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. 2010 Jul;43(1):17-25.
doi: 10.1165/rcmb.2009-0091OC. Epub 2009 Jul 31.

Functional role of canonical transient receptor potential 1 and canonical transient receptor potential 3 in normal and asthmatic airway smooth muscle cells

Jun-Hua Xiao  1 , Yun-Min Zheng, Bo Liao, Yong-Xiao Wang
Affiliations

Functional role of canonical transient receptor potential 1 and canonical transient receptor potential 3 in normal and asthmatic airway smooth muscle cells

Jun-Hua Xiao et al. Am J Respir Cell Mol Biol. 2010 Jul.

Abstract

Canonical transient receptor potential (TRPC)-encoded nonselective cation channels (NSCCs) are crucial for many cellular responses in a variety of cells; however, their molecular expression and functional roles in airway smooth muscle cells (ASMCs) remain obscure. The objective of this study was to determine whether TRPC1 and TRPC3 molecules could be important molecular constituents of native NSCCs controlling the resting membrane potential (Vm) and [Ca(2+)](i) in freshly isolated normal and ovalbumin (OVA)-sensitized/-challenged mouse ASMCs. Western blotting, RT-PCR, single-channel recording, whole-cell current-clamp recording, and a fluorescence imaging system were used to determine TRPC expression, NSCC activity, resting Vm, and resting [Ca(2+)](i). Specific individual TRPC antibodies and siRNAs were applied to test their functional roles. TRPC1 and TRPC3 proteins and mRNAs were expressed in freshly isolated ASM tissues. TRPC3 antibodies blocked the activity of NSCCs and hyperpolarized the resting Vm in ASMCs, whereas TRPC1 antibodies had no effect. TRPC3, but not TRPC1 gene silencing, largely diminished NSCC activity, hyperpolarized the resting Vm, lowered the resting [Ca(2+)](i), and inhibited methacholine-induced increase in [Ca(2+)](i). In OVA-sensitized/-challenged ASMCs, NSCC activity was greatly augmented, resting Vm was depolarized, and TRPC3 protein expression was increased. TRPC1 and TRPC3 antibodies blocked the increased activity of NSCCs and membrane depolarization in OVA-sensitized/-challenged cells. TRPC3 is an important molecular component of native NSCCs contributing to the resting Vm and [Ca(2+)](i) in normal ASMCs, as well as membrane depolarization and hyperresponsiveness in OVA-sensitized/-challenged cells, whereas TRPC1-encoded NSCCs are only activated in OVA-sensitized/-challenged airway myocytes.

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Figures

Figure 1.
Figure 1.
Canonical transient receptor potential 1 (TRPC1) and TRPC3 proteins and mRNAs are expressed in freshly isolated airway smooth muscle cells (ASMCs). (A) Western blot results shown are the representatives of at least three different experiments. Heart muscle and brain tissues were used as controls for TRPC1 and TRPC3, respectively. (B) Gel electrophoresis showed RT-PCR products for TRPC1 and TRPC3 mRNAs. Similar results were obtained from five independent experiments.
Figure 2.
Figure 2.
Native constitutivelyactive nonselective cation channels (NSCCs) are present in freshly isolated ASMCs. (A) An original recording shows the activity of single NSCCs in a cell membrane patch. The channel activity was recorded at −50 mV using the inside-out patch clamp configuration. The inset illustrates the channel recording at an extended time scale. (B) Graph displaying the mean current–voltage relationship of NSCC currents, which yielded three unitary conductances of 15, 37, and 57 pS. Each data point was obtained from at least five cells from five different animals. (C) Summary of the open probability (NPo) of single NSCCs at −50, −30, 30, and 50 mV. The numbers in parentheses indicates the numbers of patches (cells) and animals investigated. *P < 0.05 compared with the corresponding negative potential. (D) The effect of 1-oleyl-2-acetyl-sn-glycerol (OAG) and high Ca2+ on the NPo of NSCCs at −50 mV. The NPo was determined before and after bath application of 0.4% DMSO, OAG (4 μM) in 0.4% DMSO physiological saline solution (PSS), PSS, or high Ca2+ (100 mM) for 3 minutes. *P < 0.05 compared with control (before application of vehicle or reagent tested).
Figure 3.
Figure 3.
TRPC3 antibodies strongly inhibit the activity of single native constitutively active NSCCs in freshly isolated ASMCs. (A) An original recording of single NSCCs in an inside-out patch at −50 mV before and after bath application of specific TRPC3 antibodies (1:200 dilution). The inserts exhibit the channel recording at an extended time scale. (B) Summary of the effect of TRPC1 and TRPC3 antibodies in the absence and presence of their antigen peptides on the activity of single NSCCs. The relative NPo is presented as the difference in the channel activity recorded before and after application of individual TRPC antibodies (Ab). *P < 0.05 compared with control cells (without application of TRPC antibodies).
Figure 4.
Figure 4.
TRPC3 gene silencing by siRNAs largely blocks the activity of native constitutively active, nonselective cation channels in ASMCs. (A) Western blotting of TRPC1 and TRPC3 protein expression in cells cultured for 72 hours alone (control) and transfected with Lipofectamine transfection reagent, nontarget TRPC1, and TRPC3 siRNAs for 72 hours. The gel was derived from different experiments, and the photo represents a composite. Bar graph summarizes TRPC1 and TRPC3 protein expression relative to control. Data in each group were obtained from three independent experiments. *P < 0.05 compared with control. (B) RT-PCR analysis of TRPC1 and TRPC3 mRNA expression in control cells and cells transfected with Lipofectamine transfection reagent, nontarget TRPC1, and TRPC3 siRNAs for 72 hours. Data in each group were derived from five separate experiments. *P < 0.05 compared with control. (C) Western blot images show the effect of TRPC1 siRNA transfection on TRPC3 expression, and the effect of TRPC3 siRNA transfection on TRPC3 expression. The results shown are the representatives of at least three individual experiments. (D) The effect of TRPC1 and TRPC3 siRNA transfection for 72 hours on the activity of NSCCs in inside-out patches at −50 mV. Data in each group were obtained from three different experiments. *P < 0.05 compared with control.
Figure 5.
Figure 5.
TRPC3-encoded channels contribute to the resting membrane potential (Vm) in ASMCs. (A) Original recordings of the resting membrane potential in cells dialyzed without and with TRPC3 antibodies (1:200 dilution). (B) Summary of the effect of intracellular dialysis of TRPC1 and TRPC3 antibodies (Ab, 1:200 dilution) on the resting Vm. *P < 0.05 compared with control (dialyzed without TRPC antibodies). (C) Effect of TRPC1 and TRPC3 siRNA transfection for 72 hours on the resting Vm. Data in each group were obtained from three independent experiments. *P < 0.05 compared with control (cells cultured for 72 h alone). (D) Effect of transfection of TRPC1 and TRPC3 siRNAs for 72 hours on the resting [Ca2+]i. Data in each group were obtained from three different experiments. *P < 0.05 compared with control. (E) Effect of TRPC1 and TRPC3 transfection for 72 hours on methacholine (100 μM)-induced increase in [Ca2+]i. *P < 0.05 compared with control.
Figure 6.
Figure 6.
TRPC3 protein expression is specifically increased in ovalbumin (OVA)-sensitized/-challenged ASMCs. (A) Western blotting determination of individual TRPC protein expression in ASM tissues from normal and OVA-sensitized/-challenged mice. (B) Bar graph shows summary of individual TRPC protein expression. TRPC expression levels were normalized to actin expression levels. Data were obtained from at least three different experiments. The mean data were taken from three separate experiments. *P < 0.05 compared with control (mice without OVA-sensitization/challenge).
Figure 7.
Figure 7.
TRPC1- and TRPC3-encoded channels contribute to the increased activity of native constitutively active cation channels and membrane depolarization in OVA-sensitized/-challenged ASMCs. (A) OVA-sensitized/challenged mice showed airway muscle hyperresponsiveness determined by the increased Penh value after aerosol application of methacholine (30 mg/ml), measured using a Buxco's unrestricted whole-body plethysmograph system. (B) Bar graph summarizes methacholine-evoked muscle contraction in isolated tracheal rings from 10 control and 12 OVA-sensitized/-challenged mice. *P < 0.05 compared with control. (C) Effect of bath application of TPPC1 and TRPC3 antibodies (Ab, 1:200 dilution) on the increased activity of NSCCs at −50 mV in outside-out patches from OVA-sensitized/-challenged mice. *P < 0.05 compared with normal cells dialyzed without TRPC antibodies; †P < 0.05 compared with OVA-sensitized/-challenged cells dialyzed without TRPC antibodies. (D) Effect of intracellular dialysis of TRPC1 and TRPC3 antibodies (Ab, 1:200 dilution) on the membrane depolarization in OVA-sensitized/-challenged ASMCs. *P < 0.05 compared with control. †P < 0.05 compared with OVA-sensitized/-challenged cells dialyzed without TRPC antibodies.
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