The plasma calcium level is one of the most precisely regulated constants of the internal environment, and the large reservoir of calcium in the skeleton is primarily responsible for this homeostasis. The experiments presented in this thesis were designed to study quantitatively the regulation of plasma calcium. Acute hypocalcemia was induced by continuous intravenous EDTA infusion (a calcium chelating agent) at a known rate, and hypercalcemia was induced by intravenous calcium gluconate infusion. The rate used in most cases was 10 mg. calcium per kg. for one hour. Both mobilization and storage of calcium appeared to depend on equilibrium with a labile calcium storage pool in bone. The rate of storage or mobilization was shown to be proportional to the amount of blood coming in contact with this labile pool in bone (bone blood flow), and the plasma/bone difference in Ca++ activity. Bone blood flow was measured using the Pick Principle for calcium storage, and it was calculated to be 6.46 ± 0.60% of the cardiac output (14 dogs). The extracellular fluid calcium was also estimated and found to be 15.73 ± 0.72 mg/kg (14 dogs), corresponding to an extracellular fluid volume of approximately 20% of body weight. Less than 5% of the injected calcium was excreted in the urine. The labile calcium storage pool in bone was estimated from the changes in the bone-blood equilibrium after calcium was injected, and was found to be 2 - 5 times greater than the extracellular calcium. The net loss of calcium from the plasma after calcium injection, which is assumed to equal the rate at which calcium is used for bone mineralization less calcium released by resorption, was estimated as 1 - 2 mg. Ca/kg/hr. or 0.15 - 0.35% of the total bone calcium per day. The methods described provide a means of assessing quantitatively the factors involved in acute regulation of the plasma calcium level. / Medicine, Faculty of / Cellular and Physiological Sciences, Department of / Graduate

Call number: LD2668 .T4 1962 U68

published_or_final_version / Biochemistry / Master / Master of Science

published_or_final_version / Physiology / Master / Master of Philosophy

published_or_final_version / Physiology / Master / Master of Philosophy

Calcium plays an essential role in the physiology and biochemistry of many biological functions, including excitation-contraction coupling, neuronal signaling, and fertilization. In mammals, the calcium content in various tissues, organs, and cell types is tightly regulated to maintain homeostasis. A chief process controlling calcium levels is absorption of the ion from the lumen by epithelial cells that line organs including the intestines and kidney. Calcium entry at the apical membrane constitutes the first step of epithelial calcium absorption. Two highly calcium-selective transient receptor potential vanilloid (TRPV) channels, TRPV5 and TRPV6, are the pore-forming subunits responsible for epithelial calcium entry in kidney and intestine, respectively. Genetic knockout of TRPV5 or TRPV6 in animals leads to phenotypes related to defective calcium homeostasis, including lowered serum calcium levels, decreased calcium absorption, reduced bone density, impaired sperm motility, and decreased maternal-fetal calcium transfer. In humans, aberrant TRPV5/6 expression is associated with preeclampsia and calcium nephrolithiasis (kidney stones). Additionally, TRPV6 expression level is upregulated in carcinomas of prostate, colon, breast, thyroid, and ovary, suggesting a role for TRPV6 in cancer survival. A detailed understanding of epithelial calcium entry is hindered by a lack of high-resolution structural information on intact channels. This dissertation presents structural analyses of the epithelial calcium channel TRPV6. We applied modern membrane protein screening and expression techniques, including fluorescence-detection size exclusion chromatography (FSEC) and baculovirus mediated mammalian cell transduction (BacMam), to identify optimal TRPV6 constructs and purification schemes for crystallization. Using a surface mutagenesis approach guided by lower-resolution structural solutions, we engineered a rat TRPV6 mutant (TRPV6cryst) that permitted solving a 3.25 Å resolution crystal structure. We used fluorescent calcium indicator assays to show that TRPV6cryst retains the permeation and ionic block properties of the wild type channel. The tetrameric structure of TRPV6cryst reveals a transmembrane domain architecture similar to voltage gated ion channels, with the ion conducting pore coincident with the overall four-fold symmetry axis. A ring of aspartate (D541) residues, shown in previous studies as a critical determinant of calcium selectivity, forms a narrow constriction at the extracellular pore entrance, or selectivity filter. Methionine (M577) side chains in the lower portion of the channel pore plug the conduction pathway and define the closed state of the channel. The ankyrin repeat domain, linker domain, N-terminal helix, and C-terminal hook form an intracellular skirt surrounding a cavity that lies beneath the pore axis. Close interactions between these domains, in large part mediated by the N-terminal helix, suggest that they are involved in allosteric modulation or concerted movements associated with channel activation. To shed light on the structural bases of permeation and ionic block, we cocrystallized TRPV6cryst with the permeant cations Ca²⁺ and Ba²⁺, and the channel blocker Gd³⁺. We identified binding sites for these cations by exploiting their anomalous scattering properties. On the basis of the cation-binding sites, we propose a permeation mechanism in which cations are recruited toward the pore by electronegative side chains in the extracellular vestibule, followed by sequential binding at least three binding sites along the central pore axis. Ca²⁺ selectivity is apparently achieved by high-affinity binding to the ring of D541 side chains in the selectivity filter. Gd³⁺ blocks permeation by similarly binding to the D541 ring and outcompeting ions of lesser charge. The results described in this dissertation provide a structural framework to further study mechanisms of epithelial calcium entry in health and disease.

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published_or_final_version / Biochemistry / Master / Master of Philosophy