Pulmonary edema resulting from high pulmonary venous pressure (PVP) is a major cause of morbidity and mortality in heart failure (HF) patients, but current treatment options demonstrate substantial limitations. Recent evidence from rodent lungs suggests that PVP-induced edema is driven by activation of pulmonary capillary endothelial transient receptor potential vanilloid 4 (TRPV4) channels. To examine the therapeutic potential of this mechanism, we evaluated TRPV4 expression in human congestive HF lungs and developed small-molecule TRPV4 channel blockers for testing in animal models of HF. TRPV4 immunolabeling of human lung sections demonstrated expression of TRPV4 in the pulmonary vasculature that was enhanced in sections from HF patients compared to controls. GSK2193874 was identified as a selective, orally active TRPV4 blocker that inhibits Ca2+ influx through recombinant TRPV4 channels and native endothelial TRPV4 currents. In isolated rodent and canine lungs, TRPV4 blockade prevented the increased vascular permeability and resultant pulmonary edema associated with elevated PVP. Furthermore, in both acute and chronic HF models, GSK2193874 pretreatment inhibited the formation of pulmonary edema and enhanced arterial oxygenation. Finally, GSK2193874 treatment resolved pulmonary edema already established by myocardial infarction in mice. These findings identify a crucial role for TRPV4 in the formation of HF-induced pulmonary edema and suggest that TRPV4 blockade is a potential therapeutic strategy for HF patients.

Large conductance Ca2+-activated K+ (BK) channels are known to be regulated by both intracellular Ca2+ 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 IC50 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 IC50 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.

Large conductance Ca 2+ -activated K + (BK) channels are known to be regulated by both intracellular Ca 2+ and voltage. Though 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. Additionally, there was an almost 10-fold reduction in the potency of the BK channel inhibitor HMIMP, the IC 50 values being 4.3±0.3 nM 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.

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 (Thorneloe et al. 2008). Urothelial cells have also been suggested to express TRPV1 channels (Birder et al., 2001). 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 EC50 of 11 nM that were completely inhibited by the TRPV4 inhibitor ruthenium red (5 µM). 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.

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 4α-phorbol 12,13-didecanoate (4α-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, 4α-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.

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.

AIMS:Auditory stimulus-induced long QT syndrome (LQTS) is almost exclusively linked to mutations in the hERG potassium channel, which generates the I Kr ventricular repolarization current. Here, a young woman with prior episodes of auditory stimulus-induced syncope presented with LQTS and ventricular fibrillation (VF) with hypomagnesaemia and hypocalcaemia after completing a marathon, followed by subsequent VF with hypokalaemia. The patient was found to harbour a KCNE2 gene mutation encoding a T10M amino acid substitution in MiRP1, an ancillary subunit that co-assembles with and functionally modulates hERG. Other family members with the mutation were asymptomatic, and the proband had no mutations in hERG or other LQTS-linked cardiac ion channel genes. The T10M mutation was absent from 578 unrelated, ethnically matched control chromosomes analysed here and was previously described only once-in an LQTS patient-but not functionally characterized.METHODS AND RESULTS:T10M-MiRP1-hERG currents were assessed using whole-cell voltage clamp of transfected Chinese Hamster ovary cells. T10M-MiRP1-hERG channels showed <or=80% reduced tail current, left-shifted steady-state inactivation, and 50% slower recovery from inactivation when compared with wild-type channels, with mixed wild-type/T10M channels displaying an intermediate phenotype. Lowering bath K+ concentration reduced wild-type and T10M currents equivalently.CONCLUSION:Data suggest a mechanism for reduced penetrance, inherited arrhythmia in which baseline I Kr current reduction by the T10M mutation is exacerbated by superimposition of arrhythmogenic substrates such as auditory stimuli, or electrolyte disturbances that reduce I Kr (hypokalaemia) or otherwise lower the ventricular threshold for fibrillation (hypomagnesaemia and hypocalcaemia). This first example of a MiRP1 mutation associated with auditory stimulus-induced arrhythmia is supportive of the hypothesis that MiRP1 regulates hERG in the human heart.

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.

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 μM) induced instantaneous hERG current with little decay at membrane potentials from -120 to -40 mV. At more positive voltages, PD-307243 induced an Ito-like upstroke of hERG current. The actions of PD-307243 on the rapid component of delayed rectifier K+ current (IKr) 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 μM, 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 μM 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.

Voltage-gated potassium (Kv) channels are targets for therapeutic drugs in the treatment of pathologic conditions including cardiac arrhythmia and epilepsy. In this study, we synthesized three classes of novel polyammonium compounds incorporating the bicyclic unit 1,4-diazabicyclo[2.2.2]octane (DABCO) and tested their action on three representative mammalian Kv channels (Kv2.1, Kv3.4, and Kv4.2) expressed in Xenopus laevis oocytes. Nonsubstituted DABCO did not block the Kv channels tested. Simple DABCO monostrings and diDABCO strings inhibited Kv2.1 and Kv3.4 channels, with potency increasing with string length for both these DABCO classes. Both Kv2.1 and Kv3.4 were most sensitive to C16 monostrings, with IC50 values of 1.9 and 0.6 microM, respectively. For compounds comprising two DABCO groups separated by an aromatic ring, inhibition depended upon relative positioning of the two DABCO groups, and only the para form (JC638.2alpha) was active, blocking Kv2.1 with an IC50 of 186 microM. Kv4.2 channels were relatively insensitive to all compounds tested. Unlike the tetraethylammonium ion (TEA), neither JC638.2alpha nor C16 monostring TA279 produced block when applied intracellularly via the recording electrode to Kv2.1 channels expressed in Chinese hamster ovary cells, suggesting against an internal site of action. However, JC638.2alpha protected an introduced cysteine (K356C) in the Kv2.1 outer pore from permanent modification by methanethiosulfonate ethyltrimethylammonium (MTSET). These data suggest that JC638.2alpha occupies an external binding site similar to that of TEA in the Kv2.1 outer pore, but with much higher affinity than TEA. These DABCO salts represent a new class of Kv channel blockers, some with higher potencies than any previously described quaternary ammonium ions. The potential for synthesis of an array of modular derivatives suggests that DABCO compounds hold promise as probes of Kv channel structure and identity and as potential therapeutic agents.

Kv2.1 is a voltage-gated potassium (Kv) channel that generates delayed rectifier currents in mammalian heart and brain. The biophysical properties of Kv2.1 and other ion channels have been characterized by functional expression in heterologous systems, and most commonly in Xenopus laevis oocytes. A number of previous oocyte-based studies of mammalian potassium channels have revealed expression-level-dependent changes in channel properties, leading to the suggestion that endogenous oocyte factors regulate channel gating. Here, we show that endogenous oocyte potassium channel KCNE ancillary subunits xMinK and xMiRP2 slow the activation of oocyte-expressed mammalian Kv2.1 channels two-to-fourfold. This produces a sigmoidal relationship between Kv2.1 current density and activation rate in oocyte-based two-electrode voltage clamp studies. The effect of endogenous xMiRP2 and xMinK on Kv2.1 activation is diluted at high Kv2.1 expression levels, or by RNAi knockdown of either endogenous subunit. RNAi knockdown of both xMiRP2 and xMinK eliminates the correlation between Kv2.1 expression level and activation kinetics. The data demonstrate a molecular basis for expression-level-dependent changes in Kv channel gating observed in heterologous expression studies.

The physiological properties of most ion channels are defined experimentally by functional expression of their pore-forming α subunits in Xenopus laevis oocytes. Here, we cloned a family ofXenopus KCNE genes that encode MinK-related peptide K+ channel β subunits (xMiRPs) and demonstrated their constitutive expression in oocytes. Electrophysiological analysis of xMiRP2 revealed that when overexpressed this gene modulates human cardiac K+ channel α subunits HERG (human ether-a-go-go-related gene) and KCNQ1 by suppressing HERG currents and removing the voltage dependence of KCNQ1 activation. The ability of endogenous levels of xMiRP2 to contribute to the biophysical attributes of overexpressed mammalian K+ channels in oocyte studies was assessed next. Injection of an xMiRP2 sequence-specific short interfering RNA (siRNA) oligo reduced endogenous xMiRP2 expression 5-fold, whereas a control siRNA oligo had no effect, indicating the effectiveness of the RNA interference technique in Xenopus oocytes. The functional effects of endogenous xMiRP2 silencing were tested using electrophysiological analysis of heterologously expressed HERG channels. The RNA interference-mediated reduction of endogenous xMiRP2 expression increased macroscopic HERG current as much as 10-fold depending on HERG cRNA concentration. The functional effects of human MiRP1 (hMiRP1)/HERG interaction were also affected by endogenous xMiRP2. At high HERG channel density, at which the effects of endogenous xMiRP2 are minimal, hMiRP1 reduced HERG current. At low HERG current density, hMiRP1 paradoxically up-regulated HERG current, a result consistent with hMiRP1 rescuing HERG from suppression by endogenous xMiRP2. Thus, endogenous Xenopus MiRP subunits contribute to the base-line properties of K+ channels like HERG in oocyte expression studies, which could explain expression level- and expression system-dependent variation in K+channel function.