Current Activation Kinetics Research Articles

The T-type Ca2+ Channel Inhibitor Mibefradil Inhibits Voltage-Dependent K+ Channels in Rabbit Coronary Arterial Smooth Muscle Cells

We examined the effects of mibefradil, a T-type Ca2+ channel inhibitor, on voltage-dependent K+ (Kv) channels in rabbit coronary arterial smooth muscle cells using the whole-cell patch clamp technique. Mibefradil reduced the Kv current amplitude in a dose-dependent manner, with an apparent Kd value of 1.08 μM. Kv current inhibition by mibefradil was highly voltage-dependent over the full activation voltage range (−30 to +10 mV). The decay rate of Kv channel inactivation was accelerated by mibefradil without altering the kinetics of current activation. The rate constants of association and dissociation were 2.23 ± 0.07 μM−1·s−1 and 2.40 ± 0.42 s−1, respectively. Mibefradil had no significant effect on the steady-state activation or inactivation curves. In the presence of mibefradil, the recovery time constant from inactivation was decreased, and the application of train pulses (1 or 2 Hz) increased mibefradil-induced Kv channel inhibition, suggesting that the inhibitory effects of mibefradil were use-dependent. The inhibitory effect of mibefradil on Kv channels was unaffected by extracellular Ca2+-free conditions. Moreover, the absence of ATP inside the pipette did not alter the blocking effect of mibefradil. Therefore, we suggest that mibefradil directly inhibited the Kv current, independently of Ca2+ channel inhibition.

Genetic Activation of Bk Currents using a Gain-Of-Function Subunit Alters Neuronal Activity

K+ channels play key roles in dampening excitability in many types of tissues. In neurons, the large conductance Ca2+-activated K+ (BK) channel inhibits excitability by participating in the fast afterhyperpolarization and limiting neurotransmitter release. Paradoxically, BK currents have also been recently linked to enhancing excitability and seizures. Using a transgenic approach, the dual nature of BK's influence on excitability was investigated. In vivo expression of a gain-of function BK channel alpha subunit (R207Q) recapitulates aspects of both the excitatory and inhibitory influence of BK currents. The direction of influence is dependent upon the relative expression level of wildtype and mutant subunits, the resulting BK current activation kinetics, and interaction with other fast-gated K+ currents.

Local Anesthetic Inhibits Hyperpolarization-Activated Cationic Currents

Systemic administration of local anesthetics has beneficial perioperative properties and an anesthetic-sparing and antiarrhythmic effect, although the detailed mechanisms of these actions remain unclear. In the present study, we investigated the effects of a local anesthetic, lidocaine, on hyperpolarization-activated and cyclic nucleotide-gated (HCN) channels that contribute to the pacemaker currents in rhythmically oscillating cells of the heart and brain. Voltage-clamp recordings were used to examine the properties of cloned HCN subunit currents expressed in Xenopus laevis oocytes and human embryonic kidney (HEK) 293 cells under control condition and lidocaine administration. Lidocaine inhibited HCN1, HCN2, HCN1-HCN2, and HCN4 channel currents at 100 μM in both oocytes and/or HEK 293 cells; it caused a decrease in both tonic and maximal current (∼30-50% inhibition) and slowed current activation kinetics for all subunits. In addition, lidocaine evoked a hyperpolarizing shift in half-activation voltage (ΔV(1/2) of ∼-10 to -14 mV), but only for HCN1 and HCN1-HCN2 channels. By fitting concentration-response data to logistic functions, we estimated half-maximal (EC(50)) concentrations of lidocaine of ∼30 to 40 μM for the shift in V(1/2) observed with HCN1 and HCN1-HCN2; for inhibition of current amplitude, calculated EC(50) values were ∼50 to 70 μM for HCN1, HCN2, and HCN1-HCN2 channels. A lidocaine metabolite, monoethylglycinexylidide (100 μM), had similar inhibitory actions on HCN channels. These results indicate that lidocaine potently inhibits HCN channel subunits in dose-dependent manner over a concentration range relevant for systemic application. The ability of local anesthetics to modulate I(h) in central neurons may contribute to central nervous system depression, whereas effects on I(f) in cardiac pacemaker cells may contribute to the antiarrhythmic and/or cardiovascular toxic action.

Fluorinated General Anesthetics Modulate Shaker Potassium Channels at Clinically Relevant Concentrations

Recent studies demonstrated that selective intra-thalamic block of the Kv1.2 potassium channels rapidly reverses the unconsciousness of inhalational anesthesia (Alkire et al., 2009). Furthermore, intra-thalamic block of Kv1.3 with selective blocker, ShK, reverses the unconsciousness effect of sevoflurane. Both findings suggestthat Shaker-related potassium channels might be key targets for anesthetics. We tested this hypothesis using patch clamp recordings of potassium currents in cell lines stably expressing homomeric human Shaker family channels. We found that at low voltages, sub-clinical and clinical doses of sevoflurane irreversibly potentiate potassium current through Kv1.3>Kv1.2>Kv1.5>Kv1.1. channels, by increasing the peak current amplitude and accelerating the current activation kinetics. At higher voltages, clinical doses of sevoflurane inhibit Kv1.4>Kv1.5.>Kv1.3 and potentiate Kv1.2>Kv1.1. Sevoflurane had no effect on lipid bilayer conductance, suggesting direct interaction of the anesthetic with potassium channel subunits. Thus, Shaker potassium channel modulation may contribute to the clinical effects of general anesthesia. Supported by the Hillblom Foundation and NIH 1P01AG032131.

Functional Ion Channels in Human Pulmonary Artery Smooth Muscle Cells: Voltage‐Dependent Cation Channels

The activity of voltage-gated ion channels is critical for the maintenance of cellular membrane potential and generation of action potentials. In turn, membrane potential regulates cellular ion homeostasis, triggering the opening and closing of ion channels in the plasma membrane and, thus, enabling ion transport across the membrane. Such transmembrane ion fluxes are important for excitation–contraction coupling in pulmonary artery smooth muscle cells (PASMC). Families of voltage-dependent cation channels known to be present in PASMC include voltage-gated K+ (Kv) channels, voltage-dependent Ca2+-activated K+ (Kca) channels, L- and T- type voltage-dependent Ca2+ channels, voltage-gated Na+ channels and voltage-gated proton channels. When cells are dialyzed with Ca2+-free K+- solutions, depolarization elicits four components of 4-aminopyridine (4-AP)-sensitive Kvcurrents based on the kinetics of current activation and inactivation. In cell-attached membrane patches, depolarization elicits a wide range of single-channel K+ currents, with conductances ranging between 6 and 290 pS. Macroscopic 4-AP-sensitive Kv currents and iberiotoxin-sensitive Kca currents are also observed. Transcripts of (a) two Na+ channel α-subunit genes (SCN5A and SCN6A), (b) six Ca2+ channel α–subunit genes (α1A, α1B, α1X, α1D, α1Eand α1G) and many regulatory subunits (α2δ1, β1-4, and γ6), (c) 22 Kv channel α–subunit genes (Kv1.1 - Kv1.7, Kv1.10, Kv2.1, Kv3.1, Kv3.3, Kv3.4, Kv4.1, Kv4.2, Kv5.1, Kv 6.1-Kv6.3, Kv9.1, Kv9.3, Kv10.1 and Kv11.1) and three Kv channel β-subunit genes (Kvβ1-3) and (d) four Kca channel α–subunit genes (Sloα1 and SK2-SK4) and four Kca channel β-subunit genes (Kcaβ1-4) have been detected in PASMC. Tetrodotoxin-sensitive and rapidly inactivating Na+ currents have been recorded with properties similar to those in cardiac myocytes. In the presence of 20 mM external Ca2+, membrane depolarization from a holding potential of -100 mV elicits a rapidly inactivating T-type Ca2+ current, while depolarization from a holding potential of -70 mV elicits a slowly inactivating dihydropyridine-sensitive L-type Ca2+ current. This review will focus on describing the electrophysiological properties and molecular identities of these voltage-dependent cation channels in PASMC and their contribution to the regulation of pulmonary vascular function and its potential role in the pathogenesis of pulmonary vascular disease.

A Minimal Cysteine Motif Required to Activate the SKOR K+ Channel of Arabidopsis by the Reactive Oxygen Species H2O2*[S

Reactive oxygen species (ROS) are essential for development and stress signaling in plants. They contribute to plant defense against pathogens, regulate stomatal transpiration, and influence nutrient uptake and partitioning. Although both Ca(2+) and K(+) channels of plants are known to be affected, virtually nothing is known of the targets for ROS at a molecular level. Here we report that a single cysteine (Cys) residue within the Kv-like SKOR K(+) channel of Arabidopsis thaliana is essential for channel sensitivity to the ROS H(2)O(2). We show that H(2)O(2) rapidly enhanced current amplitude and activation kinetics of heterologously expressed SKOR, and the effects were reversed by the reducing agent dithiothreitol (DTT). Both H(2)O(2) and DTT were active at the outer face of the membrane and current enhancement was strongly dependent on membrane depolarization, consistent with a H(2)O(2)-sensitive site on the SKOR protein that is exposed to the outside when the channel is in the open conformation. Cys substitutions identified a single residue, Cys(168) located within the S3 α-helix of the voltage sensor complex, to be essential for sensitivity to H(2)O(2). The same Cys residue was a primary determinant for current block by covalent Cys S-methioylation with aqueous methanethiosulfonates. These, and additional data identify Cys(168) as a critical target for H(2)O(2), and implicate ROS-mediated control of the K(+) channel in regulating mineral nutrient partitioning within the plant.

An investigation of Pt alloy oxygen reduction catalysts in phosphoric acid doped PBI fuel cells

A study of a phosphoric acid doped polybenzimidazole (PBI) membrane fuel cell using commercial carbon supported, Pt alloy oxygen reduction catalysts is reported. The cathodes were made from PTFE bonded carbon supported Pt alloys without PBI but with phopshoric acid added to the electrode for ionic conductivity. Polarisation data for fuel cells with cathodes made with alloys of Pt with Ni, Co, Ru and Fe are compared with those with Pt alone as cathode at temperatures between 120 and 175 °C. With the same loading of Pt enhancement in cell performance was achieved with all alloys except Pt–Ru, in the low current density activation kinetics region of operation. The extent of enhancement depended upon the operating temperature and also the catalyst loading. In particular a Pt–Co alloy produced performance significantly better than Pt alone, e.g. a peak power, with low pressure air, of 0.25 W cm −2 with 0.2 mg Pt cm −2 of a 20 wt% Pt–Co catalyst.

Open channel block of Kv1.5 currents by citalopram

To examine whether selective serotonin reuptake inhibitor citalopram interacts with Kv1.5, one of the cardiovascular-specific Kv channel isoforms. The interaction between citalopram and Kv1.5 expressed in Chinese hamster ovary cells was studied using the whole-cell patch-clamp technique. Citalopram reduced Kv1.5 whole-cell currents in a reversible concentration-dependent manner, with an IC(50) value and a Hill coefficient of 2.8+/-1.1 micromol/L and 0.8+/-0.3, respectively. Citalopram-induced inhibition of Kv1.5 is associated with time-dependent development of block without modifying the kinetics of current activation. The inhibition increased steeply between -30 and 0 mV, which corresponded with the voltage range for channel opening. In the voltage range positive to 0 mV, inhibition displayed an additional voltage dependence, consistent with an electrical distance delta of 0.19. Citalopram slowed the deactivation time course, resulting in a tail crossover phenomenon when the tail currents, recorded in the presence and absence of citalopram, were superimposed. Inhibition of Kv1.5 by citalopram was use-dependent. The present results suggest that citalopram acts on Kv1.5 currents as an open-channel blocker, and much caution about arrhythmogenic risk is required when using citalopram in the treatment with depressed patients.

HL-1 cells express an inwardly rectifying K+ current activated via muscarinic receptors comparable to that in mouse atrial myocytes

An inwardly rectifying K+ current is present in atrial cardiac myocytes that is activated by acetylcholine (IKACh). Physiologically, activation of the current in the SA node is important in slowing the heart rate with increased parasympathetic tone. It is a paradigm for the direct regulation of signaling effectors by the Gβγ G-protein subunit. Many questions have been addressed in heterologous expression systems with less focus on the behaviour in native myocytes partly because of the technical difficulties in undertaking comparable studies in native cells. In this study, we characterise a potassium current in the atrial-derived cell line HL-1. Using an electrophysiological approach, we compare the characteristics of the potassium current with those in native atrial cells and in a HEK cell line expressing the cloned Kir3.1/3.4 channel. The potassium current recorded in HL-1 is inwardly rectifying and activated by the muscarinic agonist carbachol. Carbachol-activated currents were inhibited by pertussis toxin and tertiapin-Q. The basal current was time-dependently increased when GTP was substituted in the patch-clamp pipette by the non-hydrolysable analogue GTPγS. We compared the kinetics of current modulation in HL-1 with those of freshly isolated atrial mouse cardiomyocytes. The current activation and deactivation kinetics in HL-1 cells are comparable to those measured in atrial cardiomyocytes. Using immunofluorescence, we found GIRK4 at the membrane in HL-1 cells. Real-time RT-PCR confirms the presence of mRNA for the main G-protein subunits, as well as for M2 muscarinic and A1 adenosine receptors. The data suggest HL-1 cells are a good model to study IKAch.

Structural Determinant of Altered Current Expression, Activation Kinetics and Beta-Subunit Interaction of the Neuronal X1 Splice Variant of the Rat BK Channel

We have identified and cloned a splice variant of the rat BK channel called X1 (Poulsen et al., 2009, Biochimica et Biophysica Acta. 1788(380-389)) which is exclusively expressed in brain or nervous tissue, which has not previously been functionally characterized. The X1 variant is different from the insert-less variant Zero by having an eight amino acid insert in the extracellular loop between S1 and S2, a four amino acid insert between C-terminal S8 and S9 (SS1) and 27 amino acids between S9 and S10. Another variant Slo27, widely expressed in brain and some vascular tissues, also contains the 27 residues between S9 and S10 but only a 3 residue insert between S8 and S9 (SS2). When expressed in Xenopus oocytes, the X1 variant shows less current expression than Slo27 or Zero and an apparently faster activation speed. We attempted to dissect the underlying mechanism by generating constructs lacking one of the insert sequences. Deletion of the eight amino acids between S1 and S2 resulted in higher current expression similar to Slo27 or Zero while retaining the fast activation speed. Deletion of the four S8-S9 residues resulted in low current expression but still fast activation. Thus the eight residue insert seems to suppress channel surface expression or channel gating at low calcium concentrations, while the structural determinator of fast activation speed is less clear.We also co-expressed the X1 variant with beta 2, which is present in nervous tissue also. Beta 2 co-expression reduced current expression further and slowed channel activation but showed no signs of inactivation (at low calcium), which is a key feature of beta 2 when co-expressed with Zero or Slo27.

Inhibitory Action of Fluvoxamine on Kv1.5 Currents

The voltage-gated K(+) channel Kv1.5 was expressed in Chinese hamster ovary cells, and its interaction with fluvoxamine was studied using a whole-cell patch-clamp technique. Fluvoxamine reduced Kv1.5 whole-cell currents in a reversible, concentration-dependent manner, with an IC(50) value of 2.0 microM, and a Hill coefficient of 0.7. Fluvoxamine-induced inhibition of Kv1.5 caused a time-dependent blockade without modifying the kinetics of current activation. Fluvoxamine additionally inhibited Kv1.5 in a closed/resting or nonconducting state after deactivation. Inhibition increased steeply between -30 and 0 mV, corresponding to the voltage range for channel opening. Inhibition displayed an additional voltage dependence at voltages greater than 0 mV, consistent with an electrical distance of 0.16. Fluvoxamine slowed the deactivation time course and a tail crossover phenomenon was observed when the tail currents, recorded in the presence and absence of fluvoxamine, were superimposed. Inhibition of Kv1.5 by fluvoxamine was use-dependent. These results suggest that fluvoxamine strongly inhibits Kv1.5 currents and the inhibition of Kv1.5 by fluvoxamine is mixed channel-state dependent.

The Guanylyl Cyclase Activator YC-1 Directly Inhibits the Voltage-Dependent K+ Channels in Rabbit Coronary Arterial Smooth Muscle Cells

We investigated the effects of YC-1, an activator of soluble guanylyl cyclase (sGC), on voltage-dependent K+ (Kv) channels in smooth muscle cells from freshly isolated rabbit coronary arteries by using the whole-cell patch clamp technique. YC-1 inhibited the Kv current in a dose-dependent fashion with an apparent Kd of 9.67 μ M. It accelerated the decay rate of Kv channel inactivation without altering the kinetics of current activation. The rate constants of association and dissociation for YC-1 were 0.36 ± 0.01 μ M −1·s−1 and 3.44 ± 0.22 s−1, respectively. YC-1 did not have a significant effect on the steady-state activation and inactivation curves. The recovery time constant from inactivation was decreased in the presence of YC-1, and application of train pulses (1 or 2 Hz) caused a progressive increase in the YC-1 blockade, indicating that YC-1–induced inhibition of Kv currents is use-dependent. Pretreatment with Bay 41-2272 (also a sGC activator), ODQ (a sGC inhibitor), or Rp-8-Br-PET-cGMPs (a protein kinase G inhibitor) did not affect the basal Kv current and also did not significantly alter the inhibitory effect of YC-1. From these results, we suggest that YC-1 directly inhibits the Kv current independently of sGC activation and in a state-, time-, and use-dependent fashion.

Ba2+ currents in inner and outer hair cells of mice lacking the voltage-dependent Ca2+ channel subunits β3 or β4

Voltage-activated Ca2+ channels comprise complexes of a pore-forming Cavα1 and auxiliary subunits Cavβ, Cavα2δ and sometimes Cavγ. The intracellular Cavβ subunit assists in trafficking and surface expression of the Cavα1 subunit and can modulate biophysical properties of the Ca2+ channel. Four genes, Cavβ1-4, exist which confer different properties to Ca2+ currents through the various Cavα1 subunits. Ca2+ currents in cochlear inner (IHC) and outer hair cells (OHC) serving synaptic transmission flow predominantly through the L type Cavα1 subunit Cav1.3, but associated Cavβ subunits are unknown. In the organ of Corti, we found mRNA and protein for all four Cavβ subunits including Cavβ2, but clear assignment of the Cavβ1 4 immunolabelling with hair cells or nerve fibers was difficult. We analyzed Cavβ3 knockout (Cavβ3 / ) and Cavβ4 mutant mice (Cavβ4lh/lh), which had normal hearing. Recording voltage-activated Ba2+ currents from hair cells of the two mouse models revealed distinct significant changes of cell size and Ba2+ current properties compared with their wildtype controls. Neonatal Cavβ4lh/lh IHCs showed reduced membrane capacitances and changes in the voltage dependence and kinetics of current activation, whereas mature IHCs had reduced peak currents compared with Cavβ4wt, altogether indicating the presence of Cavβ4 in IHCs. Ba2+ currents of Cavβ3 / OHCs showed largely reduced amplitudes, changes in the voltage dependence and kinetics of Ba2+ current activation, and increased inactivation compared with Cavβ3wt, pointing to a role of Cavβ3 for OHCs. These results indicate that neither Cavβ3 nor Cavβ4 are indispensable for hair cell Ca2+ currents but contribute to the overall current properties.

CAMP-dependent kinase does not modulate the Slack sodium-activated potassium channel

The Slack gene encodes a Na +-activated K + channel and is expressed in many different types of neurons. Like the prokaryotic Ca 2+-gated K + channel MthK, Slack contains two ‘regulator of K + conductance’ (RCK) domains within its carboxy terminal, domains likely involved in Na + binding and channel gating. It also contains multiple consensus protein kinase C (PKC) and protein kinase A (PKA) phosphorylation sites and although regulated by protein kinase C (PKC) phosphorylation, modulation by PKA has not been determined. To test if PKA directly regulates Slack, nystatin-perforated patch whole-cell currents were recorded from a human embryonic kidney (HEK-293) cell line stably expressing Slack. Bath application of forskolin, an adenylate cyclase activator, caused a rapid and complete inhibition of Slack currents however, the inactive homolog of forskolin, 1,9-dideoxyforskolin caused a similar effect. In contrast, bath application of 8-bromo-cAMP did not affect the amplitude nor the activation kinetics of Slack currents. In excised inside-out patch recordings, direct application of the PKA catalytic subunit to patches did not affect the open probability of Slack channels nor was open probability affected by direct application of protein phosphatase 2B. Preincubation of cells with the protein kinase A inhibitor KT5720 also did not change current density. Finally, mutating the consensus phosphorylation site located between RCK domain 1 and domain 2 from serine to glutamate did not affect current activation kinetics. We conclude that unlike PKC, phosphorylation by PKA does not acutely modulate the function and gating activation kinetics of Slack channels.

Effect of psoralen on the cloned Kv3.1 currents

The psoralen, a furocoumarin derivative, on the cloned neuronal rat Kv3.1 channels stably expressed in Chinese hamster ovary cells was investigated using the whole-cell patch-clamp technique. Psoralen reduced Kv3.1 whole-cell currents in a reversible concentration-dependent manner, with an IC50 value and a Hill coefficient of 2.3 +/- 0.03 microM and 0.9 +/- 0.08, respectively. Psoralen accelerated the decay rate of inactivation of Kv3.1 currents without modifying the kinetics of current activation. The psoralen-induced inhibition of Kv3.1 channels was voltage-dependent, with a steep increase over the voltage range of channel opening. However, the inhibition exhibited voltage independence over the voltage range in which channels are fully activated. Psoralen slowed the deactivation time course, resulting in a tail crossover phenomenon when the tail currents, recorded in the presence and absence of psoralen, were superimposed. Inhibition of Kv3.1 by psoralen was use-dependent at a frequency of 1 Hz. The present results suggest that psoralen acts on Kv3.1 currents as an open-channel blocker.

Soluble β-amyloid oligomers alter biophysical properties of Kv1.3 channels

The aggregation of amyloid peptides in brain tissue is a hallmark of Alzheimer's disease. Amyloid beta (Aβ) exists in several assembly states, which may play different physiological or pathophysiological roles. The effects of Aβ on voltage-dependent ion channels in neurons and microglia were implicated in early stages of neurodegeneration. We tested the effect of soluble oligomers (Aβ1-42) of amyloid precursor protein (APP) on voltage-dependent potassium channels Kv1.3. Potassium current was measured during whole-cell recording from L929 cells, stably expressing Kv1.3. Acute application of Aβ1-42 reversibly reduced peak current amplitudes and affected kinetics of current activation, inactivation and deactivation in a voltage- and a dose-dependent manner. The time constant of K+ current activation during depolarizing voltage pulses from the holding membrane potential of −80 mV was reduced by Aβ1-42 at low (<10 mV) voltages, but was not changed at higher voltages. Slow C-type inactivation of K+ current was significantly faster in the presence of Aβ1-42 with the effect being most prominent at −20 mV (lowest voltage measured) and diminishing with increasing voltage. The time constant of K+ current deactivation was significantly reduced by Aβ1-42, and the effect progressively increased with voltage increase. Under the same conditions, the voltage sensitivity of Kv1.3 conductance was not significantly changed by Aβ1-42. Our results reveal acute effects of biologically active soluble β-amyloid oligomers on voltage-dependent potassium channels Kv1.3. Faster inactivation and deactivation of K+ current in the presence of Aβ could impair regulation of the membrane potential, ultimately leading to pathophysiological changes in the cell.Supported by the Hillblom Foundation.

Calcium current kinetics in young and aged human cultured myotubes

There is evidence that the complex process of sarcopenia in human aged skeletal muscle is linked to the modification of mechanisms controlling Ca(2+) homeostasis. To further clarify this issue, we assessed the changes in the kinetics of activation and inactivation of T- and L-type Ca(2+) currents in in vitro differentiated human myotubes, derived from satellite cells of healthy donors aged 2, 12, 76 and 86 years. The results showed an age-related decrease in the occurrence of T- and L-type currents. Moreover, significant age-dependent alterations were found in L-(but not T) type current density, and activation and inactivation kinetics, although an interesting alteration in the kinetics of T-current inactivation was observed. The T- and L-type Ca(2+) currents play a crucial role in regulating Ca(2+) entry during satellite cells differentiation and fusion into myotubes. Also, the L-type Ca(2+) channels underlie the skeletal muscle excitation-contraction coupling mechanism. Thus, our results support the hypothesis that the aging process could negatively affect the Ca(2+) homeostasis of these cells, by altering Ca(2+) entry through T- and L-type Ca(2+) channels, thereby putting a strain on the ability of human satellite cells to regenerate skeletal muscle in elderly people.

Does the voltage‐gated calcium channel α2δ‐1 subunit play a dual function in skeletal muscle?

The dihydropyridine receptor (DHPR), a high voltage-activated calcium (Ca2+) channel, represents a key element in the excitation–contraction (EC) coupling machinery of muscle cells. In skeletal muscle, the DHPR fulfils two known functions: (i) it controls the ryanodine receptor activity, which makes it commonly referred to as the voltage-sensor for EC coupling and (ii) it supports an L-type voltage-activated Ca2+ current. The DHPR is a macromolecular complex composed of the Cav1.1 pore-forming subunit, surrounded by β1a, γ1 and α2δ-1 auxiliary subunits. Whereas knock-out animals have clearly demonstrated the essential role of Cav1.1 and β1a subunits in EC coupling and in the control of the Ca2+ conductance, the functional importance of α2δ-1 in skeletal muscle remains largely elusive, despite the lethal phenotype of α2δ-1-null embryos. Interestingly, down-regulation of the α2δ-1 protein in the BC3H1 muscle cell line with siRNA only resulted in acceleration of Ca2+ current activation kinetics with no effect on both EC coupling and Cav1.1 trafficking (Obermair et al. 2005). However, it is hard to understand how alteration of Ca2+ current kinetics could be the cause of the lethal phenotype observed in α2δ-1-null mice embryos; instead α2δ-1 is most likely to be involved in other cell functions independent of the DHPR activity. Previous observations suggested such a function: (i) α2δ-1 appears early during muscle development, at a period when the Cav1.1 subunit is not yet expressed, and (ii) sequence analysis of α2δ-1 revealed the presence of consensus domains, known to be involved in cell adhesion and protein–protein interactions (see Fig. 1 for structural organization of the α2δ-1 subunit). In a recent issue of The Journal of Physiology, Garcia et al. raised the possibility that α2δ-1 could mediate the interaction of muscle cells with the extracellular matrix (Garcia et al. 2008). Using immunohistochemistry and a siRNA knock-down strategy, the authors provided new insights into the functional involvement of α2δ-1 in skeletal muscle cells.

Endotoxin reduces availability of voltage-gated human skeletal muscle sodium channels at depolarized membrane potentials*

Critical illness myopathy is a common cause for difficulties in weaning from the respirator and prolonged rehabilitation of patients recovering from sepsis. Several studies have shown that the primary cause of acute generalized muscle weakness is loss of muscle membrane excitability. This study was designed to investigate a potential direct interaction of lipopolysaccharides from Escherichia coli with voltage-gated human skeletal muscle sodium channels (NaV1.4) in vitro. In vitro laboratory investigation. University laboratory. NaV1.4 sodium channel alpha-subunits stably expressed in human embryonic kidney (HEK293) cells. We investigated the effect of lipopolysaccharide on voltage-dependent sodium channel gating by using two distinct modes of application: 1) acute perfusion (pharmacologic lipopolysaccharide concentrations between 5 ng/mL and 50 microg/mL) in order to establish a concentration-effect relationship; and 2) incubation with a clinically relevant concentration of lipopolysaccharide (300 pg/mL). Lipopolysaccharide did not alter the kinetics of sodium current activation or inactivation when depolarizations were started from hyperpolarized holding potentials. However, when either fast or slow inactivation was induced by membrane depolarization before the test pulse, lipopolysaccharide reversibly reduced channel availability during the test pulse at concentrations of > or = 50 ng/mL revealed by a maximum hyperpolarizing shift of -25 mV in the voltage dependence of fast and slow inactivation, respectively. Incubation with a lipopolysaccharide concentration of 300 pg/mL for 1 hr reproduced the effects on slow but not on fast inactivation. After 20 hrs of low-dose lipopolysaccharide, the peak sodium current was significantly reduced. Our results show that lipopolysaccharide interacts with voltage-gated sodium channels, reducing channel availability at depolarized membrane potentials during acute application, independent of the membrane potential after chronic exposure. These effects may contribute to reduced muscle membrane excitability in sepsis.

Open Channel Block of Kv3.1 Currents by Fluoxetine

The action of fluoxetine, a serotonin reuptake inhibitor, on the cloned neuronal rat Kv3.1 channels stably expressed in Chinese hamster ovary cells was investigated using the whole-cell patch-clamp technique. Fluoxetine reduced Kv3.1 whole-cell currents in a reversible, concentration-dependent manner, with an IC50value and a Hill coefficient of 13.4 μ M and 1.4, respectively. Fluoxetine accelerated the decay rate of inactivation of Kv3.1 currents without modifying the kinetics of current activation. The inhibition increased steeply between 0 and +30 mV, which corresponded with the voltage range for channel opening. In the voltage range positive to +30 mV, inhibition displayed a weak voltage dependence, consistent with an electrical distance δ of 0.38. The binding (k+1) and dissociation (k−1) rate constants for fluoxetine-induced block of Kv3.1 were 5.7 μ M−1s−1and 53.5 s−1, respectively. The theoretical KDvalue derived by k− 1/k+1yielded 9.3 μ M. Fluoxetine did not affect the ion selectivity of Kv3.1. Fluoxetine slowed the deactivation time course, resulting in a tail crossover phenomenon when the tail currents, recorded in the presence and absence of fluoxetine, were superimposed. Inhibition of Kv3.1 by fluoxetine was use-dependent. The present results suggest that fluoxetine acts on Kv3.1 currents as an open-channel blocker.