UVM Theses and Dissertations
Format:
Print
Author:
Ponissery Saidu, Samsudeen
Dept./Program:
Biology
Year:
2010
Degree:
PhD
Abstract:
Binding of odorant molecules to receptors on the cilia of olfactory sensory neurons (OSNs) starts a signal transduction cascade that causes an increase in intracellular Ca². Ca²⁺ not only excites the cell by opening Ca²⁺-activated cr channels but it also negatively feeds back on the transduction pathway. The feedback by Ca²⁺ is mediated through different molecular mechanisms, all of which leads to adaptation of the cell to the stimulating odorant. To recover from Ca²⁺-dependent adaptation, Ca²⁺ must be cleared from the cilia and the dendritic knob of the neuron. Mammalian cells employ multiple mechanisms including the Na⁺/Ca²⁺ exchanger (NCX), sarco/endoplasmic reticulum Ca²⁺ ATPase (SERCA) and plasma membrane calcium ATPases (PMCA) to remove high intracellular Ca²⁺. Ca²⁺ clearance mechanisms in OSNs are not very well studied except for the NCX, the presence and functional importance of which has been demonstrated.
However, the activity of NCX alone cannot account for the low resting levels of Ca² observed in OSNs. Another mechanism with a higher affinity for Ca²⁺, like PMCA, must be functional. The work presented in this thesis focuses on the other major Ca²⁺ clearance mechanism in mammalian cells -- the plasma membrane calcium ATPases (PMCA) --and its contribution to Ca²⁺ clearance from OSNs after stimulation. By pharmacologically inhibiting PMCAs with carboxyeosin (CE) and through the use of a genetic knockout of a single isoform of PMCA (PMCA2KO) in mice, we demonstrate that loss of PMCA activity significantly reduces the rate of Ca²⁺ clearance from OSNs after stimulation with either odorants or IBMXlForskolin.
We also show that the NCX and the SERCA pump contribute significantly to the clearance process. To test whether the impaired Ca² extrusion in the PMCA2KO or PMCA-inhibited cells has an effect on prolonging the duration of adaptation induced by a sustained stimulation, we compared the amplitude of Ca²⁺ responses from wild type (WT), PMCA2KO and CEtreated OSNs in response to two stimuli at varying intervals. Our results indicate that compared to the normal adaptation of WT OSNs, the PMCA2KO and CE-treated OSNs take longer to recover from the first stimulus. Also, we demonstrate that the adaptation induced with 8 sec application of IBMXlForskolin is mediated through Ca²⁺-calmodulin dependent kinase II (CaMKII).
However, the activity of NCX alone cannot account for the low resting levels of Ca² observed in OSNs. Another mechanism with a higher affinity for Ca²⁺, like PMCA, must be functional. The work presented in this thesis focuses on the other major Ca²⁺ clearance mechanism in mammalian cells -- the plasma membrane calcium ATPases (PMCA) --and its contribution to Ca²⁺ clearance from OSNs after stimulation. By pharmacologically inhibiting PMCAs with carboxyeosin (CE) and through the use of a genetic knockout of a single isoform of PMCA (PMCA2KO) in mice, we demonstrate that loss of PMCA activity significantly reduces the rate of Ca²⁺ clearance from OSNs after stimulation with either odorants or IBMXlForskolin.
We also show that the NCX and the SERCA pump contribute significantly to the clearance process. To test whether the impaired Ca² extrusion in the PMCA2KO or PMCA-inhibited cells has an effect on prolonging the duration of adaptation induced by a sustained stimulation, we compared the amplitude of Ca²⁺ responses from wild type (WT), PMCA2KO and CEtreated OSNs in response to two stimuli at varying intervals. Our results indicate that compared to the normal adaptation of WT OSNs, the PMCA2KO and CE-treated OSNs take longer to recover from the first stimulus. Also, we demonstrate that the adaptation induced with 8 sec application of IBMXlForskolin is mediated through Ca²⁺-calmodulin dependent kinase II (CaMKII).