Ca²⁺ release from the sarcoplasmic reticulum is required for sustained TRPM4 activity in cerebral artery smooth muscle cells

Abstract

The melastatin transient receptor potential (TRP) channel TRPM4 is a critical regulator of vascular smooth muscle cell membrane potential and contractility. Activation of the channel is Ca²⁺-dependent, but prolonged exposure to high (>1 μM) levels of intracellular Ca²⁺ causes rapid (within ∼2 min) desensitization of TRPM4 currents under conventional whole cell and inside-out patch-clamp conditions. The goal of the present study was to establish a novel method to record sustained TRPM4 currents in smooth muscle cells under near-physiological conditions. Using the amphotericin B-perforated patch-clamp technique, we recorded and characterized sustained (up to 30 min) transient inward cation currents (TICCs) in freshly isolated cerebral artery myocytes. In symmetrical cation solutions, TICCs reversed at 0 mV and had an apparent unitary conductance of 25 pS. Replacement of extracellular Na⁺ with the nonpermeable cation N-methyl-d-glucamine abolished the current. TICC activity was attenuated by the TRPM4 blockers fluflenamic acid and 9-phenanthrol. Selective silencing of TRPM4 expression using small interfering RNA diminished TICC activity, suggesting that the molecular identity of the responsible ion channel is TRPM4. We used the perforated patch-clamp method to test the hypothesis that TRPM4 is activated by intracellular Ca²⁺ signaling events. We found that TICC activity is independent of Ca²⁺ influx and ryanodine receptor activity but is attenuated by sarco(endo)plasmic reticulum Ca²⁺-ATPase inhibition and blockade of inositol 1,4,5-trisphosphate receptor-mediated Ca²⁺ release from the sarcoplasmic reticulum. Our findings suggest that TRPM4 channels in cerebral artery myocytes are regulated by Ca²⁺ release from inositol 1,4,5-trisphosphate receptor on the sarcoplasmic reticulum.

the melastatin transient receptor potential (TRP) channel TRPM4 is a crucial mediator of pressure-induced vascular smooth muscle membrane depolarization and vasoconstriction (8), but regulation of the channel is poorly understood. Single-channel (8, 17, 25) and whole cell patch-clamp recordings (7, 25) of TRPM4 activity in human embryonic kidney cell expression systems and cerebral artery myocytes show that the level of intracellular Ca²⁺ required for channel activation (1–10 μM) is greater than average cytosolic or “global” Ca²⁺ concentration ([Ca²⁺]) in arterial myocytes (100–300 nM) (15). However, TRPM4 also undergoes a fast desensitization, leading to decreased activity within 2 min following exposure to high global Ca²⁺ (7, 8, 16, 24, 27). We propose that desensitization under these conditions is an artifact of prolonged exposure to high levels of intracellular Ca²⁺, rather than an inherent property of the channel itself. To overcome this limitation and to limit disruption of native Ca²⁺ signaling pathways, we used the amphotericin B-perforated patch-clamp configuration to study TRPM4 activity in freshly isolated cerebral artery myocytes.

Intracellular Ca²⁺ in smooth muscle cells is not uniformly distributed. Localized, transient elevations in intracellular Ca²⁺ arise from Ca²⁺ influx via persistent activity of L-type Ca²⁺ channels (Ca²⁺ sparklets) (21, 37) and Ca²⁺ release from sarcoplasmic reticulum (SR) (Ca²⁺ sparks, puffs, and waves) (4, 14, 20, 22, 43). Temporal and spatial separation of SR Ca²⁺ release events results from the proximity of the SR and the plasma membrane (34) and strong intrinsic cytosolic Ca²⁺ buffering mechanisms (18). This architecture allows for Ca²⁺-sensitive ion channels located at the plasma membrane to be activated by intracellular Ca²⁺ release events (14, 23). For example, Ca²⁺ sparks result from Ca²⁺ release events from ryanodine receptors (RyRs) on the SR located close to the plasma membrane (22). These events cause local [Ca²⁺] to be elevated for a short period of time and open multiple large-conductance Ca²⁺-activated K⁺ (BK_Ca) channels, giving rise to spontaneous transient outward currents (STOCs) (12, 23). We hypothesize that TRPM4 channels may also be activated by intracellular Ca²⁺ signaling in smooth muscle cells.

Using the amphotericin B-perforated patch-clamp configuration, we recorded and characterized persistent, rapidly opening and closing cation currents from native vascular smooth muscle cells. We call these events “transient inward cation currents” (TICCs). TICC activity was diminished in cells isolated from cerebral arteries treated with small interfering RNA (siRNA) against TRPM4, suggesting that TICCs are formed by TRPM4 channels. TICC activity is reduced by inhibition of the sarco(endo)plasmic reticulum Ca²⁺-ATPase (SERCA) and blockade of inositol 1,4,5-trisphosphate receptor (IP₃R)-mediated Ca²⁺ release from the SR. Our findings indicate that, in cerebral artery myocytes, TRPM4 channels are regulated by SR Ca²⁺ release from IP₃R.

MATERIALS AND METHODS

Animals.

Male Sprague-Dawley rats (250–350 g body wt; Harlan) were deeply anesthetized with pentobarbital sodium (50 mg ip) and euthanized by exsanguination according to a protocol approved by the Institutional Animal Care and Use Committees of Colorado State University. Brains were isolated in cold MOPS-buffered saline [in mM: 3 MOPS (pH 7.4), 145 NaCl, 5 KCl, 1 MgSO₄, 2.5 CaCl₂, 1 KH₂PO₄, 0.02 EDTA, 2 pyruvate, and 5 glucose and 1% bovine serum albumin]. Cerebral and cerebellar arteries were dissected from the brain, cleaned of connective tissue, and stored in MOPS-buffered saline prior to further manipulation.

Isolated cerebral artery smooth muscle cell preparation.

Vessels were placed in the following cell isolation solution (in mM): 60 NaCl, 80 Na-glutamate, 5 KCl, 2 MgCl₂, 10 glucose, and 10 HEPES (pH 7.2). Arterial segments were initially incubated in 1.2 mg/ml papain (Worthington) and 2.0 mg/ml dithioerythritol for 17 min at 37°C and then in 1.0 mg/ml type II collagenase (Worthington) for 15 min at 37°C. The digested segments were washed three times in ice-cold cell isolation solution and incubated on ice for 30 min. After this incubation period, vessels were triturated to liberate smooth muscle cells and stored in ice-cold cell isolation solution for use. Smooth muscle cells were studied within 6 h following isolation.

RNA interference and reverse permeabilization.

siRNA against TRPM4 was used to downregulate expression of the channel in isolated cerebral arteries. siRNA molecules [catalog nos. 1027280 (AllStars negative control), SI02868292 (Rn_Trpm4_1), and SI02868313 (Rn_Trpm4_4)] were purchased from Qiagen and dissolved as instructed at a concentration of 20 μM in siRNA suspension buffer. A reversible permeabilization procedure was used to introduce control siRNA or TRPM4 siRNA molecules into intact cerebral arteries. To permeabilize the arteries, segments were first incubated for 20 min at 4°C in the following solution (in mM): 120 KCl, 2 MgCl₂, 10 EGTA, 5 Na₂ATP, and 20 TES (pH 6.8). Arteries were then placed in a similar solution containing siRNA (40 nM) for 3 h at 4°C and transferred to a third siRNA-containing solution with elevated MgCl₂ (10 mM) for 30 min at 4°C. For reversal of permeabilization, the arteries were placed in a MOPS-buffered physiological siRNA-containing solution consisting of (in mM) 140 NaCl, 5 KCl, 10 MgCl₂, 5 glucose, and 2 MOPS (pH 7.1, 22°C) for 30 min at room temperature. Ca²⁺ was gradually increased in the latter solution from nominally Ca²⁺-free to 0.01, 0.1, and 1.8 mM over a 45-min period. After the reversible permeabilization procedures, arteries were organ cultured for 2–3 days in DMEM-F-12 culture medium supplemented with 2 mM l-glutamine (GIBCO) and 0.5% penicillin-streptomycin (GIBCO). Arteries were then used for smooth muscle cell isolation or real-time RT-PCR.

Real-time RT-PCR.

Arteries containing siRNA were enzymatically dissociated as described above, and RNA was immediately isolated and purified using an RNeasy Protect Mini Kit (Qiagen). mRNA was synthesized into cDNA with the aid of an Omniscript reverse transcriptase kit (Qiagen) using 100 ng of RNA per reaction. Downregulation of TRPM4 was detected using a real-time SYBR Green detection assay (Bio-Rad), QuantiTect primers spanning intron/exon boundaries of TRPM4 (Qiagen), and an iQ5 multicolor real-time PCR detection system (Bio-Rad). Samples were normalized to β-actin, and cycling parameters were selected on the basis of the protocol for QuantiTect primer assays (Qiagen). β-Actin primers were designed on the basis of a published sequence (NM_031144) and were purchased from Integrated DNA Technologies. Sequences were as follows: 5′-TTGCTGACAGGATGCAGAAGGAGA-3′ (forward) and 5′-ACTCCTGCTTGCTGATCCACATCT-3′ (reverse). Downregulation of TRPM4 mRNA was calculated according to the Pfaffl method (33).

Immunocytochemistry.

Cells were enzymatically dissociated from siRNA-treated vessels as described above and allowed to adhere to glass slides for 20 min at 4°C. Cells were fixed with 4% formaldehyde for 10 min, permeabilized with cold methanol (−80°C), blocked with 2% bovine serum albumin, and incubated with primary antibodies overnight at 4°C. The following primary antibodies were used: rabbit anti-TRPM4 (1:250 dilution; ab63080, Abcam), rabbit anti-TRPM4 (1:100 dilution; sc-67125, Santa Cruz Biotechnology), sheep anti-TRPC3 (1:250 dilution; ab63012, Abcam), and goat anti-TRPC6 (1:100 dilution; sc-19197, Santa Cruz Biotechnology). Cells were subsequently washed and incubated with appropriate fluorescent secondary antibody for 2 h at room temperature. The following secondary antibodies were used at 1:1,000 dilution: anti-rabbit conjugated to Texas Red (sc-2780, Santa Cruz Biotechnology), anti-sheep conjugated to FITC (sc-2704, Santa Cruz Biotechnology), and anti-goat conjugated to Texas Red (sc-2783, Santa Cruz Biotechnology). To test specificity of the antibody, the antigenic blocking peptides (ab65597, Abcam) was incubated with the primary antibody for 10 min prior to use. Immunofluorescence images were obtained using a laser scanning confocal microscope (Fluoview 1000, Olympus) and a ×60, 1.4 numerical aperture oil immersion objective, with the pinhole diameter set for 1 Airy unit. Excitation of Texas Red was by illumination with the 543-nm line set at 74% transmission, and emission was collected using a variable band-pass filter set to 555–655 nm. Excitation for FITC was accomplished by illumination with the 488-nm line set at 1.4% transmission, and emission was collected using a variable band-pass filter set at 500–540 nm. All images were acquired at 1,024 × 1,024 pixels at 4.0 μs/pixel and were analyzed in ImageJ version 1.42q (National Institutes of Health). Membrane fluorescence (F_M) was determined using the mean fluorescence of a region of interest isolating the membrane, and total fluorescence (F_T) was determined using the mean fluorescence of the region of interest for the cytosol of the total cell.

Electrophysiological recordings.

Isolated smooth muscle cells were placed into a recording chamber (Warner Instruments) and allowed to adhere to glass coverslips for 20 min at room temperature. Whole cell currents were recorded using an AxoPatch 200B amplifier equipped with an Axon CV 203BU headstage (Molecular Devices). Recording electrodes (1–3 MΩ) were pulled, polished, and coated with wax to reduce capacitance. Gigaohm seals were obtained in a Mg²⁺-based physiological saline solution containing (in mM) 5 KCl, 140 NaCl, 2 MgCl₂, 10 HEPES, and 10 glucose. Amphotericin B (40 μM) was included in the pipette solution to perforate the membrane. Perforation was deemed acceptable if series resistance was <50 MΩ. STOC and TICC activities were recorded in normal external bathing solution containing (in mM) 134 NaCl, 6 KCl, 1 MgCl₂ 2 CaCl₂, 10 HEPES, and 10 glucose, with pH adjusted to 7.4 with NaOH. The pipette solution contained (in mM) 110 K-aspartate, 1 MgCl₂, 30 KCl, 10 NaCl, 10 HEPES, and 5 μM EGTA, with pH adjusted to 7.2 with NaOH. Additional external solutions include Na⁺-free external solution containing (in mM) 134 N-methyl-d-glucamine (NMDG), 1 MgCl₂, 2 CaCl₂, 6 KCl, 10 HEPES, and 10 glucose, with pH adjusted to 7.4 with KOH, and Ca²⁺ free external solution containing 134 NaCl, 6 KCl, 1 MgCl₂ 0.73 CaCl₂, 1 EGTA, 10 HEPES, and 10 glucose, with pH adjusted to 7.4 with NaOH. Currents were filtered at 1 kHz, digitized at 40 kHz, and stored for subsequent analysis. Clampex and Clampfit version 10.2 (Molecular Devices) were used for data acquisition and analysis, respectively. For most experiments, isolated smooth muscle cells were held at a membrane potential of +20 or −70 mV, and all recordings were performed at room temperature (22°C). In our recording solutions, the calculated reversal potentials for total monovalent cations are −1.8 and −30.6 mV for monovalent anions (Cl⁻). STOCs were defined as transient events >10 pA (>1 BK_Ca channel), and the frequency was calculated by dividing the number of events by the time between the first and last event. TICC activity at −70 mV was calculated as the sum of the open channel probability (NP_o) of multiple open states of 1.75 pA. This value was based on the reported unitary conductance of TRPM4 (25 pS). NP_o was calculated using the following equation: NP_o = ∑_{j = 1}^N[(t_j·j)/T], where t_j is time spent (in seconds) with j = 1,2,..,N channels open, N is maximum number of channels observed, and T is duration of measurement.

Calculations and statistics.

All data are means ± SE; n is the number of cells for immnocytochemistry and patch-clamp experiments. Data were compared using paired t-tests. P ≤ 0.05 was accepted as statistically significant for all experiments. All histograms were constructed in Origin 8.1 (OriginLab).

RESULTS

Sustained transient cation channel activity in freshly isolated smooth muscle cells.

The amphotericin B-perforated patch-clamp configuration, which allows for membrane voltage control with minimal disruption of intracellular Ca²⁺ dynamics (11), was used to record TRPM4 currents in native cerebral artery myocytes. After seal formation and perforation, cells were voltage-clamped at +20 mV and STOCs were recorded to demonstrate cell viability (Fig. 1A). STOC frequency was 2.8 ± 0.7 Hz, with an average amplitude of 22.7 ± 2.4 pA (Fig. 1A). Cells were then voltage-clamped at −70 mV for recording of inward currents. Under these conditions, we observed rapidly opening and closing, or “flickery,” currents (TICCs) with an average amplitude of −8.6 ± 1.6 pA (n = 8) and a frequency of 19.5 ± 2.9 Hz (n = 5; Fig. 1B). When physiological intracellular Ca²⁺ signaling activity is maintained, we are able to resolve single-channel TRPM4 currents using this method. The high TICC frequency suggests that, on average and under these conditions, at least one TRPM4 channel is open. TICCs could be recorded for as long as seal viability could be maintained, up to 30 min for some cells. The total mean NP_o for TICCs recorded under control conditions was 0.75 ± 0.08 and passed the Shapiro-Wilk test for normality (P = 0.184). A peak amplitude histogram was constructed (Fig. 1C) using the measured amplitude of channel(s). Seven peaks (solid lines) were identified and are consistent with calculated current amplitude for multiple (e.g., 1, 2, 3) channels opening. The apparent unitary TICC amplitude at a holding potential of −70 mV was −1.7 ± 0.1 pA (n = 5) and is consistent with the reported unitary conductance for TRPM4 channels (25 pS; Fig. 1C) (6, 17). Additionally, we observed two peaks (dashed lines) that did not coincide with any predicted TRPM4 current amplitude peak. These peaks occur at −2.7 and −4.2 pA at −70 mV and are suggestive of the presence of ∼38- and ∼60-pS channels. The reported unitary conductances for TRPC6 and TRPC3 are 35 pS (10) and 66 pS (45), respectively, and could account for these peaks. These currents account for a minor portion (∼16%) of TICC activity. TICCs were eliminated when Na⁺ in the bathing solution was replaced with the nonpermeable cation NMDG (Fig. 1D). To rule out the influence of BK_Ca channels, recordings were made in the presence of the selective BK_Ca channel blocker paxilline (5 μM). Paxilline abolished STOCs, but TICC activity was not changed (n = 3), demonstrating that these currents are independent of BK_Ca channel activity.

Fig. 1.

Sustained transient cation channel activity in freshly isolated smooth muscle cells. A and B: representative perforated patch-clamp recordings of spontaneous transient outward currents (STOCs) and transient inward cation currents (TICCs) in the same cell. Traces are representative of ∼30 cells. Insets show expanded time scale. V_H, holding potential. C: peak amplitude histogram of TICC activity recorded from freshly isolated smooth muscle (0.2-pA bins). Data are fitted with multiple Gaussian functions. Solid-line peaks match calculated current amplitude for TRPM4 channel openings. Dashed-line peaks do not match calculated current amplitude for TRPM4 channel openings. D: representative trace and summary data of TICC activity with Na⁺ replaced with the nonpermeable cation N-methyl-d-glucamine (NMDG, n = 3). NP_o, open channel probability. *P ≤ 0.05 vs. control.

The mean peak amplitude (pA) of TICC activity was recorded in the presence of paxilline at different holding potentials between −80 and +80 mV to examine the current-voltage (I-V) relationship. TICCs displayed modest inward rectification and, consistent with cation current activity, reversed at ∼0 mV (Fig. 2). The calculated ionic reversal potential for the solutions used for this study (symmetrical for total cations) is −1.8 mV vs. −30.6 mV for Cl⁻. When Na⁺ is replaced by the nonpermeable cation NMDG, and in the presence of paxilline, TICCs are abolished. This lack of channel activity at different holding potentials suggests that we have isolated a Na⁺-dependent current. These findings demonstrate that perforated patch-clamp configurations can be used to record sustained cation currents in freshly isolated cerebral smooth muscle cells.

Fig. 2.

Current-voltage (I-V) relationship for TICC activity in freshly isolated smooth muscle cells. Sample traces of TICC recordings were obtained at holding potentials from +80 to −70 mV. Average current amplitude-voltage (I-V) relationship is shown for cells (n = 3) with symmetrical total cation solutions under perforated patch-clamp conditions.

TICC activity was recorded in the presence of reported pharmacological inhibitors of TRPM4. Fluflenamic acid has been reported to inhibit TRPM4 (EC₅₀ = 2.8 μM) and TRPM5 (EC₅₀ = 24.5 μM) currents (40). TICC activity was absent following the administration of 10 μM fluflenamic acid (n = 5; Fig. 3A). The hydroxytricyclic derivative 9-phenanthrol has also been shown to specifically inhibit TRPM4 and not the closely related channel TRPM5 (9). TICC activity was diminished following the administration of 100 μM 9-phenanthrol (n = 3; Fig. 3B). These findings demonstrate that TICC activity is suppressed by compounds known to block TRPM4, suggesting that, in smooth muscle cells, TICCs are whole cell TRPM4 currents under near-physiological conditions.

Fig. 3.

TICC activity is blocked by TRPM4 inhibitors. A: representative trace and summary data of TICC activity in the presence of 10 μM fluflenamic acid (n = 5). B: representative traces of TICC activity in the presence of 100 μM 9-phenanthrol (n = 3).

TRPM4 siRNA selectively silences TRPM4 expression.

To test the hypothesis that TRPM4 expression is required for TICC activity, we suppressed expression of the channel using RNA interference (RNAi) technology. A reversible permeabilization technique (41) was used to introduce TRPM4-specific or control siRNA molecules into isolated cerebral arteries. Arteries were cultured for 2–3 days after siRNA treatment to allow for downregulation before smooth muscle cells were enzymatically dispersed from the vessels. In some experiments, Alexa 555-tagged control siRNA was introduced into permeabilized arteries. Using fluorescence confocal microscopy, we found that ∼70–80% of the isolated smooth muscle cells contained tagged siRNA (Fig. 4A), consistent with the findings of a prior study (5). Quantitative real-time RT-PCR was used to evaluate the effects of TRPM4 siRNA on TRPM4 mRNA levels. We found that TRPM4 mRNA levels were 60–70% less in arteries treated with siRNA against TRPM4 than in negative siRNA-treated vessels (Figs. 4B).

Fig. 4.

Small interfering RNA (siRNA)-mediated knockdown of melastatin transient receptor potential (TRPM4) expression in freshly isolated smooth muscle cells. A: freshly isolated rat cerebral artery smooth muscle cells from arteries treated with control siRNA tagged with Alexa 555 (top) or untagged siRNA (bottom). Scale bar, 11 μm. B: summary data for siRNA-mediated downregulation of TRPM4 mRNA levels in cerebral arteries as determined through real-time quantitative RT-PCR (n = 3). C: frequency histogram of normalized membrane fluorescence (F_M/F_T, where F_M is membrane fluorescence and F_T is total fluorescence) from cells with no primary antibody (no 1° control, n = 10), cells isolated from TRPM4 siRNA-treated vessels (n = 30), and control siRNA-treated vessels (n = 30). Surface plot is representative of images obtained for each treatment (right). Scale bar, 10 μm. D: summary data of TRPM4 siRNA vs. control siRNA treatment on total cell fluorescence labeling for TRPM4 (n = 40), TRPC3 (n = 30), and TRPC6 (n = 20). *P ≤ 0.05 vs. control siRNA. Right: representative images of total fluorescence for TRPM4, TRPC3, and TRPC6. Scale bar, 10 μm.

A quantitative immunocytochemical approach was used to characterize the effects of TRPM4 siRNA on TRPM4 protein expression (Fig. 4C). To quantify our images, all immnocytochemistry experiments were performed using the same protocol, and all fluorescence images were blinded and acquired under identical parameters. Cell fluorescence was normalized as a ratio of F_M to F_T, where F_M/F_T significantly >1.0 indicates increased labeling near the plasma membrane. In control cells not exposed to primary antibodies, F_M/F_T = 1.1 (n = 10). In cells isolated from vessels treated with control siRNA and incubated with anti-TRPM4 primary antibody (ab65597, epitope on the NH₂ terminus), F_M/F_T = 4.1 (n = 30), which is comparable to F_M/F_T = 5.4 (n = 15) in cells from freshly isolated cerebral arteries. In TRPM4 siRNA-treated vessels, there are two distinct populations of fluorescently labeled isolated cells. Most (23 of 29, 79.3%) of the cells from this group exhibit only background fluorescence levels (F_M/F_T = 1.2; Fig. 4C), suggesting that downregulation of TRPM4 expression is nearly complete in cells that take up the siRNA. A second, smaller population of cells (6 of 29, 20.7%) exhibit fluorescence similar to that of cells isolated from vessels treated with control siRNA (F_M/F_T = 4.2; Fig. 4C). Similar results were obtained using a different anti-TRPM4 primary antibody (sc-67125, Santa Cruz Biotechnology) targeting an epitope on the COOH terminus of TRPM4 (see Supplemental Material, Supplemental Fig. S1). Additionally, we performed immunocytochemistry experiments using the antigenic blocking peptides specific for the NH₂ terminus antibody (ab65597, Abcam). Total cell fluorescence decreased in the presence of the antigenic blocking peptide (see Supplemental Fig. S2). These findings suggest that silencing of TRPM4 expression is nearly complete in the ∼70–80% of cells that take up siRNA.

To test whether TRPM4 siRNA alters the relative protein expression levels of other TRP channels, we used the same quantitative immunocytochemical approach. TRPC3 and TRPC6 channels are present in cerebral artery smooth muscle cells and influence membrane potential and vascular tone (36, 38, 41). Cells were isolated from vessels treated with TRPM4 or control siRNA and fluorescently labeled for TRPC3 or TRPC6. Treatment with TRPM4 siRNA had no effect on total TRPC3 or TRPC6 cell fluorescence compared with cells isolated from control siRNA-treated vessels (Fig. 4D). These findings demonstrate that our siRNA procedures diminish TRPM4 mRNA and protein levels and do not alter expression of other TRP channels involved in smooth muscle cell function.

TRPM4 expression is required for TICC activity.

To further examine the hypothesis that TRPM4 expression is required for TICC activity, we studied the effects of TRPM4 siRNA on STOCs and TICCs. Smooth muscle cells enzymatically isolated from siRNA-treated cerebral arteries were patch-clamped in the perforated patch configuration, and STOC and TICC activity were recorded in the same cells at +20 and −70 mV, respectively (Fig. 5, A and B). The frequency and average amplitude of STOCs did not differ between cells isolated from cerebral arteries treated with TRPM4 siRNA and those treated with control siRNA (Fig. 5C). These findings show that the siRNA treatment does not disrupt BK_Ca channel activity or intracellular Ca²⁺ release events that generate STOCs. In contrast, TRPM4 siRNA nearly abolished TICC activity (Fig. 5, A, B, and D). Although some channel activity was observed in cells isolated from TRPM4 siRNA-treated arteries, these events were less frequent and smaller in amplitude than TICC recordings from control siRNA-treated vessels (Fig. 5, A and B, right). Total NP_o for TICC activity in smooth muscle cells dispersed from cerebral arteries treated with TRPM4 siRNA was significantly less than that in cells from vessels treated with control siRNA (Fig. 5D). These findings support the hypothesis that the molecular identity of the channel responsible for TICCs recorded from cerebral artery myocytes under perforated patch-clamp conditions is TRPM4.

Fig. 5.

TRPM4 expression is required for TICC activity. A: representative recordings of STOC and TICC activity from the same cell isolated from arteries treated with control siRNA. B: representative recordings of STOC and TICC activity from the same cell isolated from arteries treated with TRPM4 siRNA. C: summary data of STOC frequency (f, top) and average STOC amplitude (bottom). D: TICC NP_o for vessels treated with control siRNA (n = 4) or TRPM4 siRNA (n = 5). *P ≤ 0.05 vs. control siRNA.

TICCs are activated by IP₃R-dependent SR Ca²⁺ release.

Previous work showed that TRPM4 activation requires intracellular [Ca²⁺] greater than resting cytosolic levels reported for native smooth muscle cells (17, 25, 26). We propose that, under physiological conditions, TRPM4 channels are activated by locally elevated levels of intracellular Ca²⁺ that result from influx of Ca²⁺ from the extracellular space or from release of Ca²⁺ from intracellular stores. To determine whether Ca²⁺ influx activates TRPM4, we recorded TICC activity during removal of extracellular Ca²⁺. Lack of extracellular Ca²⁺ did not acutely (within 2 min) disrupt TICC activity, but prolonged (>5 min) exposure to a Ca²⁺-free bathing solution eventually resulted in decreased TICC activity (Fig. 6A). These findings suggest that TICC activity is not dependent on Ca²⁺ influx per se, but Ca²⁺ influx may be important in maintaining SR Ca²⁺ stores at levels sufficient to support channel activity. To examine this idea, we recorded TICC activity following disruption of SR Ca²⁺ stores and found that inhibition of the SERCA pump with cyclopiazonic acid (10 μM) attenuated TICC activity (Fig. 6B).

Fig. 6.

TICCs are activated by inositol 1,4,5-trisphosphate receptor (IP₃R)-dependent sarcoplasmic reticulum (SR) Ca²⁺ release. A: representative trace and summary data of TICC activity in the presence of extracellular Ca²⁺ (n = 6) and at 30 s (n = 6) and ∼5 min (n = 6) after extracellular Ca²⁺ is removed. B: representative trace and summary data of TICC activity in the presence of the sarco(endo)plasmic Ca²⁺-ATPase pump blocker cyclopiazonic acid (CPA, 10 μM, n = 5). *P < 0.05 vs. control.

To further examine the role of SR Ca²⁺ stores in the activation of TRPM4 in smooth muscle cells, we used ryanodine to disrupt RyR function, and the selective IP₃R blocker xestospongin C to inhibit SR Ca²⁺ release events. Ryanodine (50 μM) eliminated STOCs (n = 4) but had no effect on the TICC activity over the same time course (Fig. 7A). In contrast, administration of xestospongin C (1 μM) did not alter STOC frequency or amplitude (n = 5), but TICC activity was greatly diminished (Fig. 7B). These findings suggest that, in native cerebral artery myocytes, TRPM4 is activated by Ca²⁺ released from the SR through IP₃R (Fig. 8).

Fig. 7.

TICCs are activated by IP₃R-dependent SR Ca²⁺ release. A: representative traces of STOC and TICC activity in the presence of the SR ryanodine receptor (RyR) antagonist ryanodine (50 μM). Summary data of TICC NP_o are shown at right (n = 4). B: representative traces of STOC and TICC activity in the presence of the IP₃R blocker xestospongin C (1 μM). Summary data of TICC NP_o are shown at right (n = 5). *P < 0.05 vs. control.

Fig. 8.

Activation of TRPM4 by IP₃R-mediated SR Ca²⁺. VGCC, voltage-gated Ca²⁺ channels; ΔV_m, change in membrane potential; TRPM4, transient receptor potential melastatin 4; PM, plasma membrane.

DISCUSSION

The present study demonstrates a new method for recording sustained TRPM4 channel activity in freshly isolated smooth muscle cells. An important feature of this technique is the ability to record cation currents with minimal disruption of intracellular Ca²⁺ signaling dynamics. This patch-clamp method was used to examine the mechanism of Ca²⁺-dependent regulation of the channel in native smooth muscle cells. Our major findings are as follows. 1) Sustained TICCs can be recorded from cerebral artery myocytes with use of the perforated patch-clamp configuration. 2) RNAi-mediated downregulation of TRPM4 expression attenuates TICC activity. 3) TRPM4-dependent TICC activity is independent of Ca²⁺ influx per se but is attenuated by blockade of SERCA pump activity and IP₃R-mediated SR Ca²⁺ release. These findings provide the first sustained whole cell recordings of TRPM4 activity in native smooth muscle cells with intact intracellular Ca²⁺ signaling and demonstrate, for the first time, regulation of TRPM4 activity by IP₃R-mediated SR Ca²⁺ release.

TRPM4 channels are necessary for pressure- and PKC-induced smooth muscle depolarization and vasoconstriction (7, 8) and the autoregulation of cerebral blood flow in vivo (35). Efforts to understand regulation of TRPM4 have been hampered by rapid (∼2 min) desensitization under conventional patch-clamp conditions when nonphysiological levels of Ca²⁺ (∼10–100 μM) are present in the intracellular solution. Inhibition of phospholipase C (PLC) prolongs TRPM4 activity under these conditions, suggesting that a Ca²⁺-activated PLC isoform is responsible for TRPM4 rundown (24, 44). PLC-dependent desensitization of TRPM4 may result from localized depletion of phosphatidylinositol 4,5-bisphosphate (24, 44). We propose that rapid desensitization of TRPM4 activity is an artifact of recording conditions, rather than an inherent property of the channel. To test this possibility, we sought a method that would allow recording of whole cell TRPM4 currents under native conditions. The amphotericin B-perforated patch-clamp configuration allows recording of whole cell currents with minimal disruption of subcellular Ca²⁺ signaling pathways (11). This technique has been successfully used to record BK_Ca channel activation in smooth muscle cells in response to Ca²⁺ sparks (23), and we reasoned that this technique could also be used to record whole cell TRPM4 currents activated by transient Ca²⁺ events. We find that sustained currents (TICCs) were recorded from native cerebral artery myocytes under these conditions. TICCs have a high frequency of occurrence (19.5 ± 2.9 Hz), but this is not unexpected. Our previous work shows that TRPM4 activity can be recorded from ∼50% of inside-out membrane patches pulled from freshly isolated smooth muscle cells, suggesting that TRPM4 channels are abundant in these cells (8). Additionally, TICCs have biophysical properties that are consistent with those of TRPM4. For example, TICCs have an apparent single-channel conductance of ∼25 pS, in agreement with the reported unitary conductance of TRPM4 (29, 31, 42). TICCs reversed at ∼0 mV in the symmetrical cationic solutions used for this study (E_Cl⁻ = −30.6 mV), and substitution of the impermeant cation NMDG for Na⁺ in the bathing solution completely blocked TICC activity. These findings are consistent with the selectivity of TRPM4 for monovalent cations (17, 25). Furthermore, TICCs were suppressed by the TRPM4 blockers fluflenamic acid and 9-phenanthrol, supporting the hypothesis that TRPM4 is the major channel responsible for this activity. Using siRNA-mediated downregulation of TRPM4 expression, we further probed the relationship between TICCs and TRPM4. The efficacy and specificity of our TRPM4 siRNA treatment were confirmed using immunocytochemistry. Our data suggest that ∼80% of smooth muscle cells take up the TRPM4 siRNA and exhibit nearly complete knockdown of TRPM4 protein expression. Additionally, cells isolated from TRPM4 siRNA vessels did not show a change in expression of TRPC3 or TRPC6 protein. TICC activity is decreased in smooth muscle cells isolated from arteries treated with TRPM4-specific siRNA compared with cells isolated from vessels treated with negative control siRNA. Thus we conclude that TICCs represent TRPM4 activity in native cerebral artery myocytes. The virtually total inhibition of TICCs following treatment with TRPM4 siRNA suggests that most of the cells used for these experiments come from the population of cells that received siRNA in which knockdown of TRPM4 expression is nearly complete. The perforated patch-clamp technique described here provides a novel method for recording sustained TRPM4 currents under near-physiological conditions and should be useful for further characterization of the channel in smooth muscle cells.

We unexpectedly found that whole cell single-channel TICC activity exhibits modest inward rectification. In previous work, single-channel recordings from inside-out patches exhibited a linear I-V relationship (8), whereas whole cell TRPM4 currents exhibited inward and outward rectification (7, 25). Dual rectification reported for conventional whole cell TRPM4 currents may be an artifact of recording conditions. Under conventional whole cell patch-clamp conditions, the cytosolic environment is disrupted when the cell is dialyzed with the pipette solution, whereas the perforated patch-clamp configuration allows the intracellular environment to remain largely intact. TRPM4 currents are blocked by the polyamine spermine (26, 28), which could be dialyzed from the cell under conventional whole cell conditions. Polyamine block is responsible for inward rectification of inwardly rectifying K⁺ channels in vascular smooth muscle cells (30, 39). We propose that this mechanism is also responsible for inward rectification of TICCs and that this mechanism contributes to the voltage dependency of TRPM4 channels under physiological conditions in vascular smooth muscle cells. Interestingly, the I-V relationship we report for TICC activity would result in increased depolarizing current in response to membrane hyperpolarization and could be important for maintaining stable membrane potential in arterial myocytes.

The level of intracellular Ca²⁺ required to activate TRPM4 channels in whole cell and inside-out patch-clamp experiments is far greater than the average cytosolic [Ca²⁺] of arterial myocytes (100–300 nM) (15). However, downregulation of TRPM4 expression impairs pressure-induced myocyte depolarization, vasoconstriction, and autoregulation of cerebral blood flow (8, 35), demonstrating that the channel is active under physiological conditions. Subcellular regions of elevated Ca²⁺ can result from Ca²⁺ influx from the extracellular space (21, 37) and from Ca²⁺ released from intracellular stores (4, 12, 14, 20, 43), and these dynamic Ca²⁺ events can further regulate the activity of Ca²⁺-sensitive ion channels (23). We tested the hypothesis that increases in intracellular Ca²⁺ activate TRPM4 in native smooth muscle cells. We found that removal of extracellular Ca²⁺ did not initially change TICC activity, but prolonged exposure to Ca²⁺-free bathing solution eventually decreased these events, possibly due to diminished refilling of SR stores. Blockade of the SERCA pump also diminished TICC activity, consistent with a role for SR Ca²⁺ release in TRPM4 regulation. RyR inhibition did not alter TICC activity, but these currents were decreased by the IP₃R blocker xestospongin C. These findings indicate that Ca²⁺ released from IP₃Rs activate TRPM4 channels in cerebral artery smooth muscle cells (Fig. 8).

Ca²⁺ release from IP₃Rs is associated with the generation of “Ca²⁺ puffs,” which have been described in Xenopus oocytes (43) and isolated smooth muscle cells (1, 4). Our data suggest that some basal level of PLC-dependent generation of IP₃ and IP₃R-mediated Ca²⁺ release is present in freshly isolated cerebral artery smooth muscle cells and support sustained TICC activity. IP₃R-mediated Ca²⁺ release is also associated with spontaneous synchronous and asynchronous Ca²⁺ waves in smooth muscle cells in intact arteries (3, 14). The physiological function of these Ca²⁺ signaling events is not fully understood. Our findings show that TRPM4 activity in native cerebral artery myocytes requires IP₃R-mediated SR Ca²⁺ release, and we propose that TRPM4-mediated membrane depolarization in response to these Ca²⁺ signals may be central to vascular tone regulation. Consistent with this hypothesis, it has been proposed that synchronous Ca²⁺ waves in intact vessels may coordinate the membrane potential of smooth muscle cells along a segment of the vascular wall to effect uniform arterial contraction (19). Our findings suggest that TRPM4 activity could translate changes in synchronized Ca²⁺ waves into changes in smooth muscle cell membrane potential (32). Alternatively, asynchronous Ca²⁺ wave frequency reportedly increases in the presence of contractile agonists (3, 14), and this response may be important for smooth muscle contraction (14). It is possible that, during agonist-stimulated increases in asynchronous Ca²⁺ wave activity, low, noncontractile amounts of Ca²⁺ are released from IP₃Rs and activate TRPM4 to depolarize the membrane and initiate Ca²⁺ influx via voltage-dependent Ca²⁺ channels.

At physiologically relevant membrane potentials, the Ca²⁺ sensitivities of smooth muscle TRPM4 and BK_Ca channels are similar (2, 7, 12, 25), but the mechanisms that link IP₃R-mediated Ca²⁺ release events to TRPM4 channels appear to be different from those that couple RyR-dependent Ca²⁺ sparks to BK_Ca channels. Ca²⁺ sparks are highly localized and are directly coupled to numerous BK_Ca channels (12, 23). During a Ca²⁺ spark event, all these BK_Ca channels are activated almost simultaneously, resulting in the generation of a large STOC at a frequency of ∼1 Hz (13). In contrast, TICCs are more frequent (∼20 Hz) and smaller in magnitude, suggesting that activation could be due to a more persistent, but less robust, Ca²⁺ signal. Alternatively, difference in STOC vs. TICC size could suggest that fewer TRPM4 channels are present and/or coupled to Ca²⁺ release events compared with BK_Ca channels. Although additional work is required to test these ideas, our findings clearly indicate a connection between IP₃R-mediated Ca²⁺ release, activation of TRPM4 channels, and regulation of arterial tone.

GRANTS

This work was supported by National Heart, Lung, and Blood Institute Grants R01 HL-091905 (S. Earley) and F31 HL-094145 (A. L. Gonzales); National American Heart Association Scientist Development Grants AHA-0535226N (S. Earley) and AHA-0635118N (G. C. Amberg), a grant from the Colorado State University College of Veterinary Medicine and Biomedical Sciences Research Council (S. Earley), and a fellowship from the McNair Scholars Foundation (A. L. Gonzales).

DISCLOSURES

No conflicts of interest, financial or otherwise, are declared by the author(s).