Research Summary
Sickle Cell Disease is characterized by cell dehydration due in part to transiently elevated intracellular calcium levels. Red blood cells originate from erythroid precursors in the bone marrow and enter the circulation as reticulocytes. Maturing reticulocytes experience a reduction in cell volume via potassium chloride cotransporters (KCC) to reach the normal volume of a mature RBC (5).  In sickle RBC, poorly regulated KCC cause cell volume to be lower than normal, concentrating the hemoglobin inside the cell.  This makes hemoglobin S (HbS) polymerization and cell sickling more likely under deoxygenated conditions, activating another mechanism of dehydration in sickle cells called the sickling-induced pathway.  This nonspecific cation transport pathway leads to K+ efflux and Na+ influx (6).  More importantly, it allows Ca++ to enter the sickle RBC (7).

Calcium plays a significant role in the pathophysiology of the sickle red blood cell (RBC). Total calcium in sickle cells is markedly increased due to the sequestration of Ca++ into vesicles within the red blood cell (3).  Steady state cytoplasmic calcium levels have been shown to be modestly increased in sickle cells (4). The introduction of Ca++ into RBC activates a Ca++-dependent K+ channel, called the Gardos channel. Efflux of K+ through this channel, followed by Cl- and water, is the major mechanism by which sickle RBC become severely  dehydrated (8).  This dehydration is thought to be a major component of sickle pathophysiology (5).

In other cell types, calcium is involved in many processes including cell motility, cell fusion, signal transduction and growth and division. However, further potential consequences (in addition to those mentioned above) of elevated calcium in sickle RBC have not been explored.  We propose activation of the cysteine protease, calpain, as a potential consequence of transiently elevated calcium in sickle RBC.  Thus, the specific aims of my project include:

1. To compare calpain activation and calpastatin (calpain inhibitor) concentration in normal RBC, calcium-loaded normal RBC, and sickle RBC subfractions. 
2. To investigate localization patterns of m-calpain, calpastatin, and the calpain activator protein in sickle cells and calcium-loaded normal cells.
   
3. To determine the substrate preference of activated µ-calpain in red blood cells.