Na-K-2Cl Cotransporter and Store-Operated Ca2+ Entry in Pacemaking by Interstitial Cells of Cajal

Abstract

Pacemaker depolarization in interstitial cells of Cajal (ICCs) is believed to be induced by Ca 2þ transients and activation of anoctamin-1 (Ano1) channels in the plasma membrane. However, block of store-operated calcium entry (SOCE) or the Na-K-2Cl cotransporter (NKCC1) terminates pacemaker activity in ICC, indicating these transporters are involved in the initiation or maintenance of pacemaker activity. We hypothesized that SOCE contributes to pacemaker depolarization by maintaining [Ca 2þ ] in the endoplasmic reticulum, which is the underlying source of Ca 2þ transients for activation of Ano1. NKCC1 maintains the Cl À gradient supporting the driving force for inward current mediated by Ano1. Currently mechanisms sustaining release of Ca 2þ and activation of Ano1 channels during the plateau phase of slow waves are unknown, but the reverse mode of the Na þ / Ca 2þ exchange may contribute. We generated a mathematical model of pacemaker activity based on current empirical observations from ICC of mouse small intestine that incorporates functions of SOCE and NKCC1. This model reproduces experimental findings, suggesting roles for SOCE and Ano1 channels: blocking of either NKCC1 or SOCE in our model terminates pacemaker activity. Direct contribution of NKCC1 to pacemaker activity in a beat-to-beat manner is not predicted by our model. Instead, NKCC1 plays a maintenance role supporting the driving force for Cl À efflux. Incorporation of SOCE allows the model to drive pacemaker activity without a diastolic depolarization, as observed in cardiac pacemaking. Further biological experiments are necessary to validate and further refine the roles of NKCC1, Na þ /Ca 2þ exchange, and Ano1 in the pacemaker mechanism of ICC.

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

“…Previously we and others have addressed the role of Ca 2+ entry and Ca 2+ release in the initiation of pacemaker activity in ICC (van Helden et al, 2000;Hirst et al, 2002;van Helden and Imtiaz, 2003;Park et al, 2006;Drumm et al, 2017), however, much about the dynamics and sources of Ca 2+ in ICC is not yet understood. Modeling of slow waves (Youm et al, 2019) and empirical data obtained in the current study suggest that NCX switches from Ca 2+ exit mode to Ca 2+ entry mode during the slow wave cycle. The Ca 2+ exit mode clears Ca 2+ from the proximity of ANO1 channels maintaining a low open probability for these channels during the inter-slow wave interval.…”

“…Efflux of Cl − would decrease during the plateau phase of slow waves because the ICC depolarize close to E Cl . To maintain Cl − homeostasis, Cl − recovery must occur, and previous studies and modeling have suggested a role for the Na-K-Cl co-transporter (NKCC1) in this process (Wouters et al, 2006;Zhu et al, 2016;Youm et al, 2019). NKCC1 utilizes energy from the Na + gradient to concentrate Cl − ions in cells (Russell, 2000) and recover Cl − lost during slow waves.…”

“…Consistent with this idea, we found that ouabain, at concentrations likely to be specific for the α2 isoform of the Na + pump, lengthened the duration of slow wave currents. For such a mechanism to function, as predicted by modeling (Youm et al, 2019), the changes in ion concentrations hypothesized would need to occur in restricted volumes (i.e., microdomains) within ICC because it is unlikely that slow wave currents would result in appreciable reduction in cytoplasmic [Cl − ] or significant increases in cytoplasmic [Na + ].…”

“…Modeling has suggested that a shift in the Na + gradient, due to Na + entry from the action of the Na + K + Cl − cotransporter (NKCC1), may facilitate slow wave currents due to the Ca 2+ entry mode of NCX (Youm et al, 2019). Restoration of the Na + gradient is likely to occur via the actions of Na + K + ATPase (Na + pump).…”

“…Ca 2+ entry via the Ca 2+ entry mode of NCX has been reported in various cells (Leblanc and Hume, 1990;Luther et al, 1992), and pacemaker activity in urethra interstitial cells and ICC in the small intestine have been suggested to be linked to NCX operating in Ca 2+ entry mode (Bradley et al, 2006;Lowie et al, 2011;Drumm et al, 2015). The function of NCX and the proximity between NCX proteins and ANO1 channels in microdomains within cells may be contributing factors in activation and deactivation of CaCC in ICC and in the sustained activation of CaCC during the slow wave plateau phase, as recent modeling of slow waves suggests (Youm et al, 2019).…”