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Na+-K+-Cl- cotransporter (NKCC) maintains the chloride gradient to sustain pacemaker activity in interstitial cells of Cajal.

ABSTRACT: Interstitial cells of Cajal (ICC) generate electrical slow waves by coordinated openings of ANO1 channels, a Ca2+-activated Cl- (CaCC) conductance. Efflux of Cl- during slow waves must be significant, as there is high current density during slow-wave currents and slow waves are of sufficient magnitude to depolarize the syncytium of smooth muscle cells and PDGFR?+ cells to which they are electrically coupled. We investigated how the driving force for Cl- current is maintained in ICC. We found robust expression of Slc12a2 (which encodes an Na+-K+-Cl- cotransporter, NKCC1) and immunohistochemical confirmation that NKCC1 is expressed in ICC. With the use of the gramicidin permeabilized-patch technique, which is reported to not disturb [Cl-]i, the reversal potential for spontaneous transient inward currents (ESTICs) was -10.5 mV. This value corresponds to the peak of slow waves when they are recorded directly from ICC in situ. Inhibition of NKCC1 with bumetanide shifted ESTICs to more negative potentials within a few minutes and reduced pacemaker activity. Bumetanide had no direct effects on ANO1 or CaV3.2 channels expressed in HEK293 cells or L-type Ca2+ currents. Reducing extracellular Cl- to 10 mM shifted ESTICs to positive potentials as predicted by the Nernst equation. The relatively rapid shift in ESTICs when NKCC1 was blocked suggests that significant changes in the transmembrane Cl- gradient occur during the slow-wave cycle, possibly within microdomains formed between endoplasmic reticulum and the plasma membrane in ICC. Recovery of Cl- via NKCC1 might have additional consequences on shaping the waveforms of slow waves via Na+ entry into microdomains.

SUBMITTER: Zhu MH

PROVIDER: S-EPMC5206290 | biostudies-literature | 2016 Dec

REPOSITORIES: biostudies-literature

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Na+-K+-Cl- cotransporter (NKCC) maintains the chloride gradient to sustain pacemaker activity in interstitial cells of Cajal.

Zhu Mei Hong MH Sung Tae Sik TS Kurahashi Masaaki M O'Kane Lauren E LE O'Driscoll Kate K Koh Sang Don SD Sanders Kenton M KM

American journal of physiology. Gastrointestinal and liver physiology 20161013 6

Interstitial cells of Cajal (ICC) generate electrical slow waves by coordinated openings of ANO1 channels, a Ca2+-activated Cl- (CaCC) conductance. Efflux of Cl- during slow waves must be significant, as there is high current density during slow-wave currents and slow waves are of sufficient magnitude to depolarize the syncytium of smooth muscle cells and PDGFRα+ cells to which they are electrically coupled. We investigated how the driving force for Cl ...[more]

PMID: 27742704

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Project description: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.

Project description:Key pointsRhythmic action potentials and intercellular Ca2+ waves are generated in smooth muscle cells of colonic longitudinal muscles (LSMC). Longitudinal muscle excitability is tuned by input from subserosal ICC (ICC-SS), a population of ICC with previously unknown function. ICC-SS express Ano1 channels and generate spontaneous Ca2+ transients in a stochastic manner. Release of Ca2+ and activation of Ano1 channels causes depolarization of ICC-SS and LSMC, leading to activation of L-type Ca2+ channels, action potentials, intercellular Ca2+ waves and contractions in LSMC. Nitrergic neural inputs regulate the Ca2+ events in ICC-SS. Pacemaker activity in longitudinal muscle is an emergent property as a result of integrated processes in ICC-SS and LSMC.AbstractMuch is known about myogenic mechanisms in circular muscle (CM) in the gastrointestinal tract, although less is known about longitudinal muscle (LM). Two Ca2+ signalling behaviours occur in LM: localized intracellular waves not causing contractions and intercellular waves leading to excitation-contraction coupling. An Ano1 channel antagonist inhibited intercellular Ca2+ waves and LM contractions. Ano1 channels are expressed by interstitial cells of Cajal (ICC) but not by smooth muscle cells (SMCs). We investigated Ca2+ signalling in a novel population of ICC that lies along the subserosal surface of LM (ICC-SS) in mice expressing GCaMP6f in ICC. ICC-SS fired stochastic localized Ca2+ transients. Such events have been linked to activation of Ano1 channels in ICC. Ca2+ transients in ICC-SS occurred by release from stores most probably via inositol trisphosphate receptors. This activity relied on influx via store-operated Ca2+ entry and Orai channels. No voltage-dependent mechanism that synchronized Ca2+ transients in a single cell or between cells was found. Nitrergic agonists inhibited Ca2+ transients in ICC-SS, and stimulation of intrinsic nerves activated nitrergic responses in ICC-SS. Cessation of stimulation resulted in significant enhancement of Ca2+ transients compared to the pre-stimulus activity. No evidence of innervation by excitatory, cholinergic motor neurons was found. Our data suggest that ICC-SS contribute to regulation of LM motor activity. Spontaneous Ca2+ transients activate Ano1 channels in ICC-SS. Resulting depolarization conducts to SMCs, depolarizing membrane potential, activating L-type Ca2+ channels and initiating contraction. Rhythmic electrical and mechanical behaviours of LM are an emergent property of SMCs and ICC-SS.

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Na+-K+-Cl- cotransporter (NKCC) maintains the chloride gradient to sustain pacemaker activity in interstitial cells of Cajal.

Publications

Na+-K+-Cl- cotransporter (NKCC) maintains the chloride gradient to sustain pacemaker activity in interstitial cells of Cajal.

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