To test whether TRPC contributes to the MS channel in skeletal muscle, we exposed membrane patches to 1-oleoyl-2-acetyl-sn-glycerol (OAG, 100–200 µM) while recording single-channel activity. OAG activates TRPC1 and 3 channels in native and recombinant systems22-24
Exposure of membrane patches to OAG added to the patch electrode had no effect on the activity of MS channel activity recorded from cell-attached patches (25/25 patches, data not shown). OAG also had no detectable effects on single-channel activity when added directly to the bathing solution. The absence of an effect of OAG suggests the skeletal muscle MS channel is not composed of TRPC subunits.
shows the effects of a variety of TRP channel antagonists on the activity of single MS ion channels. Two aminoethoxydiphenyl borate (2-APB) blocks recombinant TRPC channels at low micromolar concentrations.26-29
In our recordings from membrane patches, 2-APB produced no discernible effect on MS channel activity in skeletal muscle at concentrations up to 1 mM in the electrode solution. The record at the top of shows a MS channel activity recorded in the presence of 500 µM 2-APB. The graph to the right of the figure is the distribution of current amplitudes plotted with the amplitude shown on the vertical axis. The record was obtained at a holding potential of –60 mV. At this potential, peak inward current was approximately 1.5–1.7 pA under control conditions in the absence of any blocker. The presence of 500 µM 2-APB had no effect on either the amplitude of the single-channel current nor on the apparent gating of the single channel over a wide range of concentrations. also shows that 200 µM of either spermidine or flufenamic acid had little effect on the activity of single MS channels. By contrast, shows that SKF-96365 and ruthenium red blocked the single-channel current. Block appeared as both a reduction in the amplitude of the single-channel current and a change in the gating transitions that occur within the open channel. Subsequent experiments examined the mechanism of block of MS channels by SKF-96365 and ruthenium red in more detail.
Figure 1. Effects of TRP channel blockers on MS channels. (Top) MS channel currents recorded with a patch electrode containing physiological saline (control) or physiological saline containing either 1000 µM 2-APB, 40 µM ruthenium (more ...)
shows the effect of the concentration of SKF-96365 added to the patch electrode on the single-channel activity. With 25 µM SKF-96365 in the electrode, there were few closures within a channel opening, similar to the channel activity recorded in the absence of blocker. The few brief closures in the presence of 25 µM SKF-96365 could represent channel blocking events or conformational transitions of the channel protein. At higher concentrations of SKF-96365, there was a clear dose-dependent increase in the number of rapid closings. Adding 100 µM to the electrode produced far more fast closures when compared with the record obtained with 50 µM SKF-96365. With 200 µM SKF-96365 in the electrode, flickery block of the open channel increased to a point where the amplitude of the unitary current was difficult to discern. These observations suggest a simple model in which SKF-96365 acts as an open channel blocker in which the open and closed times represent the entry and exit of the drug from the channel pore.30,31
The predictions of the open channel block model were tested in the next set of experiments.
Figure 2. Records of single MS channel currents in the presence of increasing concentrations of SKF-96365. Records show single MS channel currents recorded in the presence of increasing concentrations of SKF-96365. SKF-96365 was added directly (more ...)
shows the analysis of the block of single MS channels in skeletal muscle by SKF-96365. shows the distribution of open and closed times within a single activation of the channel in the presence of either 50 µM (A) or 100 µM (B) SKF-9635 in the patch recording electrode. The histograms of open and closed times were fit with a single exponential, consistent with the presence of a single open and single closed state. shows the inverse of the mean open time (blocking rate) plotted as a function of the concentration of SKF-96365 in the patch electrode. The inverse of the mean open time increased with concentration of SKF-96365 in the electrode. As predicted by a simple two-state blocking model, the blocking rate depended linearly on the concentration of SKF-96365.
Figure 3. Analysis of the effect SKF-96365 on the mean open and closed times within a single opening. Histograms of open and closed times measured from records of single-channel activity recorded in the presence of either 50 µM (A) or (more ...)
The slope of the relation between the inverse of the mean open time and the concentration of SKF-96365 gave a second-order rate coefficient kon = 13.3x106 M−1s−1. As shown in , the brief closed times (unblocking rate) decreased somewhat with increasing concentration of blocker. However, resolution of rapid closures was difficult and there was considerable scatter in the measured values of blocked times. A linear fit to the data gave mean blocked time koff = 1609 sec−1. This value is not precise as there was considerable variability in the measured durations of blocked time, particularly at lower blocker concentrations where channel closing events outnumber blockages. The ratio of unblocking and blocking rates, koff/kon, gives a dissociation constant, KD = ~124 µM. The results support the interpretation that SKF-96365 produces the rapid transitions between open and closed channel levels by acting as an open channel blocker in which it rapidly enters and exits the channel pore.
In the next set of experiments, we examined the blocking actions of ruthenium red on MS channel in skeletal muscle. shows records of MS channel activity in the presence of different concentrations of ruthenium red. Ruthenium red had complex effects on the single-channel current. Increasing the concentration of ruthenium red from 5 µM (top record) to 40 µM (bottom record) clearly reduced the amplitude of the single-channel current. The amplitude of the single-channel current, however, was not constant but varied between several distinct levels. We interpreted this as ruthenium red causing a change in the probability of occupancy of the subconductance levels.
Figure 4. Records of single MS channel currents in the presence of increasing concentrations of ruthenium red. Ruthenium red was added directly to the patch electrode filling solution. Each record is from a different patch. The concentration of (more ...)
The effects of ruthenium red on MS channels resemble the block of MS channels by aminoglycoside antibiotics, in which blocker reduced both the amplitude of the single-channel current and increased occupancy of a subconductance level.32
Block differed, however, in that aminoglycosides cause channels to fluctuate between the fully open state and a single subconductance level. By contrast, gating in the presence of ruthenium red appeared to involve transitions between several subconductance levels. We analyzed the subconductance behavior in the presence of ruthenium red by analyzing the distribution of current amplitudes open channel current, excluding all transitions to the fully closed state. The results of this analysis are shown in .
Figure 5. Analysis of the effect of ruthenium red on the amplitude of the single-channel current. (A) Single channel currents recorded in the presence of either 10 µM (left) or 40 µM (right) ruthenium red. The graph below each (more ...)
shows single-channel currents recorded in the presence of either 10 µM (left) or 40 µM (right) ruthenium red. In the presence of 40 µM ruthenium red, the single-channel current is reduced, but the open channel current varies between several levels. We formed histograms of the amplitude of the open channel current by setting cursors within a channel opening event that excluded transitions to the fully closed, zero current level. Data points for the histograms were obtained from multiple channel openings within an experiment. The distribution of the amplitudes of the open channel current were skewed and could not be fit with a single Gaussian, as would be expected for a single open state with constant conductance. The skew can be seen in for the amplitude distribution for single-channel activity with 10 µM ruthenium red, where there is a “tail” extending toward zero current. We chose to fit the amplitude distribution of the open channel current as a sum of three Gaussian components, representing the fully open state (O) and two subconductance levels (S1 and S2). These levels are indicated with dotted lines in the records of single-channel activity. The fit to a sum of three Gaussian components was made using a maximum likelihood fitting routine.
shows that increasing the concentration of ruthenium red from 10–40 µM, reduced both the amplitude of the fully open state O as well as the probability of being in O, which was measured as the integral of the single Gaussian fit to O divided by the total area. We measured the distribution of current amplitudes for a number of experiments in which the patch electrode contained either 10, 15, 20 or 40 µM ruthenium red, as shown on the x-axis of . shows a plot of the probability of occupancy of O and the subconductance levels (S1 and S2) as a function of the ruthenium red concentration. The data shows that occupancy of state O decreases with blocker concentration, while there is an increase in the occupancy of S1 and S2. (inset) shows the amplitude of the fully open state plotted as a function of ruthenium red concentration. The fit data were fit with a simple expression for binding to a single site with a K1/2
of 49 µM. These results show ruthenium red blocks MS channels by both reducing occupancy probability of state O with increased probability of S1 and S2 as well as reducing the amplitude of the fully open state. Similar behavior has been observed for the block of MS channels by aminoglycoside antibiotics.32
Ruthenium red differs from aminoglycosides, such as neomycin, in having a greater potency (~49 µM vs. 200–300 µM, respectively. Thus, ruthenium red is a high affinity antagonist for MS channels in skeletal muscle.
shows that subconductance fluctuations in the presence of ruthenium red depend on membrane potential. In this experiment, the patch electrode contained 20 µM ruthenium red. Single channel activity was recorded at a constant holding potential of either –70, -50 or –30 mV. The amplitude distributions of the open channel current were obtained at each holding potential and the distribution fit as the sum of three Gaussian components. Inspection of the amplitude distributions measured at each voltage in the presence of a constant concentration of ruthenium red shows progressive reduction in the occupancy of the fully open state O, with a corresponding increase in occupancy of the next sublevel, S1. The results suggest subconductance fluctuations produced by ruthenium red are voltage-dependent and likely contribute to the blocking mechanism at depolarized membrane potentials. Further studies are required to analyze the precise mechanism of block of MS channels by ruthenium red in skeletal muscle and are not considered further in this paper.
Figure 6. Voltage-dependence of the subconductance transitions in the presence of ruthenium red. Experiment showing the block of the single-channel current produced by 20 µM ruthenium red at a constant holding potential of either –70, (more ...)
The pharmacological analysis shows MS channels in skeletal muscle are insensitive to 2-APB, but blocked by SKF-96365 and ruthenium red, properties consistent with TRPV channels. The absence of a potentiating effect of 2-APB on MS channels suggests MS channels are not TRPV1, 2 or 3. A possible candidate is TRPV4, which shows mechanosensitive gating in mammalian cells.33,34
In addition, TRPV4 knockout mice have reduced sensitivity to pressure stimuli,33
and minor abnormalities in auditory processing,36
but are otherwise phenotypically normal. The defects in various forms of mechanosensation in the TRPV4 knockout suggests TRPV4 may function as a mechanosensitive ion channel. We recorded single-channel activity from skeletal muscle fibers from TRPV4 knockout mice to test the hypothesis TRPV4 contributes to MS channels.
FDB fibers were isolated from TRPV4 knockout mice and single-channel activity recorded from membrane patches. Recordings were made with physiological saline in the patch electrode at a holding potential of –60 mV. Patch currents were recorded for 1–5 min at –60 mV and then the holding potential was changed in 10 mV increments from –60 to –10 mV. We recorded patch currents at each holding potential for 1–3 min. MS channel open probability increases with depolarization6,7
and so recordings at potentials more positive than –60 would increase the likelihood of observing channel activity. Despite the long recording times at both negative and more depolarized voltages, we failed to detect either spontaneous MS channel activity or activity induced by applying suction to the patch electrode in 21 out of 21 recordings from membrane patches on FDB fibers from TRPV4−/−
mice. Previous work shows ~70% of recordings from membrane patches on wild-type FDB fibers show robust MS channel activity.7
Therefore, the probability of observing no MS channel activity in our experiments if channels were present is negligibly small.
Vriens et al. showed that hypotonic solutions activate TRPV4 in response to cell swelling.34
Skeletal muscle fiber volume increases linearly with the inverse of the extracellular osmotic strength over the range 77–1100 mOsms, indicating fibers behave as a freely distensible, semipermeable bag containing a fixed amount of solute.37
There is evidence hypotonic solutions increase single channel activity in normal and dystrophic human myotubes.38
Therefore, we performed experiments examining the effects of hypotonic solutions on the activity of single MS channels in skeletal muscle.
shows an example of an experiment where the normal extracellular solution was switched to a hypotonic solution (250 mOsms). In these experiments, muscle fibers were exposed to the hypotonic solution while recording from membrane patches. shows reducing the osmolarity from 350–250 mOsms was associated with the appearance of a 25 pS channel. In this experiment, the channel showed prolonged open times, consistent with a hypo-osmotic activation mechanism. This behavior was observed in 3 out of 10 patches. In some of the experiments, exposure to a hypotonic solution was associated with the appearance of a larger conductance channel of ~35–38 pS (data not shown). The results suggest that MS channels may be activated by hypotonic solutions.
Figure 7. Hypo-osmotic activation of the MS channel in skeletal muscle. Experiment showing the effect of hypotonic solution on MS channels in skeletal muscle fibers. Recordings was made from a cell-attached patch and the extracellular solution (more ...)