• Intracellular calcium regulation in vagal sensory neurons

      Auston, Darryl Allen; Kao, Joseph P. Y. (2007)
      Ca2+ regulates many cellular processes. An important mechanism for increasing cytosolic Ca2+ concentration ([Ca2+] i) is release from endoplasmic reticulum (ER) stores gated by ryanodine receptors (RyRs) and IP3 receptors (IP3Rs). The released Ca2+ can be taken up by mitochondria. Mitochondrial Ca 2+ uptake is believed to serve two functions. First, mitochondria act as a reversible store for Ca2+. Second, Ca2+ sequestration into mitochondria can regulate energy production. I have investigated the nature of the intracellular Ca2+ stores in nodose ganglion neurons (NGNs)---specifically whether the stores are functionally distinct. I have also examined the relationship between mitochondria and the intracellular Ca2+ stores. To determine if intracellular Ca2+ stores in NGNs are physiologically distinct, I used microfluorimetry, together with caffeine, a classic RyR agonist, and intracellular photorelease of caged IP3 (cIP3) to show that depletion of the RyR-gated store does not prevent Ca2+ release from the IP3R-gated store. Likewise, depletion of the IP3R-gated store does not prevent release from the RyR-gated store. Moreover, thapsigargin (TG) and cyclopiazonic acid (CPA), two inhibitors of the sarcoplasmic/endoplasmic reticulum Ca2+-ATPases, are able to deplete the RyR-gated Ca2+ store more effectively than the IP3R-gated Ca2+ store. These results indicate that the two Ca2+ stores are functionally distinct. Furthermore, using photorelease of cIP3 and BiNiX, a caged agonist of RyRs, I demonstrated the presence of functional IP3Rs and RyRs in the neurite extensions of cultured and acutely-dissociated NGNs. In examining mitochondria's role in Ca2+ homeostasis, I used microfluorimetry to show mitochondria can buffer increases in [Ca 2+]i caused by CICR, but not Ca 2+ influx through voltage-dependent Ca2+ channels. Simultaneous imaging of NADH/FAD+ fluorescence and intra-mitochondrial Ca2+ concentration ([Ca2+] m) revealed that mitochondrial buffering of CICR causes concomitant rises in [Ca2+]m and mitochondrial metabolism. I provide evidence that suggests a constitutive ER-to-mitochondria Ca 2+ flux. Importantly, variations of this ER-to-mitochondria Ca 2+ flux cause corresponding changes in energy metabolism, as reflected by changes in mitochondrial NADH levels. These results indicate that mitochondria are able to monitor and respond to the energy needs of the cell by buffering Ca2+ release through RyRs and by sensing the ER-to-mitochondria Ca2+ flux.
    • Mitochondrial regulation of Ca2+ released from the intracellular stores

      Li, Gong; Kao, Joseph P. Y. (2012)
      Ca2+ is a ubiquitous second messenger that can be released from intracellular Ca2+ stores in the endoplasmic reticulum (ER) through ryanodine receptor (RyR) channels and IP3 receptor (IP3R) channels. The released Ca2+ is a cytosolic Ca2+ signal, which can regulate diverse cellular functions. Ca2+ clearance mechanisms are important because they directly affect the spatio-temporal characteristic of the Ca+ signal. Mitochondria have the capacity to take up cytosolic Ca2+ and are thus a key Ca2+ clearance mechanism. Mitochondrial Ca2+ uptake impacts cellular physiology in two ways. The first is the effect of mitochondria in sculpting cytosolic Ca2+ signals. The second is the effect of Ca2+ uptake on energy production by the mitochondria. In the present studies, I examined the relationship between mitochondria and Ca2+ released from the ER through both RyRs and IP3Rs in vagal sensory neurons. In the course of the work, diverse techniques were used, including patch clamp electrophysiology, focal photolysis of caged molecules to achieve intracellular generation of second messengers, as well as confocal imaging of fluorescent indicators of cytosolic and mitochondrial Ca2+ concentration, fluorescent organellar stains, and fluorescent indicators of mitochondrial membrane potential. The key findings of the studies are: 1) Mitochondria in the sub-plasma-membrane region preferentially take up Ca2+ released from ER through RyR channelscompared with mitochondria in the cell interior. 2) When sub-plasma-membrane RyRs are activated locally, the resulting cytosolic Ca2+ signal decays back to baseline more slowly than Ca2+ signals resulting from activation of RyRs in the cell interior; this is a consequence of differential Ca2+ handling by mitochondria near the plasma membrane. 3) Preferential Ca2+ uptake by sub-plasma-membrane mitochondria is a consequence of these mitochondria having a more hyperpolarized membrane potential. 4) Upon metabotropic activation of IP3 production and consequent IP3R-mediated Ca2+ release, sub-plasma-membrane mitochondria show preferential Ca2+uptake. 5) Increase in mitochondrial Ca2+concentration following uptake drives increased NADH production.