Alterations in membrane transport in response to environment and disease
Proteins in the membranes of cells regulate the intracellular composition, and allow the passage of nutrients and wastes. When the function of these pathways is disturbed, cellular function can be impaired and disease states can arise.
Transport can be modulated by a number of variables: we have at various times studied temperature (important during hibernation), salt adaptation, uraemia, malaria, sickle cell disease, cancer and hypertension. At present we are concentrating on three of these:
Sickle cell disease: The dehydration of sickle cells via loss of KCl leads to deoxyHbS polymerization and sickling. KCl loss occurs via the Gardos channel+ band3; KCC1 and KCC3; and a NSCC Psickle. In all cases an anomalous response to deoxygenation is a key signalling component of the transporter activation pathway in sickle cells. Thus in normal HbA red cells, deoxygenation leads to inhibition of KCC and no activation of other cation permeability pathways. In HbS cells, KCC is very active in deoxy conditions, and additionally Psickle as an NSCC is activated. Calcium entering via Psickle promotes the Gardos channel. We have shown that the oxygen effect is due to membrane-associated not bulk haemoglobin, and that Cl-sensitive kinases are involved.
Malaria: In its intraerythrocytic form the parasite induces a transport pathway for ions and small solutes which is involved in nutrition, waste removal and volume regulation. The origin (parasite or host) and nature of this pathway is controversial, but it represents a prime target for novel malaria chemotherapy, and Trojan horse delivery of antimalarials.
Cancer: Epithelial cancers upregulate a number of important membrane transporters, which are critical for progression of cell cycle, cell growth and volume regulation. The KCC family of cation chloride cotransporters is vital in this respect. Our Taiwanese collaborators have shown KCC3 and 4 are essential for invasion and proliferation of cervical cancer cells, and intracellular Cl represents a key control element.
Current Research Programme
Sickle cell disease
Using whole-cell patchclamp and tracer flux methodology we are looking at oxygen sensitivity of transport pathways in HbS cells. We are also using other manoeuvres such as peroxynitrite or phenazine methosulphate treatment to induce Psickle-like pathways in normal cells. We are screening Psickle inhibitors which have a potential as a treatment for sickling crises, and are setting up clinical trials with potential inhibitors. Ameliorating sickle crises is important in this debilitating disease, and we have a promising avenue for reducing their frequency. The likely membrane binding site for HbS is on the C-terminus of Band 3, and we are looking at this with small interfering peptides, and antibodies. Hb binding is also determined spectroscopically.
Using patchclamp methodology and growth assays we are screening inhibitors of the NPPs (parasite induced transport pathways) as potential antimalarials. We are also looking at delivery of toxic molecules (e.g. azaserine) via the NPPs. We are trying to identify the nature of the NPPs and the role of haemoglobinopathies in malaria growth.
We are looking at the expression of particular candidate kinases (SPAK and WNK) as regulators of KCC. We are also looking at other transport proteins (NKCC, NHE etc) to see if they are involved in tumour growth. To test the role of KCC3 in cancer we intend to look at tumour development in KCC3 knockout mice. If intracellular Cl is the key controller of kinase activity, we intend to use antibodies to phosphorylation to test Cl-dependence and try to establish the role for chloride in signalling in cancer cell lines.
We are also working on the role of arginine transport in uremia; glucose transport (GLUT1) mutants in children; transport alteration in Band 3 mutants; production of reconstitutable dried red cells; and aquaporin modification by peroxynitrite.