Synaptotagmin I binds intestinal epithelial NHE3 and mediates cAMP- and Ca2+-induced endocytosis by recruitment of AP2 and clathrin

Mark W. Musch, Donna L. Arvans, Margaret M. Walsh-Reitz, Kazuhiko Uchiyama, Mitsunori Fukuda, Eugene B. Chang

Research output: Contribution to journalArticlepeer-review

33 Citations (Scopus)


Apical membrane sodium hydrogen exchanger 3 (NHE3), a major pathway for non-nutrient-dependent intestinal Na+ absorption, is tightly regulated by second messenger systems that affect its functional activity and membrane trafficking. However, the events and components involved in NHE3 regulation are only partially understood. We report that the adaptor protein synaptotagmin I (Syt I) plays a pivotal role in cAMP- and Ca2+- induced cargo recognition of NHE3 and initiation of its endocytosis. Both mouse small intestine (jejunum) and Caco-2BBe Syt I coimmunoprecipitated with NHE3, particularly following increases in cellular cAMP or Ca2+. Following short interfering RNA (siRNA) suppression of Syt I expression, cAMP- and Ca 2+-induced inhibition of NHE3 activity were still observed but NHE3 endocytosis was blocked, as assessed by 22Na influx and apical membrane biotin labeling, respectively. Similar effects on NHE3 inhibition and endocytosis were observed by siRNA suppression of either the μ-subunit of the adaptor protein 2 (AP2) complex or the heavy chain of clathrin. Coimmunoprecipitation analyses of NHE3 with these adaptor proteins revealed that cAMP- and Ca2+-induced NHE3-Syt I interaction preceded and was required for recruitment of AP2 and the clathrin complex. Confocal microscopy confirmed both the time sequence and protein associations of these events. We conclude that Syt I plays a pivotal role in mediating cAMP- and Ca 2+-induced endocytosis of NHE3 (but not in inhibition of activity) through cargo recognition of NHE3 and subsequent recruitment of AP2-clathrin assembly required for membrane endocytosis.

Original languageEnglish
Pages (from-to)G1549-G1558
JournalAmerican Journal of Physiology - Gastrointestinal and Liver Physiology
Issue number6
Publication statusPublished - 2007 Jun


  • Adaptor protein 2
  • Caco-2BBe cells
  • Calcium
  • Cyclic nucleotides
  • Diarrheal diseases
  • Intestinal Na absorption
  • Membrane trafficking
  • Sodium/hydrogen exchange
  • Water and electrolyte transport


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