Calcium & Calcium-sensing receptors

Transcription

1 Calcium & Calcium-sensing receptors Prof Arthur D Conigrave School of Life & Environmental Sciences, University of Sydney Department of Endocrinology, Royal Prince Alfred Hospital

2 Collaborators Sydney Ryan Mun Alice Huang Vimesh Avlani Kim Woollett Sarah Brennan Rebecca Mason Mahvash Goolam Mark Rybchyn Alice Brown Leigh Delbridge Kim Edwards Rory Clifton-Bligh EU Multifaceted CaSR Consortium Enikő Kallay et al. Raj Thakker et al Daniela Riccardi et al. Frank Bruggeman et al. Romuald Mentaverri. Maria-Luisa Brandi et al. Monash (Melbourne) Katie Leach Anna Cook Patrick Sexton Arthur Christopoulos Manchester (UK) Donald Ward USA Edward Brown Qing Fan Wenhan Chang

3 The calcium-sensing receptor G-protein coupled receptor (Family C) mediates feedback regulation of calcium homeostasis

4 G-protein coupled receptors: Class C Taste Receptors

5 Regulation of plasma Ca 2+ by parathyroid hormone Parathyroid Plasma Ca 2+ PTH PTH Receptor Kidney Ca 2+ Reabsorption

6 Roles of calcium-sensing receptors in calcium metabolism Parathyroid X PTH Ca 2+ Kidney X X FILTRATE 2+ Ca 2+ Ca URINE

7 Regulation of renal calcium excretion by calcium-sensing receptors Lumen Interstitium Ca 2+ 3 K + 6 Cl - 3 Na + 5 K + 3Na + 2K + CaSR Ca 2+ 6 Cl - + Thick Ascending Limb 0 (cortical) High plasma Ca 2+ levels suppress: electrical gradient for Ca 2+ transport (acute signaling effect) divalent cation selectivity of paracellular path* *controls claudin-14 expression via mirna Gong Y et al. (2012) EMBO J. 31:

8 CaSR: calcium-sensing receptor CaSR expression reduced in Primary Hyperparathyroidism

9 Low CaSR expression in primary hyperparathyroidism: a low Ca 2+ sensitivity state Remnant normal cells CaSR expression Adenomatous cells Parathyroid adenoma with remnant normal tissue Kifor et al. JCEM 1996

10 Impaired Ca 2+ -mediated feedback in adenomatous human parathyroid cells HC Mun et al. (2009) JCEM 94,

11 Mutations of the CaSR underlie disorders of calcium metabolism 1. Inactivating mutations Familial Hypocalciuric Hypercalcemia (benign) Neonatal Severe Hyperparathyroidism (severe) 2. Activating mutations Autosomal Dominant Hypocalcemia

12 CaSR mutations explain inherited disorders of calcium: FHH and NSHPT FHH Usually benign Incidental finding in adults Hypercalcemia (Ca > 2.6 mm) Hypocalciuria (uca < 1 mmol d -1 ) PTH Normal NSHPT Life-threatening disease of neonates Severe Hypercalcemia (plasma total Ca 2+ > 3.5 mm) Primary Hyperparathyroidism (PTH High) Demineralization Pathological Fractures Inactivating mutations of CaSR FHH: Familial Hypocalciuric Hypercalcemia NSHPT: Neonatal Severe Hyperparathyroidism

13 CaSR mutations explain inherited disorders of calcium: FHH and NSHPT * Mutations: Family J R795W; Family E E297K; Family N R185E FHH: Familial Hypocalciuric Hypercalcemia NSHPT: Neonatal Severe Hyperparathyroidism Pollak et al. (1993) Cell 75,

14 CaSR mutations explain inherited disorders of calcium: ADH Autosomal Dominant Hypocalcemia Typically benign Hypocalcemia variable (can be impressive) Total Ca < 2.0 mm Ionized Ca < 1.0 mm Low or low-normal serum magnesium Normal-high serum phosphate PTH normal or just below normal Childhood Seizures (some cases) Hypercalciuria (uca > 6 mmol/d) in presence of calcitriol Nephrocalcinosis and Renal Failure

15 A family with hypocalcemia

16 Proband (II.3) Clinical Features 33 year old man (Townsville) : hypocalcemic on family screening Infrequent muscle cramps Paresthesiae on fine finger movements No seizures

17 Biochemical results Serum total calcium: 1.71 mmol/l ( mmol/l) Serum ionized calcium: 0.77 mmol/l ( mmol/l) Serum inorganic phosphate: 1.3 mmol/l ( mmol/l) Serum PTH: 1.0 pmol/l ( pmol/l) Serum magnesium: 0.66 mmol/l ( mmol/l) 24 h urinary calcium excretion: 0.7 ( mmol/day) Urinary Ca: creat ratio: 0.04 ( mmol/mmol)

18 Initial Treatment Caltrate 2 daily, calcitriol 0.25 µg bd 24 h U.Ca excretion: 9.4 ( mmol/day) Urine Ca:creat ratio: 0.46 ( mmol/mmol) Thiazide used to lower urinary calcium excretion

19 Other family members Hypocalcemia Mother (I.2) Two male siblings (II.2 and II.4) His son (III.2) One nephew (III.1) and one niece (III.3) Profile of Clinical features Childhood seizures (II.4, III.1, III.2, III.3) Infrequent muscle cramps only (I.1, II.2 II.3)

20 Inheritance of hypocalcemia in Family A I.1* II.1 * II.2 * II.3 * II.4 * CS II.5 * III.1 CS III.2 * CS III.3 CS CS: Childhood Seizures * DNA sample obtained

21 Structure of CaSR gene ATG 7A product G2182A PCR and restriction digest analysis exon 7 (7A primers) Wild-type: Taq1 Mutant: Taq1 site lost Before Taq1 After Taq1

22 Genotyping family members: PCR-restriction digest analysis Taq1-treated DNA Uncut Hypocalcemic A B C D E F G H Two unaffected family members

23 Molecular Analysis and Conclusions G2182A encodes missense mutation E604K E604K results in enhanced CaSR sensitivity to Ca 2+ Sensitized CaSRs shift the parathyroid set-point to: Low PTH levels are inadequate to maintain normal plasma Ca 2+ Urinary calcium excretion is inappropriately high

24 Conclusions and therapeutic considerations Hypocalcemic seizures in infants and children a significant risk Calcium supplements and calcitriol to adjust plasma ionized calcium to mmol/l Avoid hypercalciuria (nephrocalcinosis and renal failure is a significant risk) - thiazides Screen at-risk members of family Offspring of heterozygotes should be screened at birth for hypocalcemia

25 A new pharmacology Pharmacological modulators: disorders of calcium and bone metabolism Pathological mediators: CaSR as target in pathogenesis and therapy Physiological modulators

26 CaSR as therapeutic target: calcimimetics Receptor response Super-sensitizing parathyroid cells to Ca 2+ o Parathyroid cells NPS R467 NPS R568 HEK-CaR cells Cinacalcet Sensipar Nemeth et al. (1998) PNAS 95,

27 Therapeutic Applications of CaSR modulators A. Type II Calcimimetics (e.g., Cinacalcet) Primary Hyperparathyroidism Secondary Hyperparathyroidism Tertiary Hyperparathyroidism B. Calcilytics (e.g., NPS 2143) Osteoporosis: clinical trials disappointing Hypoparathyroid states (ADH) NEW Asthma: nebulised calcilytic effective in animal models* *Yarova PL et al. (2015) Sci Trans Med 7(284):284ra60

28 Cationic activators of the calcium-sensing receptor: OMG! Inorganic cations Calcium Magnesium Strontium Organic cations Neomycin Polyamines Cationic proteins Myelin Basic protein; Eosinophilic Cationic protein; Amyloid-beta proteins

29 Cationic activators of the calcium-sensing receptor: OMG! Inorganic cations Calcium Magnesium Strontium Organic cations Neomycin Polyamines Cationic proteins Myelin Basic protein; Eosinophilic Cationic protein; Amyloid-beta proteins

30 G-protein coupled receptors: Class C Nutrient receptors Taste Receptors

31 Is the CaSR an L-amino acid-sensing receptor?

32 Cytoplasmic Free [Ca 2+ ](nm) Receptor Activation by Ca 2+ and L-amino acids Experiment Time (min) Human parathyroid cells

33 PTH Secretion (fg min -1 cell -1 ) Impact of Amino Acids on Parathyroid Hormone Secretion Experiment Time (min)

34 Generation of amino acid signals in GIT

35 Expression of CaSRs in enteroendocrine cells CaSR Gastrin Gastrin release stimulated by : Calcium, L-Phe & other AAs, peptone, Neutral ph CaSR: a multimodal sensor for G-cell growth and gastrin secretion CaSR: calcium-sensing receptor Ray JM et al. (1997) JCI 99, Feng J et al. (2010) PNAS 107:

36 Amino acid sensors: mrna expression in CCK-secreting cells CaSR: calcium-sensing receptor CCK: Cholecystokinin Liou A P et al. Am J Physiol Gastrointest Liver Physiol 2011;300:G538-G546

37 Effects of amino acids on CCK secretion Isolated I cells Wang Y et al. Am J Physiol Gastrointest Liver Physiol 2011;300:G528-G537

38 Crystal structure: AA and calcium binding sites

39 Y. Geng et al. (2016) Elife 5, pii: e13662

40 Y. Geng et al. (2016) Elife 5, pii: e13662

41 L-amino acids bind in the VFT domain orthosteric site L-Trp binds in CaSR VFT domain cleft and facilitates closure Y. Geng et al. (2016) Elife 5, pii: e13662

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46 Conclusions CaSRs mediate feedback regulation of calcium metabolism: parathyroid, kidney CaSRs modulate bone metabolism: osteoblasts, osteoclasts, metastases in bone CaSRs modulate vitamin D metabolism Impaired expression contributes to Primary Hyperparathyroidism Inactivating mutations: Hypercalcemic states (FHH, NSHPT) Activating mutations: Hypocalcemic states (ADH) CaSRs: targets in pharmacotherapy of calcium metabolism disorders Hyperparathyroidism Hypoparathyroidism Osteoporosis Promiscuity for organic cations - pathophysiological mediators e.g., asthma Role in L-amino acid sensing (aromatics) nutrient control of hormone release Roles in coupling protein and calcium metabolism Binding sites for L-amino acids and calcium identified in extracellular domains

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