Earl Gordon

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

Abstract The transient receptor potential vanilloid 4 (TRPV4) member of the TRP superfamily has recently been implicated in numerous physiological processes. Here we describe a small molecule TRPV4 channel activator, GSK1016790A, which we have utilized as a valuable tool in investigating the role of TRPV4 in the urinary bladder. GSK1016790A elicited Ca 2+ influx in mouse and human TRPV4 expressing HEK cells (EC 50 values of 18 and 2.1 nM, respectively), and 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 TRPV4The 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.

GSK1016790A, a Novel and Potent TRPV4 Channel Agonist Induces Urinary Bladder Contraction and Hyperactivity: Part I

Abstract The transient receptor potential vanilloid 4 (TRPV4) member of the TRP superfamily has recently been implicated in numerous physiological processes. Here we describe a small molecule TRPV4 channel activator, GSK1016790A, which we have utilized as a valuable tool in investigating the role of TRPV4 in the urinary bladder. GSK1016790A elicited Ca 2+ influx in mouse and human TRPV4 expressing HEK cells (EC 50 values of 18 and 2.1 nM, respectively), and 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 TRPV4The 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.

RNA interference reveals that endogenous Xenopus MinK-related peptides govern mammalian K+ channel function in oocyte 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.

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 (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.

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 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.

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 (α subunit, α+β1 and α+β4 subunits). HMIMP blocked all of these channels with an IC50 of ∼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 NaV1.5 (1 μM), CaV3.2, L-type Ca2+, 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.

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 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.