Integrated calcium and proton signaling during a rhythmic behavior in C. elegans

Thesis (Ph.D.)--University of Rochester. School of Medicine & Dentistry. Dept. of Pharmacology and Physiology, 2014.Biological rhythms are intrinsic to nearly all organisms; yet, they can also be very complex. The genetic model organism C. elegans exhibits several behavioral rhythms that have been studied using integrative physiology. One rhythm in particular, the defecation motor program (DMP), has been shown to be timed by cell-autonomous calcium oscillations. Subsequent trans-epithelial proton fluxes contribute to the DMP’s behavioral output as well as to physiologic events such as development and lifespan. In addition to defining mechanisms that contribute to oscillatory calcium signaling, studies focused on this behavior have emphasized the importance of pH homeostasis and have led to the identification of a novel role for protons in signaling. The work presented here demonstrates that the establishment of an apical intestinal proton gradient, driven by a proton V-ATPase, is necessary for nutrient uptake and development. Proton movement across the basolateral membrane, through the Na+/H+ exchanger NHX-7, is also critical for proper execution of the DMP but not required for pH homeostasis or development. This flux signals posterior body wall muscle contraction and coincides with the underlying IP3R-mediated calcium wave, suggesting calcium-dependent regulation. Structure-function analyses of NHX-7 revealed that its activity is impacted by calcium signaling and feedback from the membrane proton/sodium gradient. Interestingly, the structure-function analysis also indicated that disruption of a consensus binding site for a calcineurin homologous protein, PBO-1, altered the distribution of NHX-7. Genetic analyses further revealed that loss of PBO-1 also affects the localization of the NHX-2 isoform in the apical membrane, which helps to reestablish pH homeostasis following defecation. This ultimately results in a phenotype that resembles a global disruption of intestinal proton transport. The localization deficit appears to be an effect on membrane stability/retention rather than forward protein trafficking. Collectively, these studies support the idea that proton and calcium oscillations synergize to execute the DMP as a means to maintain the intestinal membrane proton motive force. Thus, in addition to contributing to our understanding of oscillatory calcium signaling, pH homeostasis, and proton signaling, our results show that the defecation motor program is an appropriate model to study and potentially target biological rhythms therapeutically