title: Direct evidence for calcineurin binding to the exon-7 loop of the sodium-bicarbonate cotransporter NBCn1.
abstract: The NaHCO3 cotransporter NBCn1 plays a role in neutralizing intracellular acid loads at the basolateral membrane in cells of the medullary thick ascending limb (mTAL). Calcineurin inhibitors (Cn-Is) are known to both downregulate NBCn1 expression in the distal nephron and cause renal tubular acidosis (RTA), a risk factor for nephrocalcinosis and nephrolithiasis. In this report, we provide a new perspective on concurrent studies of NBCn1 in various tissues by using cell-free binding assays to investigate the interaction of NBCn1 with the calcineurin (Cn) isoform PPP3CA. Surface plasmon resonance (SPR) analyses show that the protein domain Exon 7 (translated from cassette II of NBCn1) binds Cn with an equilibrium dissociation constant (KD) of 30 +/- 15 nm. Linked-reaction tests suggest that the binding involves a conformational change. Nested PCR reactions were used to show that NBCn1-Exon 7 splice variants with alternative N-termini regions are expressed in the kidney, as well as other tissues. Additionally, we discuss NBCn1-Exon 7 implication in acid-base balance and calcium crystallization in the kidney.
authors: 1. Department of Medicine, The George Washington University, Washington, DC 20052 ; 2. Division of Renal Diseases and Hypertension, The George Washington Medical Faculty Associates, Washington, DC 20037.
authors: Harindarpal S
authors: HS
authors: Gill
authors: 3. GE Healthcare, Piscataway, NJ 08554.
authors: Eric D
authors: ED
authors: Roush
authors: 4. Hospital of University of Pennsylvania, Philadelphia, PA 19104.
authors: Lauren
authors: L
authors: Dutcher
authors: 2. Division of Renal Diseases and Hypertension, The George Washington Medical Faculty Associates, Washington, DC 20037.
authors: Samir
authors: S
authors: Patel
mesh_terms: D001665:Binding Sites; D019703:Calcineurin / Q000378:metabolism*; D002474:Cell-Free System; D005091:Exons; D006801:Humans; D007668:Kidney / Q000378:metabolism; D020033:Protein Isoforms; D017434:Protein Structure, Tertiary; D027982:Sodium-Bicarbonate Symporters / Q000737:chemistry / Q000378:metabolism*; D020349:Surface Plasmon Resonance
chemical_list: D020033:Protein Isoforms; C422022:SLC4A7 protein, human; D027982:Sodium-Bicarbonate Symporters; D019703:Calcineurin
keywords: bicarbonate; calcineurin; renal physiology; surface plasmon resonance; transporters