Distribution of Calretinin, Calbindin D28k, and Parvalbumin in Subcellular Fractions of Rat Cerebellum: Effects of Calcium

Abstract

The distribution of calretinin, calbindin D28k, and parvalbumin was examined in subcellular fractions prepared from rat cerebellum and analyzed by immunoblot. Calretinin was also quantified by radioimmunoassay. As expected, all three soluble, EF‐hand calcium‐binding proteins were predominantly localized in the cytosolic fraction. Calretinin and calbindin D28k were also detected in membrane fractions. Calretinin was more abundant in synaptic membrane than in microsomal fractions. The cerebellar microsomal fraction contained the greatest concentration of membrane‐associated calbindin D28k. The association of calretinin and calbindin D28k with membrane fractions was decreased in samples prepared or incubated in low calcium. Quantification of calretinin in subcellular fractions of rat cerebellum revealed a greater amount of calretinin in cytosolic fractions prepared or incubated in low calcium and reduced amounts of calretinin in all membrane fractions incubated in low calcium with the exception of the mitochondrial fraction. These results imply that calretinin and calbindin D28k might have physiological target molecules that are associated with, or are components of, brain membranes.

Calbindin D28k targets myo -inositol monophosphatase in spines and dendrites of cerebellar Purkinje neurons

“…7) and Ran-binding protein M (11). Together with older reports on enzyme regulation (12)(13)(14)(15) and membrane͞cytoskeleton targeting of CB (16,17), these data suggest that CB not only acts as a potent Ca 2ϩ buffer but also as a Ca 2ϩ sensor. So far, however, no in situ data on the interaction of CB with one of its targets have been reported.…”

Calbindin D28k Exhibits Properties Characteristic of a Ca2+ Sensor

“…Although most reports deal with the Ca 2ϩ -buffering function of calbindin D 28k , several lines of evidence suggest that the protein also acts as a Ca 2ϩ sensor. Spectroscopic investigations (19) and in vitro studies using antibodies (20) have shown that calbindin D 28k undergoes a conformational shift upon Ca 2ϩ binding. Additionally, a number of putative Ca 2ϩ -dependent interactions with target proteins or brain membrane fractions have been reported (20 -23), and erythrocyte membrane Ca 2ϩ -ATPase and 3Ј,5Ј-cyclic nucleotide phosphodiesterase have been shown to be stimulated in a dose-dependent, saturable manner with calbindin D 28k (24).…”

“…Additionally, a number of putative Ca 2ϩ -dependent interactions with target proteins or brain membrane fractions have been reported (20 -23), and erythrocyte membrane Ca 2ϩ -ATPase and 3Ј,5Ј-cyclic nucleotide phosphodiesterase have been shown to be stimulated in a dose-dependent, saturable manner with calbindin D 28k (24). Moreover, the finding that a fraction of calbindin D 28k (9 -55%) (20,(25)(26)(27)) is specifically associated with particulate structures in the cell indicates that the protein plays other roles in addition to its function as a mobile Ca 2ϩ buffer.…”

myo-Inositol Monophosphatase Is an Activated Target of Calbindin D28k

“…It is expressed in many tissues including brain, intestine, kidney, and pancreas. Many reports have focused on the presence of calbindin in specific neuronal cell types in the brain and sensory system (1)(2)(3). It constitutes as much as 0.1-1.5% of the total soluble protein in brain (4), and in some cells it may be present at intracellular concentrations of up to 2 mM (5).…”