Ca2+ signaling driving pacemaker activity in submucosal interstitial cells of Cajal in the colon

Interstitial cells of Cajal (ICC) generate pacemaker activity responsible for phasic contractions in colonic segmentation and peristalsis. ICC along the submucosal border (ICC-SM) contributing to mixing and more complex patterns of colonic motility. We show the complex patterns of Ca2+ signaling in ICC-SM and the relationship between ICC-SM Ca2+ transients and activation of SMCs using optogenetic tools. ICC-SM displayed rhythmic firing of Ca2+ transients ∼15 cpm and paced adjacent SMCs. The majority of spontaneous activity occurred in regular Ca2+ transients clusters (CTCs) that propagated through the network. CTCs were organized and dependent upon Ca2+ entry through voltage-dependent Ca2+ conductances, L- and T-type Ca2+ channels. Removal of Ca2+ from the external solution abolished CTCs. Ca2+ release mechanisms reduced the duration and amplitude of Ca2+ transients but did not block CTCs. These data reveal how colonic pacemaker ICC-SM exhibit complex Ca2+ firing patterns and drive smooth muscle activity and overall colonic contractions. Synopsis How Ca2+ signaling in colonic submucosal pacemaker cells couples to smooth muscle responses is unknown. This study shows how ICC modulate colonic motility via complex Ca2+ signaling and defines Ca2+ transients’ sources using optogenetic techniques.


Global Ca 2+ firing patterns in ICC-SM 141
Movement generated by muscle contractions is always an issue when imaging cells in 142 muscle preparations in situ. Therefore, we prepared and utilized submucosal tissues separated 143 from the muscle strips in most experiments. At low magnification (10x), rhythmic Ca 2+ waves 144 occurred and spread through ICC-SM networks in isolated submucosal layer preparations. The 145 Ca 2+ waves occurred at 8-22 cycle min −1 (Fig. 3 A-G) and averaged 14.9 ± 1.9 cycle min −1 146 Ca 2+ waves swept through ICC-SM networks (Fig. 6 E&J). Also apparent from the occurrence 181 maps was that the firing sequence of sites changed from Ca 2+ wave to Ca 2+ wave. Not all firing 182 sites discharged Ca 2+ transients during each wave cycle, and some sites fired more than once. 183 From particle analysis the average particle area/frame of Ca 2+ transients averaged 3.2 ± 0.4 184 µm 2 (Fig. 6 H; n = 25) and particle count/frame averaged 0.27 ± 0.1 (Fig. 6 I; n = 25). 185 Particle analysis also showed that Ca 2+ firing sites were most active during the first ~256 186 ms of a Ca 2+

Molecular expression of Ca 2+ entry channels in ICC-SM 215
The apparent dependence on the Ca 2+ gradient to maintain pacemaker function in ICC-216 SM suggests that Ca 2+ entry mechanisms are critical for initiation and organization of CTCs. 217 Therefore, we examined expression of several Ca 2+ channels that might convey Ca 2+ entry in 218 ICC-SM (Fig. 8). After isolation of the submucosal layer from the proximal colon of Kit +/copGFP 219 mice and subsequent cell dispersion, we sorted copGFP-positive ICC-SM with fluorescence 220 activated cell-sorting (FACS) and evaluated the expression of voltage-dependent and -221 independent Ca 2+ channels by qPCR (Fig. 8 A&B). First, we confirmed the purity of sorted ICC-222 SM with cell-specific markers (Fig. 8 A). Kit receptors and ANO1 channels are signatures of 223 ICC throughout the GI tract and enrichment of Kit and Ano1 expression was observed in sorted 224 ICC-SM compared to unsorted cells (Kit expression was 0.21 ± 0.014; Ano1 expression was 0.14 225 ± 0.02 relative to Gapdh). The expression levels of Myh11 a smooth muscle cell marker and 226 Uch11 a panneuronal marker encoding PGP9.5 were minimal (Myh11 expression was 0.03 ± 227 0.002; Uch11 expression was 0.002 ± 0.0001 relative to Gapdh). confirming the purity of ICC-228 SM sorted by FACS. 229 ICC-SM expressed L-type voltage-dependent Ca 2+ channels encoded by Cacna1c and 230 Cacna1d abundantly (CaV 1.2 and CaV 1.3 channels, respectively). Cacna1c showed a 0.012 ± 231 0.0005 and Cacna1d showed 0.066 ± 0.008 relative to Gapdh (Fig. 8 B; n = 4). ICC-SM also 232 expressed Cacna1h (CaV 3.2) and to a lesser extent Cacna1g (CaV 3.1), both T-type voltage-233 dependent Ca 2+ channels (Fig. 8 B; n = 4). Cacna1h expression was abundant in ICC-SM (0.07 234 ± 0.003 relative to Gapdh). Cacna1g expression was less than Cacna1h 0.0014 ± 0.0001 relative 235 to Gapdh (Fig. 8 B;  10.5 ± 4.7% (Fig. 9 E-G; n = 8) and PTCL count was reduced to 12.3 ± 4.8% (Fig. 9 H; n = 8). 252 The number of firing sites also decreased to 8.4 ± 3% in the presence of nicardipine ( presence of isradipine as shown in the firing sites occurrence maps (Supplemental Fig. 1 C&D). 256 PTCL area was reduced to 18 ± 5 % (Supplemental Fig. 1 E-G; n = 7), and PTCL count was 257 reduced to 19.5 ± 6 % (Supplemental Fig. 1 H; n = 7). The number of firing sites was inhibited 258 by isradipine to 21.5 ± 6% (Supplemental Fig. 1 I;  Cacna1g. Therefore, the role of T-type Ca 2+ channels in modulating Ca 2+ signaling in ICC-SM 268 was evaluated with the specific T-type channel antagonists NNC 55-0396 (10 M), TTA-A2 (10 269 M) and Z-944 (1 M). NNC 55-0396 reduced Ca 2+ transient firing ( Fig. 10 A&B), Ca 2+ 270 transients firing sites occurrence maps (Fig. 10 C&D) and firing sites. PTCL area and PTCL 271 count traces show a reduction in Ca 2+ transient firing (Fig. 10 E&F). PTCL area was reduced to 272 31.7 ± 3.3 % (Fig. 10 E-G; n = 9) and PTCL count was reduced to 35.6 ± 4.5 % (Fig. 10 H;  The effects of nicardipine were tested in the presence of pinacidil. Under these conditions 304 nicardipine significantly reduced Ca 2+ transients in ICC-SM (Fig. 12 G&H; n = 5). PTCL area 305 was reduced to 38.5 ± 7.6 % (Fig. 12 G; n = 5) and PTCL count was reduced to 42.3 ± 9.1 % 306 ( Fig. 12 H; n = 5). In some regards these results were surprising as membrane potential 307 hyperpolarization might reduce contributions from L-type Ca 2+ channels (CaV 1.2). One 308 explanation is that CaV 1.3, which are abundant in ICC-SM and activate at more negative 309 potentials than CaV 1.2 [64] may contribute to Ca 2+ entry at more hyperpolarized potentials. 310 Further addition of NNC 55-0396 (10 M) inhibited Ca 2+ transients in ICC-SM to a greater 311 extent. PTCL area was reduced to 9.0 ± 2 % (Fig. 12 G; n = 5), and PTCL count was reduced to 312 11.2 ± 1.9 % (Fig. 12 H; n = 5). The utilization of two Ca 2+ conductances with different ranges 313 of voltage-dependent activation for the initiation of CTCs provides a safety factor that insures 314 persistence of pacemaker activity over a broad range of membrane potentials. 315 Inhibiting voltage dependent Ca 2+ channels in the presence of pinacidil unmasked 316 underlying Ca 2+ transients that occurred more randomly than the clustered transients occurring 317 normally (Fig. 12F&I). We tabulated the number of Ca 2+ events in the intervals between CTCs 318 (calculated from a period of 2s before the onset a CTC). Underlying Ca 2+ events were more 319 frequent in the presence of pinacidil and nicardipine and increased again upon addition of NNC 320 55-0396 (

Contributions of intracellular Ca 2+ stores and release channels in ICC-SM Ca 2 activity 324
Previous studies have demonstrated that Ca 2+ signaling in ICC-MY in the small intestine 325 depends not only on Ca 2+ influx but also on Ca 2+ release from intracellular stores [44]. Ca 2+ 326 release from stores is also critical for generation of pacemaker currents and slow waves [65][66][67]. 327 The role of Ca 2+ release mechanisms in Ca 2+ signaling in ICC-SM was also evaluated. 328 Thapsigargin (1 M; A SERCA pump antagonist) reduced, but did not block, Ca 2+ transient 329 firing in ICC-SM ( Fig. 13 A-D). PTCL area was reduced to 29 ± 12% ( The effects of 2-APB could be non-specific and may include effects on store-operated 364  Fig. 8 B), so the role of SOCE in 367 maintenance of Ca 2+ transients was examined using an Orai antagonist. GSK 7975A (10 M; An 368 Orai antagonist) reduced the firing frequency of CTCs (Fig. 16 A&B). Firing site occurrence 369 ( Fig. 16 C&D) and PTCL counts and areas were reduced (Fig. 16 E&F). Ca 2+ PTCL area was 370 reduced to 42.4 ± 9.4 % (Fig. 16 E-G; n = 7) and PTCL count was reduced to 48 ± 7 % ( Fig. 16  371 H; n = 7). The number of firing sites was also inhibited by GSK 7975A to 47.5 ± 4.1% (Fig. 16  In this study we developed a new preparation in which ICC-SM adherent to the 415 submucosa was used to allow very high-resolution imaging without complications from 416 muscular contractions. Preparations of this type may be valuable for future studies of cellular 417 mechanisms responsible for pacemaker activity and factors that regulate or degrade pacemaker 418 activity in pathophysiological conditions. 419 The pacemaker function of ICC-SM was demonstrated in a novel manner by 420 simultaneous two color optogenetic imaging with green (GCaMP6f) and red (RCaMP1 The importance of Ca 2+ entry as the primary means of activation and organization of 496 pacemaker activity in ICC-SM was shown by the dyscoordination of Ca 2+  four important functions in the pacemaker activity of ICC-SM: i) Propagation of activity within 554 the ICC-SM network depends upon voltage-dependent Ca 2+ entry, and the functions and voltage-555 dependent properties of three types of Ca 2+ conductances appear to provide a safety factor that 556 tends to preserve pacemaker activity over a broad range of membrane potentials (see Fig. 17). ii) 557 Ca 2+ entry is the mechanism that organizes Ca 2+ release events into CTCs. These events 558 constitute the Ca 2+ waves that propagate through ICC-SM networks, and presumably by 559 activation of Ano1 channels cause slow wave depolarizations. iii) Ca 2+ entry also appears to 560 contribute to refilling of stores, as pacemaker activity was not as immediately dependent upon 561 SOCE as in other ICC [49,81]. iv) The observations that treatments expected to reduce Ca 2+ 562 release from stores and reduce coupling between Ca 2+ entry and CICR reduced, but did not block 563 Technology, Belfast, UK) to increase laser intensity and uniformity throughout the imaging field 645 of view (FOV). The system also has two high-speed electron multiplying charged coupled 646 devices (EMCCD) cameras (Andor iXon-Ultra 897 EMCCD Cameras; ANDOR Technology, 647 Belfast, UK) to allow dual-color imaging simultaneously and maintain sensitive and fast speed 648 acquisition at full frame of 512x512 active pixels as previously described (Baker et al., 2015). 649 Briefly, images were captured, and image sequences were collected at 33 to 50 fps using       release from stores is not the primary pacemaker event but a secondary response to Ca 2+ entry.

ICC-SM IHC ICC-SM Cop-kit
Inhibition of Ca 2+ release from stores would lead to reduced probability of CICR and decrease the frequency of CTCs. Our hypothesis is that Ca 2+ entry and/or release from stores activates Ca 2+ -dependent Clcurrent due to ANO1 in the plasma membrane. Active propagation between cells in ICC networks (Phase 2) was inhibited by blocking voltage-dependent Ca 2+ channels.
Active propagation may also require or depend upon amplification of Ca 2+ in microdomains by CICR. The duration of Ca 2+ entry is likely to be brief due to voltage-dependent inactivation of L-and T-type Ca 2+ channels. The duration of CTCs appears to be enhanced by CICR (Phase 3).
Our data show that the duration of CTCs is reduced by several manipulations known to inhibit Ca 2+ release from stores. In Phase 4 store reloading may occur by multiple mechanisms and may include: i) transient Ca 2+ entry via sparklets, ii) activation of SOCE via STIM/ORAI interactions, and iii) the increase in Ca 2+ entry that occurs via depolarization and activation of Ca 2+ entry at