An extensive class of proteins binds the Ca2+-binding protein calmodulin (CaM) through what are termed """"""""IQ domains"""""""". The biological functions of many in this class, including Na+, K+, and Ca2+ channels, G protein modulators and unconventional myosins are regulated by Ca2+ via the CaM-IQ domain complex, yet little is known about how these important molecular switches function. We propose to help fill this gap in knowledge by deriving detailed transient and steady-state kinetic mechanisms for Ca2+-dependent switching in the CaM-IQ domain complexes in neuromodulin and PEP-19, which serve as CaM buffers and/or stores in neurons. Both are of fundamental interest because they represent two major classes of IQ domains: A glycine residue found in half of all IQ domains is present in neuromodulin, but has been replaced by a lysine in PEP-19. Structural information indicates that this difference should significantly affect interactions with CaM. These proteins are also of interest because of the control they appear to exert over the distribution of CaM among its many targets in the cell. Knowledge of the mechanisms determining how they interact with CaM will provide insights to this control process, and hence to the way in which the network of CaM targets is orchestrated. The actions of this network are a major determinant of how neurons and other cells respond to Ca2+ signals.
SPECIFIC AIM 1 : Derive steady-state kinetic mechanisms for Ca2+-dependent switching in the complexes between CaM and the IQ domains in neuromodulin and PEP-19. This will be performed using native and mutant CaMs, and fluorescent protein reporters based on neuromodulin and PEP-19. The final steady state mechanisms will include the kinetic parameters governing formation of the major switching intermediates.
SPECIFIC AIM 2 : Expand the steady-state mechanisms derived under Aim 1 to encompass the transient kinetic behaviors of the complexes between CaM and the IQ domains in neuromodulin and PEP-19. Mechanisms derived for Ca2+-dependent switching in the two CaM complexes will be rigorously tested and refined by fitting them to the responses exhibited by the fluorescent reporters over a range of transient and steady-state conditions. The health-related benefits of this research program derive from the direct relationship between mutations in proteins containing IQ domains and numerous human diseases: Truncations and other mutations in the ASPM protein, which contains multiple IQ domain repeats, are associated with microcephaly, and the number of repeats appears to be positively correlated with brain size. Mutations in the Na+ channel IQ domain cause irregularities in cardiac electrical activity, and defects in other ion channels regulated via CaM-IQ domain switches are responsible for a host of cardiovascular and neurological disorders. Mutations in unconventional myosins result in immune-deficiencies and neurological impairment, and are the most frequent cause of deafness-blindness in humans.
