlate with extensive electrophysiological data and provide a structural basis for understanding rectification. The channel's extracellular surface, with large structured turrets and an unusual selectivity filter entryway, might explain the relative insensitivity of eukaryotic inward rectifiers to toxins. These same surface features also suggest a possible approach to the development of inhibitory agents specific to each member of the inward-rectifier K+ channel family.

Diastolic Intracellular Calcium-Membrane Voltage Coupling Gain and Postshock Arrhythmias

Recurrent ventricular arrhythmias after initial successful defibrillation are associated with poor clinical outcome. We tested the hypothesis that postshock arrhythmias occur because of spontaneous sarcoplasmic reticulum Ca release, delayed afterdepolarization (DAD), and triggered activity (TA) from tissues with high sensitivity of resting membrane voltage (V(m)) to elevated intracellular calcium (Ca(i)) (high diastolic Ca(i)-voltage coupling gains). We simultaneously mapped Ca(i) and V(m) on epicardial (n=14) or endocardial (n=14) surfaces of Langendorff-perfused rabbit ventricles. Spontaneous Ca(i) elevation (SCaE) was noted after defibrillation in 32% of ventricular tachycardia/ventricular fibrillation at baseline and in 81% during isoproterenol infusion (0.01 to 1 micromol/L). SCaE was reproducibly induced by rapid ventricular pacing and inhibited by 3 mumol/L of ryanodine. The SCaE amplitude and slope increased with increasing pacing rate, duration, and dose of isoproterenol. We found TAs originating from 6 of 14 endocardial surfaces but none from epicardial surfaces, despite similar amplitudes and slopes of SCaEs between epicardial and endocardial surfaces. This was because DADs were larger on endocardial surfaces as a result of higher diastolic Ca(i)-voltage coupling gain, compared to those of epicardial surfaces. Purkinje-like potentials preceded TAs in all hearts studied (n=7). I(K1) suppression with CsCl (5 mmol/L, n=3), BaCl(2) (3 micromol/L, n=3), and low extracellular potassium (1 mmol/L, n=2) enhanced diastolic Ca(i)-voltage coupling gain and enabled epicardium to also generate TAs. Higher diastolic Ca(i)-voltage coupling gain is essential for genesis of TAs and may underlie postshock arrhythmias arising from Purkinje fibers. I(K)(1) is a major factor that determines the diastolic Ca(i)-voltage coupling gain.

Ito Channels Are Octomeric Complexes with Four Subunits of Each Kv4.2 and K+ Channel-interacting Protein 2

Mammalian voltage-gated K+ channels are assemblies of pore-forming alpha-subunits and modulating beta-subunits. To operate correctly, Kv4 alpha-subunits in the heart and central nervous system require recently identified beta-subunits of the neuronal calcium sensing protein family called K+ channel-interacting proteins (KChIPs). Here, Kv4.2.KChIP2 channels are purified, integrity of isolated complexes confirmed, molar ratio of the subunits determined, and subunit valence established. A complex has 4 subunits of each type, a stoichiometry expected for other channels employing neuronal calcium sensing beta-subunits.

Blocker-resistant Ca 2+ currents in rat CA1 hippocampal pyramidal neurons

Ca 2+ currents resistant to organic Ca 2+ channel antagonists are present in different types of central neurons. Here, we describe the properties of such currents in CA1 neurons acutely dissociated from rat hippocampus. Blocker-resistant Ca 2+ currents were isolated by combined application of N-, P/Q- and L-type Ca 2+ current antagonists (ω-conotoxin GVIA 2 μM; ω-conotoxin MVIIC 3 μM; ω-agatoxin IVA 200 nM; nifedipine 10 μM) and constituted approximately 21% of the total Ba 2+ current. The blocker-resistant current showed properties similar to R-type currents in other cell types, i.e. voltages of half-maximal inactivation and activation of −76 and −17 mV, respectively, and strong inactivation during the test pulse. In addition, blocker-resistant Ca 2+ currents in CA1 neurons displayed a characteristically rapid deactivation. Application of mock action potentials revealed that charge transfer through blocker-resistant Ca 2+ channels is highly sensitive to action potential shape and changes in resting membrane voltage. Pharmacological experiments showed that these currents were highly sensitive to the divalent cation Ni 2+ (half-maximal block at 28 μM), but were relatively resistant to the spider toxin SNX-482 (8% and 52% block at 0.1 and 1 μM, respectively). In addition to the functional analysis, we examined the expression of pore-forming and accessory Ca 2+ channel subunits on the messenger RNA level in isolated CA1 neurons using quantitative real-time polymerase chain reaction. Of the pore-forming α subunits encoding high-threshold Ca 2+ channels, Ca v2.1, Ca v2.2 and Ca v2.3 messenger RNA levels were most prominent, corresponding to the high proportion of N-, P/Q- and R-type currents in these neurons. In summary, CA1 neurons display blocker-resistant Ca 2+ currents with distinctive biophysical and pharmacological properties similar to R-type currents in other neuron types, and express Ca 2+ channel messenger RNAs that give rise to R-type Ca 2+ currents in expression systems.

Structural Basis of Inward Rectification: Cytoplasmic Pore of the G Protein-Gated Inward Rectifier GIRK1 at 1.8 Å Resolution

Inward rectifier K + channels govern the resting membrane voltage in many cells. Regulation of these ion channels via G protein-coupled receptor signaling underlies the control of heart rate and the actions of neurotransmitters in the central nervous system. We have determined the protein structure formed by the intracellular N- and C termini of the G protein-gated inward rectifier K + channel GIRK1 at 1.8 Å resolution. A cytoplasmic pore, conserved among inward rectifier K + channels, extends the ion pathway to 60 Å, nearly twice the length of a canonical transmembrane K + channel. The cytoplasmic pore is lined by acidic and hydrophobic amino acids, creating a favorable environment for polyamines, which block the pore. These results explain in structural and chemical terms the basis of inward rectification, and they also have implications for G protein regulation of GIRK channels.

Voltage-dependent, slowly activating K+ current (I(Ks)) and its augmentation by carbachol in rat pancreatic acini.

Acetylcholine-stimulated exocrine secretion of Cl- and water requires the concomitant activation of K+ channels. However, there has not been much investigation of the carbachol- (CCH-) activated K+ channel of rodent pancreatic acini. Here, in a study of rat pancreatic acini, we characterize a voltage-dependent, slowly activating outward current (I(Ks)) that is augmented by CCH. Intact acini were obtained by enzymatic digestion and fast-whole-cell patch-clamp was applied. With symmetrical [Cl-] (32 mmol/l) in the pipette and bath solution, acinar cells had resting membrane voltages of -45+/-0.8 mV (n=97) under current-clamp conditions. CCH (10 micromol/l), which is known to activate Cl- channels via a Ca2+-mediated pathway, sharply depolarized the membrane to -4+/-0.5 mV, which was more negative than E(Cl) (0 mV), and reversed it to -41+/-0.9 mV (n=83) by washout. A clamp voltage of 0 mV activated I(Ks) under control conditions (91+/-8.6 pA, n=83). During CCH application an increase of outward current was observed at 0 mV, and at -50 mV a marked increase of inward Cl current occurred. In the presence of CCH the slow activation of I(Ks) was rarely distinguishable because of interference by the huge Cl- conductance. During CCH washout and decrease of inward current, a persistent augmentation of I(Ks) was revealed (486+/-36.3 pA, n=83). I(Ks) and its augmentation were abolished by substituting K+ in the pipette solution with Cs+. Augmentation of I(Ks) was mimicked by applying ionomycin (0.1 micromol/l), a Ca2+ ionophore. Pharmacological blockers were tested. The chromanol 293B and clotrimazole blocked I(Ks) at micromolar concentrations (IC50=3 micromol/l and 9 micromol/l, respectively) and Ba2+ was a poor blocker (IC50=3 mmol/l). In the presence of CCH (0.2 micromol/l), the membrane was depolarized to around -20 mV and the addition of 293B (10 micromol/l) further depolarized the membrane by 11+/-3 mV (n=5). These data suggest the presence of I(Ks) channels in rat pancreatic acini and their muscarinic activation.