Single LED visible light positioning system based on image sensor and calculated azimuth angle.

A single-lamp visible light positioning (VLP) system based on an optical camera is proposed and demonstrated. The system uses a circular light-emitting diode (LED) to transmit ID data, and a common smartphone camera for positioning. First, we propose a scheme to calculate the azimuth angle using a marked lamp bead, avoiding the use of a magnetometer, which can improve the accuracy of the azimuth angle. Second, we propose a new positioning method using a chord in the image plane, which passes through the midpoints of two chords parallel to the LED plane. The experimental results show that when the experimental area is 1.8m×3.6m, the three-dimensional (3D) average positioning errors are 8.0, 7.5, and 7.2 cm for the heights of 1.75, 1.45, and 1.2 m.

Human-induced pluripotent stem cell-derived cardiomyocytes exhibit temporal changes in phenotype.

Human-induced pluripotent stem cell-derived cardiomyocytes (hiPS-CMs) have been recently derived and are used for basic research, cardiotoxicity assessment, and phenotypic screening. However, the hiPS-CM phenotype is dependent on their derivation, age, and culture conditions, and there is disagreement as to what constitutes a functional hiPS-CM. The aim of the present study is to characterize the temporal changes in hiPS-CM phenotype by examining five determinants of cardiomyocyte function: gene expression, ion channel functionality, calcium cycling, metabolic activity, and responsiveness to cardioactive compounds. Based on both gene expression and electrophysiological properties, at day 30 of differentiation, hiPS-CMs are immature cells that, with time in culture, progressively develop a more mature phenotype without signs of dedifferentiation. This phenotype is characterized by adult-like gene expression patterns, action potentials exhibiting ventricular atrial and nodal properties, coordinated calcium cycling and beating, suggesting the formation of a functional syncytium. Pharmacological responses to pathological (endothelin-1), physiological (IGF-1), and autonomic (isoproterenol) stimuli similar to those characteristic of isolated adult cardiac myocytes are present in maturing hiPS-CMs. In addition, thyroid hormone treatment of hiPS-CMs attenuated the fetal gene expression in favor of a more adult-like pattern. Overall, hiPS-CMs progressively acquire functionality when maintained in culture for a prolonged period of time. The description of this evolving phenotype helps to identify optimal use of hiPS-CMs for a range of research applications.

Characterizing the role of Thr352 in the inhibition of the large conductance Ca2+-activated K+ channels by 1-[1-hexyl-6-(methyloxy)-1H-indazol-3-yl]-2-methyl-1-propanone.

Large conductance Ca(2+)-activated K(+) (BK) channels are known to be regulated by both intracellular Ca(2+) and voltage. Although BK channel modulators have been identified, there is a paucity of information regarding the molecular entities of this channel that govern interaction with blockers and activators. Using both whole-cell and single-channel electrophysiological studies we have characterized the possible role that a threonine residue in the pore region of the channel has on function and interaction with BK channel modulators. A threonine-to-serine substitution at position 352 (T352S) resulted in a 59-mV leftward shift in the voltage-dependent activation curve. Single-channel conductance was 236 pS for the wild-type channel and 100 pS for the T352S mutant, measured over the range -80 mV to +80 mV. In addition, there was an almost 10-fold reduction in the potency of the BK channel inhibitor 1-[1-hexyl-6-(methyloxy)-1H-indazol-3-yl]-2-methyl-1-propanone (HMIMP), the IC(50) values being 4.3 +/- 0.3 and 38.2 +/- 3.3 nM for wild-type and mutant channel, respectively. There was no significant difference between wild type and the mutant channel in response to inhibition by iberiotoxin. The IC(50) was 8.1 +/- 0.3 nM for the wild type and 7.7 +/- 0.3 nM for the mutant channel. Here, we have identified a residue in the pore region of the BK channel that alters voltage sensitivity and reduces the potency of the blocker HMIMP.

2-[2-(3,4-dichloro-phenyl)-2,3-dihydro-1H-isoindol-5-ylamino]-nicotinic acid (PD-307243) causes instantaneous current through human ether-a-go-go-related gene potassium channels.

Long and short QT syndromes associated with loss and gain of human ether-a-go-go-related gene (hERG) channel activity, respectively, can cause life-threatening arrhythmias. As such, modulation of hERG channel activity is an important consideration in the development of all new therapeutic agents. In the present study, we investigated the mechanisms of action of 2-[2-(3,4-dichloro-phenyl)-2,3-dihydro-1H-isoindol-5-ylamino]-nicotinic acid (PD-307243), a known hERG channel activator, on hERG channels stably expressed in Chinese hamster ovary (CHO) cells using the patch-clamp technique. In the whole-cell recordings, the extracellular application of PD-307243 concentration-dependently increased the hERG current and markedly slowed hERG channel deactivation and inactivation. PD-307243 had no effect on the selectivity filter of hERG channels. The activity of PD-307243 was use-dependent. PD-307243 (3 and 10 muM) induced instantaneous hERG current with little decay at membrane potentials from -120 to -40 mV. At more positive voltages, PD-307243 induced an I(to)-like upstroke of hERG current. The actions of PD-307243 on the rapid component of delayed rectifier K(+) current (I(Kr)) in rabbit ventricular myocytes were similar to those observed in hERG channel-transfected CHO cells. Inside-out patch experiments revealed that PD-307243 increased hERG tail currents by 2.1 +/- 0.6 (n = 7) and 3.4 +/- 0.3-fold (n = 4) at 3 and 10 muM, respectively, by slowing the channel deactivation but had no effect on channel activation. During a voltage-clamp protocol using a prerecorded cardiac action potential, 3 muM PD-307243 increased the total potassium ions passed through hERG channels by 8.8 +/- 1.0-fold (n = 5). Docking studies suggest that PD-307243 interacts with residues in the S5-P region of the channel.

A critical GxxxA motif in the gamma6 calcium channel subunit mediates its inhibitory effect on Cav3.1 calcium current.

The eight members of the calcium channel gamma subunit family are integral membrane proteins that regulate the expression and behaviour of voltage and ligand gated ion channels. While a subgroup consisting of gamma(2), gamma(3), gamma(4) and gamma(8) (the TARPs) modulate AMPA receptor localization and function, the gamma(1) and gamma(6) subunits conform to the original description of these proteins as regulators of voltage gated calcium channels. We have previously shown that the gamma(6) subunit is highly expressed in atrial myocytes and that it is capable of acting as a negative modulator of low voltage activated calcium current. In this study we extend our understanding of gamma(6) subunit modulation of low voltage activated calcium current. Using engineered chimeric constructs, we demonstrate that the first transmembrane domain (TM1) of gamma(6) is necessary for its inhibitory effect on Cav3.1 current. Mutational analysis is then used to identify a unique GxxxA motif within TM1 that is required for the function of the subunit strongly suggesting the involvement of helix-helix interactions in its effects. Results from co-immunoprecipitation experiments confirm a physical association of gamma(6) with the Cav3.1 channel in both HEK cells and atrial myocytes. Single channel analysis reveals that binding of gamma(6) reduces channel availability for activation. Taken together, the results of this study provide both a molecular and a mechanistic framework for understanding the unique ability of the gamma(6) calcium channel subunit to modulate low voltage activated (Cav3.1) calcium current density.

1-[1-Hexyl-6-(methyloxy)-1H-indazol-3-yl]-2-methyl-1-propanone, a potent and highly selective small molecule blocker of the large-conductance voltage-gated and calcium-dependent K+ channel.

The large-conductance voltage-gated and calcium-dependent K(+) (BK) channels are widely distributed and play important physiological roles. Commonly used BK channel inhibitors are peptide toxins that are isolated from scorpion venoms. A high-affinity, nonpeptide, synthesized BK channel blocker with selectivity against other ion channels has not been reported. We prepared several compounds from a published patent application (Doherty et al., 2004) and identified 1-[1-hexyl-6-(methyloxy)-1H-indazol-3-yl]-2-methyl-1-propanone (HMIMP) as a potent and selective BK channel blocker. The patch-clamp technique was used for characterizing the activity of HMIMP on recombinant human BK channels (alpha subunit, alpha+beta1 and alpha+beta4 subunits). HMIMP blocked all of these channels with an IC(50) of approximately 2 nM. The inhibitory effect of HMIMP was not voltage-dependent, nor did it require opening of BK channels. HMIMP also potently blocked BK channels in freshly isolated detrusor smooth muscle cells and vagal neurons. HMIMP (10 nM) reduced the open probability significantly without affecting single BK-channel current in inside-out patches. HMIMP did not change the time constant of open states but increased the time constants of the closed states. More importantly, HMIMP was highly selective for the BK channel. HMIMP had no effect on human Na(V)1.5 (1 microM), Ca(V)3.2, L-type Ca(2+), human ether-a-go-go-related gene potassium channel, KCNQ1+minK, transient outward K(+) or voltage-dependent K(+) channels (100 nM). HMIMP did not change the action potentials of ventricular myocytes, confirming its lack of effect on cardiac ion channels. In summary, HMIMP is a highly potent and selective BK channel blocker, which can serve as an important tool in the pharmacological study of the BK channel.

N-((1S)-1-{[4-((2S)-2-{[(2,4-dichlorophenyl)sulfonyl]amino}-3-hydroxypropanoyl)-1-piperazinyl]carbonyl}-3-methylbutyl)-1-benzothiophene-2-carboxamide (GSK1016790A), a novel and potent transient receptor potential vanilloid 4 channel agonist induces urinary bladder contraction and hyperactivity: Part I.

The transient receptor potential (TRP) vanilloid 4 (TRPV4) member of the TRP superfamily has recently been implicated in numerous physiological processes. In this study, we describe a small molecule TRPV4 channel activator, (N-((1S)-1-{[4-((2S)-2-{[(2,4-dichlorophenyl)sulfonyl]amino}-3-hydroxypropanoyl)-1-piperazinyl]carbonyl}-3-methylbutyl)-1-benzothiophene-2-carboxamide (GSK1016790A), which we have used as a valuable tool in investigating the role of TRPV4 in the urinary bladder. GSK1016790A elicited Ca2+ influx in mouse and human TRPV4-expressing human embryonic kidney (HEK) cells (EC50 values of 18 and 2.1 nM, respectively), and it evoked a dose-dependent activation of TRPV4 whole-cell currents at concentrations above 1 nM. In contrast, the TRPV4 activator 4alpha-phorbol 12,13-didecanoate (4alpha-PDD) was 300-fold less potent than GSK1016790A in activating TRPV4 currents. TRPV4 mRNA was detected in urinary bladder smooth muscle (UBSM) and urothelium of TRPV4+/+ mouse bladders. Western blotting and immunohistochemistry demonstrated protein expression in both the UBSM and urothelium that was absent in TRPV4-/- bladders. TRPV4 activation with GSK1016790A contracted TRPV4+/+ mouse bladders in vitro, both in the presence and absence of the urothelium, an effect that was undetected in TRPV4-/- bladders. Consistent with the effects on TRPV4 HEK whole-cell currents, 4alpha-PDD demonstrated a weak ability to contract bladder strips compared with GSK1016790A. In vivo, urodynamics in TRPV4+/+ and TRPV4-/- mice revealed an enhanced bladder capacity in the TRPV4-/- mice. Infusion of GSK1016790A into the bladders of TRPV4+/+ mice induced bladder overactivity with no effect in TRPV4-/- mice. Overall TRPV4 plays an important role in urinary bladder function that includes an ability to contract the bladder as a result of the expression of TRPV4 in the UBSM.

Systemic activation of the transient receptor potential vanilloid subtype 4 channel causes endothelial failure and circulatory collapse: Part 2.

The transient receptor potential (TRP) vanilloid subtype 4 (V4) is a nonselective cation channel that exhibits polymodal activation and is expressed in the endothelium, where it contributes to intracellular Ca2+ homeostasis and regulation of cell volume. The purpose of the present study was to evaluate the systemic cardiovascular effects of GSK1016790A, a novel TRPV4 activator, and to examine its mechanism of action. In three species (mouse, rat, and dog), the i.v. administration of GSK1016790A induced a dose-dependent reduction in blood pressure, followed by profound circulatory collapse. In contrast, GSK1016790A had no acute cardiovascular effects in the TRPV4-/- null mouse. Hemodynamic analyses in the dog and rat demonstrate a profound reduction in cardiac output. However, GSK1016790A had no effect on rate or contractility in the isolated, buffer-perfused rat heart, and it produced potent endothelial-dependent relaxation of rodent-isolated vascular ring segments that were abolished by nitric-oxide synthase (NOS) inhibition (N-nitro-L-arginine methyl ester; L-NAME), ruthenium red, and endothelial NOS (eNOS) gene deletion. However, the in vivo circulatory collapse was not altered by NOS inhibition (L-NAME) or eNOS gene deletion but was associated with (concentration and time appropriate) profound vascular leakage and tissue hemorrhage in the lung, intestine, and kidney. TRPV4 immunoreactivity was localized in the endothelium and epithelium in the affected organs. GSK1016790A potently induced rapid electrophysiological and morphological changes (retraction/condensation) in cultured endothelial cells. In summary, inappropriate activation of TRPV4 produces acute circulatory collapse associated with endothelial activation/injury and failure of the pulmonary microvascular permeability barrier. It will be important to determine the role of TRPV4 in disorders associated with edema and microvascular congestion.

Acetic acid opens large-conductance Ca2+-activated K+ channels in guinea pig detrusor smooth muscle cells.

Acetic acid was found to have actions on urinary bladder smooth muscle in our routine ion channel screening assays. Numerous studies have examined the mechanisms of bladder irritation by acetic acid; however, the direct effect of acetic acid on ion channels in detrusor smooth muscle cells has not been evaluated. We used whole-cell patch-clamp techniques to examine the effect of acetic acid on large-conductance Ca2+-activated K+ channels (BKCa) from guinea pig detrusor smooth muscle cells and CHO cells expressing recombinant human BKCaalphabeta1 (CHO BKCaalphabeta1) and human BKCaalpha (CHO BKCaalpha). Acetic acid activated BKCa currents in a concentration-dependent (0.01% to 0.05% v/v) manner in all the cell systems studied. Acetic acid (0.05%) increased BKCa current at +30 mV by 2764+/-918% (n=8) in guinea pig detrusor smooth muscle cells. Acetic acid (0.03%) shifted the V1/2 of conductance-voltage curve by 64+/-14 (n=5), 128+/-14 (n=5), and 126+/-12 mV (n=4) in CHO BKCaalpha, CHO BKCaalphabeta1 and detrusor smooth muscle cells, respectively. This effect of acetic acid was found to be independent of pH and was also not produced by its salt form, sodium acetate. Automated patch-clamp experiments also showed similar activation of CHO BKCaalphabeta1 by acetic acid. In conclusion, acetic acid directly activates BKCa channels in detrusor smooth muscle cells. This novel study necessitates caution while interpreting the results from acetic acid bladder irritation model.

Functional TRPV4 channels and an absence of capsaicin-evoked currents in freshly-isolated, guinea-pig urothelial cells.

Previously we have shown that the transient receptor potential vanilloid 4 (TRPV4) channel regulates urinary bladder function, and that TRPV4 is expressed in both smooth muscle and urothelial cell types within the bladder wall.(1) Urothelial cells have also been suggested to express TRPV1 channels.(2) Therefore, we enzymatically isolated guinea-pig urothelial cells in an attempt to record TRPV4 and TRPV1-mediated currents. The identity of the isolated cells was confirmed by quantitative PCR for the urothelial marker uroplakin 1A. Whole-cell patch-clamp recordings with the TRPV4 agonist, GSK1016790A, activated urothelial currents with an EC(50) of 11 nM that were completely inhibited by the TRPV4 inhibitor ruthenium red (5 microM). Urothelial currents were also activated by challenge with hypotonic extracellular solution (220 mOsm) known to activate TRPV4 channels. However, the TRPV1 agonist capsaicin, which activated TRPV1 currents in HEK cells expressing TRPV1, was unable to evoke current in these freshly isolated guinea-pig urothelial cells. We demonstrate that TRPV4 channels are functionally expressed at the plasma membrane of freshly isolated, guinea-pig urothelial cells, further supporting the important role of TRPV4 in urinary bladder physiology.

Mallotoxin is a novel human ether-a-go-go-related gene (hERG) potassium channel activator.

Human ether-a-go-go-related gene (hERG) encodes a rapidly activating delayed rectifier potassium channel that plays important roles in cardiac action potential repolarization. Although many drugs and compounds block hERG channels, activators of the channel have only recently been described. Three structurally diverse synthetic compounds have been reported to activate hERG channels by altering deactivation or inactivation or by unidentified mechanisms. Here, we describe a novel, naturally occurring hERG channel activator, mallotoxin (MTX). The effects of MTX on hERG channels were investigated using the patch-clamp technique. MTX increased both step and tail hERG currents with EC(50) values of 0.34 and 0.52 microM, respectively. MTX leftward shifted the voltage dependence of hERG channel activation to less depolarized voltages ( approximately 24 mV at 2.5 microM). In addition, MTX increased hERG deactivation time constants. MTX did not change the half-maximal inactivation voltage of the hERG channel, but it reduced the slope of the voltage-dependent inactivation curve. All of these factors contribute to the enhanced activity of hERG channels. During a voltage-clamp protocol using prerecorded cardiac action potentials, 2.5 microM MTX increased the total potassium ions passed through hERG channels by approximately 5-fold. In conclusion, MTX activates hERG channels through distinct mechanisms and with significantly higher potency than previously reported hERG channel activators.