Myosin light chain kinase-independent inhibition by ML-9 of murine TRPC6 channels expressed in HEK293 cells

Abstract

Background and purpose: Myosin light chain kinase (MLCK) plays a pivotal role in regulation of cellular functions, the evidence often relying on the effects of extracelluarly administered drugs such as ML-9. Here we report that this compound exerts non-specific inhibitory actions on the TRPC6 channel, a transient receptor potential (TRP) protein.

Experimental approach: Macroscopic and single channel currents were recorded from transfected HEK293 cells by patch-clamp techniques.

Key results: Cationic currents elicited by carbachol (CCh; 100 microM) in HEK293 cells overexpressing murine TRPC6 (I(TRPC6)) were dose-dependently inhibited by externally applied ML-9 (IC(50)=7.8 microM). This inhibition was voltage-dependent and occurred as fast as external Na(+) removal. Another MLCK inhibitor, wortmannin (3 microM), and MLCK inhibitory peptides MLCK-IP(11-19) (10 microM) and -IP(480-501) (1 microM) showed little effects on I(TRPC6) density and the inhibitory efficacy of ML-9. The extent of the inhibition also unchanged with co-expression of wild-type or a dominant negative mutant of MLCK. Inhibitory effects of ML-9 on I(TRPC6) remained unaffected whether TRPC6 was activated constitutively or by a diacylglycerol analogue OAG (100 microM). Similar rapid inhibition was also observed with a ML-9 relative, ML-7. Intracellular perfusion of ML-9 via patch pipette, dose-dependently suppressed I(TRPC6). In inside-out patch configuration, bath application of ML-9 (and ML-7) rapidly diminished approximately 35pS single TRPC6 channel activities. Contrarily, currents due to TRPC7 expression were rapidly enhanced by externally applied ML-9 and ML-7, which was not prevented by MLCK inhibitory peptides.

Conclusion and implications: These results strongly suggest that ML compounds inhibit TRPC6 channels via a mechanism independent of inhibition of MLCK activity.

Figure 1

Inhibitory effects of ML-9 on TRPC6 current (I_TRPC6). Nystatin-perforated recording in normal external solution with a holding potential: −60 mV. (a) Typical traces for the inhibitory effects of ML-9 of 10 μM (upper) and 100 μM (lower) on CCh (100 μM)-evoked I_TRPC6. (b) Relationship between ML-9 concentration and relative I_TRPC6 amplitude after inhibition. Relative amplitude is calculated as the ratio of current amplitudes 5 s after, to just before, application of ML-9. Data points are fitted by the Hill equation with non-linear least-square routine (IC₅₀=7.8 μM, n_H=1.0, n=4–6; see the Methods). (c) Expanded time courses of the effects of Na⁺ removal (NMDG substitution) and 100 μM ML-9 on I_TRPC6. Representative of separate six experiments. Each current trace is normalized to its amplitude just before drug application and then superimposed. The time courses of the currents are fitted by an exponential function: I=I₀ exp(−(t−t₀)/τ)+C (smooth curves), where I₀, t₀, τ and C denote the amplitude of drug-inhibited component, the onset of drug application, time constant of drug's effect and the steady level reached after drug application, respectively, and I₀+C is set to be −1. τ Values (in seconds) in the figure indicate the results of best fit by non-linear least-square routine. CCh, carbachol; ML-9, [1-(5-chloronaphthalene-1-sulphonyl)homopiperazine, HCl]; NMDG, N-methyl D-glucamine.

Figure 2

Voltage-dependency of ML-9's effects on I_TRPC6. (a) Current–voltage (I–V) relationships of I_TRPC6 before (bold curve) and during (dotted curve) application of 10 μM ML-9. These curves were constructed by applying a 2 s rising ramp voltage, which can be seen as short vertical deflections in Figure 1a. (b) Voltage-dependent effects of ML-9. The fraction of I_TRPC6 remaining in the presence of 10 μM ML-9 is calculated from the I–V curves in (a), and plotted against the membrane potential. Values near the reversal potential are omitted because of large scattering due to ‘division by zero'. (c) The fractions of I_TRPC6 remaining after ML-9 inhibition at −100 and 100 mV averaged from seven different experiments. Paired t-test (n=7). I_TRPC6, TRPC6 current; ML-9, [1-(5-chloronaphthalene-1-sulphonyl)homopiperazine, HCl].

Figure 3

Wortmannin was ineffective on I_TRPC6. Nystatin-perforated recording at −60 mV. (a) Actual trace of I_TRPC6. Wortmannin (WT) (3 μM) was introduced into the bath 5–10 min before application of CCh. In this experiment, WT was further applied after activation of I_TRPC6 by CCh (100 μM), but no discernible change was observed. (b) Current density of I_TRPC6 with and without pretreatment with 3 μM WT. P>0.05 with unpaired t-test (n=6). CCh, carbachol; I_TRPC6, TRPC6 current.

Figure 4

Intracellular perfusion of MLCK-inhibitory peptides and dominant negative inhibition of MLCK did not affect the inhibitory efficacy of ML-9 on I_TRPC6. Conventional whole-cell recording at −60 mV. To reduce the degree of desensitization, Ca²⁺-free rather than normal external solution was used as the bathing solution. (a) Representative trace for the extracellular ML-9 (10 μM) effects on I_TRPC6 in the presence of 10 μM MLCK IP_11–19 peptide in the pipette. (b) Summary of the effects of 10 μM MLCK IP_11–19 and 1 μM MLCK IP_480–501 on I_TRPC6 density (n=7–11). (c) ML-9 concentration–inhibition curves for I_TRPC6 with (solid) and without (nystatin-perforated recording; dotted; taken from Figure 1b) inclusion of MLCK inhibitory peptides (10 μM MLCK IP_11–19 or 1 μM MLCK IP_480–501). The solid curve is drawn according to the best fit of the data points with Hill equation (see Methods). IC₅₀=10.1 μM; n_H=1.0 (n=4–6). There was no statistically significant difference between two curves with ANOVA. (d and e) Influence of overexpression of wild type (wt-MLCK) or a dominant-negative mutant of MLCK (mut-MLCK) on current density of I_TRPC6 (d; n=15–18) and inhibitory efficacy of ML-9 for I_TRPC6 (e; n=8–11). (d) ^*P<0.05 from other columns with ANOVA and Bonferroni's t-test. ANOVA, analysis of variance; IP, inhibitory peptides; ML-9, [1-(5-chloronaphthalene-1-sulphonyl)homopiperazine, HCl]; MLCK, myosin light chain kinase.

Figure 5

Activation mode of TRPC6 did not affect the efficacy of ML-9 and ML-7. Conventional whole-cell recording at −60 mV with Ca²⁺-free external solution. (a) Representative traces showing the effects of ML-9 and ML-7 on basal (upper) and OAG (100 μM; lower)-induced I_TRPC6. (b and c) Summary of the effects of ML-9 and ML-7 on basal I_TRPC6 at 10 μM (b) and on OAG-induced I_TRPC6 at three different concentrations (1, 10, 100 μM) (c). n=6–7. (d) Inhibitory efficacy of ML-7 for I_TRPC6 with overexpression of wild type (wt-MLCK) or a dominant negative mutant of MLCK (mut-MLCK). n=8–11. ^*P<0.05. ML-7, [1-(5-iodonaphthalene-1-sulphonyl)homopiperazine, HCl]; ML-9, [1-(5-chloronaphthalene-1-sulphonyl)homopiperazine, HCl]; MLCK, myosin light chain kinase.

Figure 6

Concentration-dependent inhibition of I_TRPC6 by intracellularly applied ML-9. ML-9 was included in the pipette under conventional whole-cell clamp (−60 mV), with bath containing Ca²⁺-free external solution. (a) Typical traces of I_TRPC6 with intrapipette inclusion of 10 μM ML-9, which abolished the inhibitory effect of extracellularly applied ML-9 at the same concentration (upper), and of 30 μM ML-9, which strongly inhibited I_TRPC6 (lower). (b) Summary of the effects of intracellularly applied ML-9 on I_TRPC6 density. ^*P<0.05 with ANOVA and Bonferroni's t-test. n=5–8. ANOVA, analysis of variance; ML-9, [1-(5-chloronaphthalene-1-sulphonyl)homopiperazine, HCl].

Figure 7

Effects of ML-9 and ML-7 on single TRPC6 channels. Inside-out (I/O) patch recording with CCh (100 μM) in the pipette. To suppress K⁺ and Cl⁻ channels, 5 mM tetraethylammonium and 100 μM DIDS were also added in the pipette. (a) Actual traces at three different potentials (−50, 0 and 50 mV). Boxes on the right indicate all-points-in-open-state amplitude histograms with the results of Gaussian fit (smooth curves). (b) I–V relationship for single TRPC6 channel. Linear regression of data points between −50 and 50 mV gives a unitary conductance of 33.4 pS (n=5–8), which is in good accordance with the value obtained previously (Shi et al., 2004). (c) Inhibition of single TRPC6 channel activity by 10 μM ML-9, which was applied rapidly into the bath at the bar. Two large vertical deflections indicate the electrical artefacts of a solenoid valve. (d) Summary of the inhibitory effects of 10 μM ML-9 and ML-7 on single TRPC6 channels. The extent of single-channel current inhibition by ML compounds was calculated as the ratio of mean currents (averaged for 2 s) 5 s after, to just before, application of the drugs. n=8–11. CCh, carbachol; DIDS, [4,4-diisothiocyanostilbene-2,2-disulphonic acid, 2Na]; ML-7, [1-(5-iodonaphthalene-1-sulphonyl)homopiperazine, HCl]; ML-9, [1-(5-chloronaphthalene-1-sulphonyl)homopiperazine, HCl].

Figure 8

Enhancing actions of ML-compounds on macroscopic TRPC7 current (I_TRPC7). Conventional whole-cell recording at −60 mV with Ca²⁺-free external solution. (a) Representative records of basal (upper) and CCh-induced (lower) I_TRPC7 demonstrating the enhancing effect of ML-9 and ML-7. (b) Fractional change in I_TRPC7 amplitude after rapid application of ML-9 and ML-7. n=6–8. (c) Ineffectiveness of MLCK inhibitory peptide (MLCK-IP_480–501=1 μM) on the effects of extracellular ML-9 and ML-7 on CCh (100 μM)-induced I_TRPC7. n=5–6. CCh, carbachol; IP, inhibitory peptides; ML-7, [1-(5-iodonaphthalene-1-sulphonyl)homopiperazine, HCl]; ML-9, [1-(5-chloronaphthalene-1-sulphonyl)homopiperazine, HCl]; MLCK, myosin light chain kinase.