Characterization of the deafwaddler mutant of the rat plasma membrane calcium-ATPase 2
A R Penheiter1, A G Filoteo, C L Croy
1Department of Biochemistry and Molecular Biology, Mayo Clinic, 200 First Street SW, Rochester, MN 55905, USA.
Abstract:
The deafwaddler mutant in mice was the first spontaneous mutant discovered in the plasma membrane Ca(2+) pump (PMCA) [Street, V.A. et al., 1998, Nat. Genet. 19, 390-394]. A nucleotide substitution in deafwaddler results in a Gly to Ser transition at amino acid 283 in the small cytoplasmic loop of PMCA isoform 2 (PMCA2). PMCA2 is abundant in the stereocilia of auditory and vestibular hair cells, neurons of the spiral ganglion, and participates in inner ear development. Mice that are homozygous for deafwaddler are deaf and have poor balance. However, the balance and hearing disorders of the deafwaddler mice appear to be less severe than homozygotes for a functionally null frameshift mutant or homozygous PMCA2 knockout mice, suggesting that deafwaddler PMCA2 retains some biological activity. To examine the enzymic effects of the deafwaddler mutant, PMCA2 wild-type and deafwaddler were produced by transient expression in COS cells as well as baculovirus-mediated expression in Sf9 insect cells. Membrane preparations were assayed for calcium transport and ATPase activity. No significant differences in the regulation by calmodulin of the wild-type and deafwaddler PMCA2b were found. Steady-state transport assays and pre-steady-state ATPase assays of these two proteins revealed that the K(0.5) for Ca(2+), K(0.5) for calmodulin, degree of activation by calmodulin and rate of activation by Ca-calmodulin were nearly identical. However, calcium transport of the deafwaddler pump was reduced to 30% of the wild-type activity. Although calcium transport activity was reduced in the deafwaddler pump, total phosphoenzyme formation from ATP was slightly higher for deafwaddler than for wild-type. 50 microM LaCl3 (which blocks the E(1)P to E(2)P conformational transition) increased the steady-state level of phosphoenzyme 3-fold for the wild-type but had no effect on the deafwaddler. Taken together, the kinetic data suggest that the deafwaddler mutation affects PMCA2 by slowing the E(1)P to E(2)P transition, resulting in approximately 70% reduction in the PMCA2-mediated Ca(2+) export.
Insights
The deafwaddler mutation in plasma membrane calcium ATPase 2 (PMCA2) slows calcium transport, reducing it by 70% while retaining some function. This impacts inner ear development, leading to hearing and balance disorders in mice.
Area of Science:
- Biochemistry
- Genetics
- Neuroscience
Background:
- The deafwaddler mutant mouse was the first identified spontaneous mutation in the plasma membrane calcium ATPase (PMCA).
- This mutation affects PMCA isoform 2 (PMCA2), which is crucial for inner ear development and function, particularly in auditory and vestibular hair cells.
Purpose of the Study:
- To investigate the enzymic effects of the deafwaddler mutation on PMCA2 activity.
- To understand how the specific Glycine to Serine substitution at amino acid 283 impacts PMCA2's calcium transport and ATPase functions.
Main Methods:
- Produced wild-type and deafwaddler PMCA2 proteins using transient expression in COS cells and baculovirus-mediated expression in Sf9 insect cells.
- Assayed calcium transport and ATPase activity in membrane preparations.
- Utilized steady-state and pre-steady-state kinetic assays, including the use of LaCl3 to probe conformational transitions.
Main Results:
- The deafwaddler PMCA2 exhibited significantly reduced calcium transport activity (approximately 30% of wild-type).
- While total phosphoenzyme formation was slightly higher in deafwaddler, the mutation did not affect calmodulin regulation or activation.
- Lanthanum chloride, which blocks the E(1)P to E(2)P transition in wild-type PMCA2, had no effect on the deafwaddler mutant, indicating a defect in this transition.
Conclusions:
- The deafwaddler mutation primarily impairs PMCA2 function by slowing the E(1)P to E(2)P conformational transition.
- This kinetic defect leads to a substantial reduction in PMCA2-mediated calcium export, explaining the observed hearing and balance deficits in deafwaddler mice.
- Despite reduced activity, the deafwaddler PMCA2 retains partial biological function, differentiating it from null mutants.
