Na+/Ca2 + Exchange and Pacemaker Activity of Interstitial Cells of Cajal

Abstract

Interstitial cells of Cajal (ICC) are pacemaker cells that generate electrical slow waves in gastrointestinal (GI) smooth muscles. Slow waves organize basic motor patterns, such as peristalsis and segmentation in the GI tract. Slow waves depend upon activation of Ca²⁺-activated Cl^- channels (CaCC) encoded by Ano1. Slow waves consist of an upstroke depolarization and a sustained plateau potential that is the main factor leading to excitation-contraction coupling. The plateau phase can last for seconds in some regions of the GI tract. How elevated Ca²⁺ is maintained throughout the duration of slow waves, which is necessary for sustained activation of CaCC, is unknown. Modeling has suggested a role for Na⁺/Ca²⁺ exchanger (NCX) in regulating CaCC currents in ICC, so we tested this idea on murine intestinal ICC. ICC of small and large intestine express NCX isoforms. NCX3 is closely associated with ANO1 in ICC, as shown by immunoprecipitation and proximity ligation assays (PLA). KB-R7943, an inhibitor of NCX, increased CaCC current in ICC, suggesting that NCX, acting in Ca²⁺ exit mode, helps to regulate basal [Ca²⁺] _i in these cells. Shifting NCX into Ca²⁺ entry mode by replacing extracellular Na⁺ with Li⁺ increased spontaneous transient inward currents (STICs), due to activation of CaCC. Stepping ICC from -80 to -40 mV activated slow wave currents that were reduced in amplitude and duration by NCX inhibitors, KB-R7943 and SN-6, and enhanced by increasing the NCX driving force. SN-6 reduced the duration of clustered Ca²⁺ transients that underlie the activation of CaCC and the plateau phase of slow waves. Our results suggest that NCX participates in slow waves as modeling has predicted. Dynamic changes in membrane potential and ionic gradients during slow waves appear to flip the directionality of NCX, facilitating removal of Ca²⁺ during the inter-slow wave interval and providing Ca²⁺ for sustained activation of ANO1 during the slow wave plateau phase.

Keywords: ANO1; Ca2+-activated Cl– current; gastrointestinal motility; slow waves; smooth muscle.

FIGURE 1

Quantitative analysis of Na⁺-Ca²⁺ exchanger (Slc8a1-3) expression in ICC and unsorted cells from small intestine and colon. (A) Summary graph showing relative expression of Slc8a1, Slc8a2, and Slc8a3 in small intestinal ICC and in unsorted intestinal cells. Note greatly increased expression of all three isoforms in ICC of small intestine relative to the unsorted cells. (B) Summary graph showing relative expression of Slc8a1, Slc8a2, and Slc8a3 in colon ICC and in unsorted colon cells. Only Slc8a3 was elevated in colonic ICC. Expression of all transcripts was normalized to Hprt.

FIGURE 2

Cl^– conductance activated by KB-R7943. (A) Cells were dialyzed with internal solution (E_Cl = –40 mV, Solution VI in Table 2) and held at –50 mV. KB-R7943 (15 μM) activated inward current. Ramp potentials from –80 to +80 mV were applied before (example a in the trace) and after (example b in the trace) addition of KB-R7943 (15 μM). (B) Shows current responses to ramp potentials (a: controls, black trace in the panel) and b: KB-R7943 (15 μM), red trace in each panel). Data taken from traces in panel A. The currents activated by KB-R7943 (15 μM) were outwardly rectifying and reversed at E_Cl. (C) Summarized data show average normalized currents (currents density, pA/pF) at 0 mV before (control) and after KB-R7943 (15 μM; n = 5). ***p < 0.001.

FIGURE 3

Effects of low [Na⁺]_o on spontaneous transient inward currents (STICs) in ICC. (A,B) Representative traces showing that replacement of [Na⁺]_o (140 mM) with equimolar Li⁺ (Solution II) increased the amplitude and frequency of STICs (holding potentials = –80 mV) in colonic (A) and small intestinal (B) ICC. Under control condition, the external solution was CaPSS (Solution I) and internal solution was Solution V (see Table 2). Insets show expanded time scale from upper panel (dotted boxes) to display how STIC amplitude and frequency were measured. Blue dots denote detection of STICs using event detection analysis (see section “Materials and Methods”). (C) Representative trace showing STICs at various potentials after 0 mM [Na⁺]_o in a colonic ICC. STICs reversed at about –29 mV (trace uncorrected for junction potential). Dotted lines denote 0 pA. (D,E) Summary data from five experiments shows the effects of low [Na⁺]_o on the frequency (D) and amplitude (E) of STICs (**p < 0.01).

FIGURE 4

Effects of Ca²⁺-activated Cl^– channel (CaCC) blockers on STICs activated by low [Na⁺]_o in colonic and small intestinal ICC. (A–C) Representative traces showing increase in STICs in response to low external [Na⁺]_o (see Solution II). (A) Low external [Na⁺]_o increased STICs for the duration of the exposure (in this 10 min; small intestinal ICC). STICs were inhibited by CaCC blockers. (B) NPPB (50 μM) inhibited STICs in colonic ICC held at –80 mV (Internal Solution V). (C) STICs were inhibited in small intestinal ICC by T16Ainh-A01 (10 μM). Dotted lines denote 0 pA. (D,E) Summary data showing the effects of low [Na⁺]_o and Ano1 blockers on the frequency (D) and amplitude (E) of STICs (*p < 0.05, **p < 0.01 vs. control; #p < 0.05, ##p < 0.01, ###p < 0.001 vs. low [Na⁺]_o).

FIGURE 5

KB-R7943 and SN-6 inhibit STICs activated by low [Na⁺]_o. Cells were held at –80 mV and STICs occurred spontaneously. (A,B) Representative traces showing that KB-R7943 (5 μM) and SN-6 (5 μM) inhibited STICs activated by 0 mM [Na⁺]_o (Solution II) in colonic ICC. (C,D) Representative traces showing that KB-R7943 (5 μM) and SN-6 (5 μM) inhibited STICs activated by 0 mM [Na⁺]_o in small intestinal ICC. Dotted lines denote 0 pA. (E,F) Summary data showing the effects of low [Na⁺]_o and Ano1 blockers on the frequency (D) and amplitude (E) of STICs. **p < 0.01 vs. control; ##p < 0.01 vs. low [Na⁺]_o.

FIGURE 6

Effects of shifting E_NCX to more negative potentials on slow wave currents in small intestinal ICC. (A,E) Representative traces showing slow wave currents in small intestinal ICC evoked by step depolarizations (500 ms) from –80 to –40 mV. Repolarization to holding potential (–80 mV) resulted in long lasting tail currents that exceeded the deactivation properties of ANO1 (45). The sustained tail currents demonstrate the autonomous nature of the slow wave currents (i.e., once activated these responses persist even after repolarization). Reduced [Na⁺]_o (to 20 mM; 115 mM Na⁺ replaced with equimolar Li⁺, Solution III) increased the amplitude of slow wave currents and the amplitude and duration of the tail currents (red traces), as compared to control currents (with 140 mM [Na⁺]_o, black traces). (A) KB-R7943 (5 μM, KBR) and (E) SN-6 (5 μM) reduced the slow wave currents and tail currents (green traces). (B–D) Summary data showing the effects of low [Na⁺]_o and KB-R7943 on the amplitude of slow wave currents (B), the peak amplitude of tail currents (C) and the duration of tail currents (D) from 5 experiments. (F–H) Summary data showing the effects of low [Na⁺]_o and SN-6 on the amplitude of slow wave currents (F), the peak amplitude of tail currents (G), and the duration of tail currents (H) from four experiments. *p < 0.05, **p < 0.01, ***p < 0.001.

FIGURE 7

Effects of NCX antagonists on ANO1 currents expressed in HEK 293 cells. (A,B) show ANO1 current responses from HEK 293 cells transfected with the AC splice variant of Ano1 to step protocols from –80 mV to +80 mV before and in the presence of SN-6 (5 μM). A symmetrical Cl^– gradient was established for these experiments (i.e., external and internal solutions were Solutions IV and VII, respectively). SN-6 had no effect on the ANO1 currents, and data from 5 cells are summarized in (C). (D,E) Show ANO1 current responses from HEK 293 cells transfected with the AC splice variant of Ano1 to step protocols from –80 mV to +80 mV before and in the presence of KB-R7943 (KBR; 5 μM). Peak currents at 400 ms during step depolarization were divided by cell capacitance and reported as current densities (pA/pF). KBR had no effect on the ANO1 currents, and data from 5 cells are summarized in (F).

FIGURE 8

Effects of NCX antagonists on T- and L-type Ca²⁺ currents. (A,C) Show current responses to step depolarizations of HEK 394 cells (–80 to –40 mV) expressing Ca_V3.2 channels before (black traces), in the presence of KB-R7943 (5 μM, KBR) or SN-6 (5 μM) (red traces), and after wash out of the drugs (green traces). (B,D) Summaries of the effects of KB-R7943 and SN-6 on peak T-type Ca²⁺ currents in 5 and 4 cells, respectively (*p < 0.05). (E,G) Show L-type Ca²⁺ currents evoked by step depolarizations of colonic SMC (–80 to 0 mV) before (black traces), in the presence of KB-R7943 (5 μM, KBR) or SN-6 (5 μM) (red traces), and after wash out of the drugs (green traces). (F,H) Summaries of the effects of KB-R7943 and SN-6 on peak L-type Ca²⁺ currents (n = 5 cells for each experiment; **p < 0.01).

FIGURE 9

Inhibiting NCX decreases the duration of Ca²⁺ transient clusters in ICC-MY networks. (A) Raw image of an ICC-MY network recorded from the jejunum of a Kit-Cre-GCaMP6f mouse (far left panel). The summated heat map of all Ca²⁺ transients that occurred in this field of view (FOV) over a 30 s recording is shown before and after addition of SN-6 (10 μM) in the middle and far right panel, respectively. (B) Traces of summated Ca²⁺ activity in the ICC-MY network before (black) and after (red) addition of SN-6 (10 μM). Occurrence maps show the activity of each individual firing site within the ICC-MY network plotted against time, with each firing site in the network plotted as a differently colored lane. The number of colored lanes (counted along the y-axis) indicates the number of active firing sites. The width of each lane (measured along the x axis) at any point indicates the duration that the site was active. Note that under control conditions, many sites fired multiple Ca²⁺ transients during each cycle, but after SN-6, the number of occurrences at each site decreased to only a single event. (C) Expanded timescale showing a single CTC highlighted in the dashed gray box in (B), before (black) and after (red) addition of SN-6 (10 μM). (D) Summary effect of SN-6 (10 μM) on CTC firing frequency, n = 6. (E) Summary effect of SN-6 (10 μM) on CTC duration, n = 6. **p < 0.01. (F) Histogram showing the distribution of values for CTC duration before (black) and after (red) addition of SN-6 (10 μM; n = 6). ****p < 0.0001.

FIGURE 10

Effects of ouabain on slow wave currents. Slow wave currents, activated by depolarizing steps from –80 to –40 mV, are shown in (A) before and in the presence of ouabain (200 nM). Ouabain increased the amplitude of the slow wave current, the amplitude of the tail current upon repolarization to –80 mV and the duration of the tail current. Summarized data from five experiments are shown in (B–D), respectively. *p < 0.05, **p < 0.01.

FIGURE 11

Protein-protein interaction between ANO1 and NCX3. (A) Co- IP of ANO1 and NCX3 from small intestine smooth muscle membrane fraction. Immunoprecipitates (IP) were obtained by elution with low pH buffer, and immunoblotting (IB) was performed using Wes with anti-ANO1 and anti-NCX3 antibodies. Lanes: 1, protein standards; 2, ANO1 in membrane fraction (2.5 mg); 3, ANO1 in ANO1 immunoprecipitate (5 ml); 4, ANO1 in non-immune rabbit IgG IP; 5, NCX3 in membrane fraction (1 mg); 6, NCX3 in NCX3 immunoprecipitate (5 ml); 7, NCX3 in non-immune rabbit IgG IP; 8, NCX3 in Ano1 immunoprecipitate (5 ml); 9, ANO1 in NCX3 immunoprecipitate (5 ml) n = 4. (B) Association between ANO1 and NCX3 occurs in situ in ICC, as demonstrated by PLA. ICC from small intestine (green, left panel) were exposed to the rabbit anti-ANO1 and goat anti-NCX3 antibodies, followed by the Duolink minus-anti rabbit IgG and plus-anti goat IgG secondary antibodies and the ligation and amplification reactions. For the PLA negative control, ICC were exposed to rabbit anti-ANO1 and mouse anti-myosin regulatory light chain antibodies (upper left panel). PLA-positive red spots were detected by ANO1-NCX3 co-localization (lower left panel). Few red spots were observed in negative controls. Nuclear staining of ICC was achieved with DAPI (blue, left panel). Intact ICC were confirmed by differential interference contrast (DIC) (right panels).

FIGURE 12

Schematic proposing how NCX contributes to ionic currents and fluxes during the slow wave cycle in ICC. This figure summarizes how the activity of NCX might be integrated with other known ionic mechanisms involved in generation of pacemaker activity in ICC. Electronmicroscopy has demonstrated close apposition between the plasma membrane and ER, and the restricted volumes formed are termed microdomains. Each frame in the schematic is a temporal representation of a typical microdomain, and ionic concentrations in the restricted volumes of the pacemaker units are hypothesized from current and previous findings. For simplicity, ion channels and transporters appear or disappear in individual frames as their role waxes and wanes. Frame 1 shows the period of the inter-slow wave interval. Stochastic Ca²⁺ release events from the ER trigger (Red arrow) activation of ANO1 channels in the plasma membrane (PM), activating STICs. Following a Ca²⁺ transient, NCX, operating in Ca²⁺ exit mode (Blue arrow heads), and SERCA (not shown) rapidly restore basal [Ca²⁺]_i within the microdomain, reducing open probability of ANO1 channels and limiting the duration of STICs. Frame 2 During the interval between slow waves, the probability of STICs increases with time (Hirst and Edwards, 2001). Summation of STDs (voltage response to STICs) causes activation of voltage-dependent (T-type) Ca²⁺ current (VDCC) (Zheng et al., 2014). Ca²⁺ entry triggers Ca²⁺-induced Ca²⁺ release in many microdomains in synchrony and activation of whole cell CaCC currents, known as the slow wave current (Zhu et al., 2009). Ca²⁺ entry and Cl^– efflux cause depolarization close to E_Cl (Kito et al., 2005). In Frame 3 Cl^– efflux slows as membrane potential approaches E_Cl, but Cl^– conductance remains high, clamping membrane potential for a second or more (plateau phase of slow wave) near E_Cl. Cl^– loss is recovered by the action of the Na⁺K⁺2Cl^– (NKCC1) cotransporter (Wouters et al., 2006). This transporter restores [Cl^–]_i in the microdomain using the energy of the Na⁺ gradient. In Frame 4 accumulation of Na⁺ in the restricted volume of the microdomain flips NCX into Ca²⁺ entry mode (Red arrow heads), and the close association between Ca²⁺ entry through NCX, ANO1 channels and Ca²⁺ release channels (IP3R and RYR) helps to sustain Ca²⁺ transients and activation of ANO1, creating the sustained depolarization of the plateau phase. In Frame 5 [Cl^–]_i is restored and the accumulated Na⁺ is eventually removed by the combined actions of NCX and the Na⁺/K⁺ ATPase (NKX). Reducing [Na⁺]_i flips NCX back into the Ca²⁺ exit mode, and causes reduced [Ca²⁺]_i in the microdomain, deactivation of ANO1 and repolarization. Reloading of ER Ca²⁺ stores (by SOCE, Zheng et al., 2018; not shown) increases the probability of STICs and recharges the next slow wave cycle.