Cell volume regulation: Function, identification and biochemical control of inorganic ion transport in red blood cells

Volume regulation is a fundamental physiological process featured by the cells of most vertebrate species. In response to swelling, cells undergo net efflux of solutes and osmotically obliged water in a process known as regulatory volume decrease (RVD). In response to shrinkage, cells undergo a net influx of solutes and osmotically obliged water in a process known as regulatory volume increase (RVI). During RVD and RVI, net fluxes of solutes and osmotically obliged water tend to restore volume-perturbed;,cells to normal resting volume. This dissertation focuses on two central topics of cell volume regulation research: (1) Identification of volume regulatory ion transporters, and (2) Biochemical control of the volume regulatory Na/H exchanger during osmotic cell shrinkage. I utilize the classical model of cell volume regulation, the red blood cell (RBC) to study cell volume regulation and volume regulatory ion transporters.; The K+ efflux pathway active during RVD in the Winter flounder RBC is K-Cl co-transport (KCC). Winter flounder KCC is functionally similar to mammalian KCC1, yet is insensitive to furosemide, and can perform K-sulfamate cotransport. The Na+ flux pathway active during RVI in the Winter flounder RBC is Na/H exchange (NHE). Winter flounder NHE is a homologue of the mammalian NHE1 isoform that is uniquely insensitive to amiloride- and benzoyl guanidine- derivatives, as verified with chimeras of human and flounder NHE1. I have further demonstrated that NHE1 can be purified and functionally reconstituted in liposomes, and hence can be used to study the protein structural basis for NHE1 function and inhibition.; In the Amphiuma RBC, I have demonstrated that net phosphorylation of NHE1 is increased during osmotic cell shrinkage, but is masked by simultaneous increases and decreases in phosphorylation at separate loci. I have dismissed the hypothesis that NHE1 activity is increased by vesicle trafficking, and provide kinetics evidence that activation of NHE1 during osmotic cell shrinkage is dependent on increased protein phosphorylation. I also provide kinetic evidence that NHE1 is activated by a shrinkage-induced kinase that is governed by cytosolic macromolecular crowding.