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. 2015 Apr 15;308(8):C608-20.
doi: 10.1152/ajpcell.00360.2014. Epub 2015 Jan 28.

Intracellular Ca(2+) Release From Endoplasmic Reticulum Regulates Slow Wave Currents and Pacemaker Activity of Interstitial Cells of Cajal

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Intracellular Ca(2+) Release From Endoplasmic Reticulum Regulates Slow Wave Currents and Pacemaker Activity of Interstitial Cells of Cajal

Mei Hong Zhu et al. Am J Physiol Cell Physiol. .
Free PMC article

Abstract

Interstitial cells of Cajal (ICC) provide pacemaker activity in gastrointestinal muscles that underlies segmental and peristaltic contractions. ICC generate electrical slow waves that are due to large-amplitude inward currents resulting from anoctamin 1 (ANO1) channels, which are Ca(2+)-activated Cl(-) channels. We investigated the hypothesis that the Ca(2+) responsible for the stochastic activation of ANO1 channels during spontaneous transient inward currents (STICs) and synchronized activation of ANO1 channels during slow wave currents comes from intracellular Ca(2+) stores. ICC, obtained from the small intestine of Kit(+/copGFP) mice, were studied under voltage and current clamp to determine the effects of blocking Ca(2+) uptake into stores and release of Ca(2+) via inositol 1,4,5-trisphosphate (IP3)-dependent and ryanodine-sensitive channels. Cyclocpiazonic acid, thapsigargin, 2-APB, and xestospongin C inhibited STICs and slow wave currents. Ryanodine and tetracaine also inhibited STICs and slow wave currents. Store-active compounds had no direct effects on ANO1 channels expressed in human embryonic kidney-293 cells. Under current clamp, store-active drugs caused significant depolarization of ICC and reduced spontaneous transient depolarizations (STDs). After block of ryanodine receptors with ryanodine and tetracaine, repolarization did not restore STDs. ANO1 expressed in ICC has limited access to cytoplasmic Ca(2+) concentration, suggesting that pacemaker activity depends on Ca(2+) dynamics in restricted microdomains. Our data from studies of isolated ICC differ somewhat from studies on intact muscles and suggest that release of Ca(2+) from both IP3 and ryanodine receptors is important in generating pacemaker activity in ICC.

Keywords: ANO1 channel; IP3 receptor; SERCA pump; ryanodine receptor.

Figures

Fig. 1.
Fig. 1.
Effects of SERCA pump inhibitors on spontaneous transient inward currents (STICs) and slow wave currents. A and C: cyclopiazonic acid (CPA; 30 μM) and thapsigargin (1 μM) inhibited the amplitude and frequency of STICs (holding potential was −80 mV). B and D: portions of the traces in A and C (designated as a and b) were expanded to show that slow wave currents, evoked by stepping from the holding potential −80 mV to −35 mV, were decreased by CPA (30 μM) and thapsigargin (1 μM). E and F: summarized effects of CPA (30 μM) and thapsigargin (1 μM) on STIC amplitude (E) and frequency (F). **P < 0.01; ***P < 0.001.
Fig. 2.
Fig. 2.
The effects of tetracaine on STICs and pacemaker currents. A: tetracaine (500 μM) inhibited the amplitude and frequency of STICs at a holding potential of −80 mV with external Ca2+-containing physiological salt solution (CaPSS) and pipette solution containing CsCl. B: portions of the trace in A (designated as a and b) were expanded to show that slow wave currents decreased after application of tetracaine. C and D: summarized data showing averaged effects of tetracaine (500 μM) on STIC amplitude (C) and frequency (D). ***P < 0.001.
Fig. 3.
Fig. 3.
The effect of ryanodine on STICs and slow wave currents. A: ryanodine (50 μM) transiently increased the amplitude and frequency of STICs, then inhibited the amplitude and frequency of STICs at a holding potential of −80 mV with external CaPSS solution and pipette solution containing CsCl. B and C: ryanodine had no significant effect on slow wave currents within the first few minutes (B), but it inhibited slow wave currents in interstitial cells of Cajal (ICC) after several minutes (C). Traces in B and C are shown with expanded time scales from A (a, b, c, d). DF: summarized data showing average effects of ryanodine (50 μM) on peak current (D), STIC amplitude (E), and STIC frequency (F). Rya1 describes STICs during the initial 3–6 min after application of ryanodine, and Rya2 indicates STICs after 6 min. **P < 0.01; ***P < 0.001; ns, not significant.
Fig. 4.
Fig. 4.
Effects of store-active drugs on anoctamin 1 (ANO1) currents. Ano1 was expressed in HEK-293 cells. Currents were recorded when cells were dialyzed with 100 nM free [Ca2+]i and stepped from −80 mV to +70 mV in 10-mV increments. Holding potential was −80 mV. A, C, and E: currents measured in control conditions. B, D, and F: store-active drugs such as ryanodine (B), CPA (D), and thapsigargin (F) had no significant direct effect on ANO1 currents.
Fig. 5.
Fig. 5.
Effects of 2-aminoethoxydiphenyl borate (2-APB) on STICs and slow wave currents of ICC. A: 2-APB (50 μM) reduced the amplitude and frequency of STICs at a holding potential of −80 mV with external CaPSS solution and pipette solution containing CsCl. B: portion of the trace in A (designated as a and b) were expanded to show that slow wave currents were decreased by 2-APB. C and D: summarized effects of 2-APB (50 μM) on STIC amplitude (C) and frequency (D). ***P < 0.001.
Fig. 6.
Fig. 6.
Effects of xestospongin C on STICs and slow wave currents of ICC. A: xestospongin C (350 nM) transiently increased the amplitude and frequency of STICs, then inhibited STICs at a holding potential of −80 mV with external CaPSS solution and pipette solution containing CsCl. B: slow wave currents evoked by stepping from −80 mV to −35 mV (holding potential was −80 mV) were reduced by xestospongin C. B: traces from A (a and b) are shown at an expanded time scale. C and D: summarized data showing effects of xestospongin C (350 nM) on average STIC amplitude (C) and frequency (D). **P < 0.01.
Fig. 7.
Fig. 7.
Effects of tetracaine on the resting membrane potential (RMP) and spontaneous transient depolarizations (STDs) of ICC in current-clamp mode. A and B: tetracaine (500 μM) induced depolarization and decreased the amplitude of STDs in ICC. After repolarization by injection of current, STDs did not cause significant recovery of STDs (B). CE: summarized effects of tetracaine on RMP (C), amplitude of STDs (D), and frequency of STDs (E). Tetracaine 1 describes STD during the initial application of tetracaine (i.e., during I = 0 conditions) and tetracaine 2 indicates the tetracaine effect on STDs during I-C conditions. **P < 0.01; ***P < 0.001.
Fig. 8.
Fig. 8.
Effects of ryanodine on RMP and STDs in ICC under current-clamp. A and B: ryanodine (50 μM) induced depolarization and decreased amplitude of STDs of ICC with external solution (CaPSS) and pipette solution (KCl). After repolarization of membrane potential by injection of current, STDs recovered and were blocked by 5-nitro-2-(3-phenylpropylamino)-benzoic acid (NPPB; B). C and D: summarized data of ryanodine effects on RMP. E and F: summarized data of ryanodine (50 μM) and NPPB (100 μM) effects on STD amplitude (E) and STD frequency (F). Rya1 describes STD during the initial application of ryanodine, during I = 0 conditions, and Rya2 indicates the ryanodine effect on STD, during I-C conditions. *P < 0.05, **P < 0.01, ***P < 0.001; ns, not significant.
Fig. 9.
Fig. 9.
Effects of CPA and thapsigargin on RMP and STDs of ICC in current-clamp (I = 0) mode. A: CPA (30 μM) induced depolarization and reduced amplitude of STDs of ICC with CaPSS as the external solution and KCl solution in the pipette. B: application of thapsigargin (1 μM) induced significant depolarization and decreased the amplitude of STDs of ICC. C and D: summarized data of CPA (30 μM) and thapsigargin (1 μM) effects on RMP and STIC amplitude. E and F: summarized effects of CPA and thapsigargin on frequency of STDs. **P < 0.01; ***P < 0.001.
Fig. 10.
Fig. 10.
Effects of 2-APB on RMP and STDs of ICC in current-clamp mode. A. 2-APB (50 μM) reduced the amplitude and induced depolarization of STDs of ICC with external solution (CaPSS) and pipette solution (KCl). BD: summarized effects of 2-APB (50 μM) on RMP (B), STD amplitude (C), and STD frequency (D). **P < 0.01; and ***P < 0.001.
Fig. 11.
Fig. 11.
Effects of different concentrations of [Ca2+]i on ANO1 currents. ICC dialyzed with solutions containing different concentrations of Ca2+ were held at −80 mV and stepped to potentials from −80 mV to −35 mV. Large-amplitude slow wave currents (ANO1 current) were activated by depolarization, and no evidence of tonic activation of ANO1 (as observed in HEK-293 cells) was observed even when high cells were dialyzed with 1 μM Ca2+. AC: traces are representative of 3 cells each dialyzed with [Ca2+]i: <10 nM (low; A), 500 nM (B), or 1 μM (C). Changing [Ca2+]i over this range did not affect the need for voltage-dependent activation of slow wave currents. D: summarized data of peak slow wave currents elicited at concentrations of [Ca2+]i.

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