Characterization of the deafwaddler mutant of the rat plasma membrane calcium-ATPase 2

The deafwaddler mutant in mice was the first spontaneous mutant discovered in the plasma membrane Ca2+ pump (PMCA) [Street, V.A. et al., 1998, Nat. Ge net. 19, 390-394]. A nucleotide substitution in deafwaddler results in a Gl y 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 ve stibular hair cells, neurons of the spiral ganglion, and participates in in ner ear development. Mice that are homozygous for deafwaddler are deaf and have poor balance. However, the balance and hearing disorders of the deafwa ddler mice appear to be less severe than homozygotes for a functionally nul l frameshift mutant or homozygous PMCA2 knockout mice, suggesting that deaf waddler PMCA2 retains some biological activity. To examine the enzymic effe cts of the deafwaddler mutant, PMCA2 wild-type and deafwaddler were produce d by transient expression in COS cells as well as baculovirus-mediated expr ession 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-s tate transport assays and pre-steady-state ATPase assays of these two prote ins revealed that the K-0.5 for Ca2+, K-0.5 for calmodulin, degree of activ ation by calmodulin and rate of activation by Ca-calmodulin were nearly ide ntical. However, calcium transport of the deafwaddler pump was reduced to 3 0% of the wild-type activity. Although calcium transport activity was reduc ed in the deafwaddler pump, total phosphoenzyme formation from ATP was slig htly higher for deafwaddler than for wild-type. 50 muM LaCl3 (which blocks the E1P to E2P conformational transition) increased the steady-state level of phosphoenzyme 3-fold for the wild-type but had no effect on the deafwadd ler. Taken together, the kinetic data suggest that the deafwaddler mutation affects PMCA2 by slowing the E1P to E2P transition, resulting in approxima tely 70% reduction in the PMCA2-mediated Ca2+ export. (C) 2001 Elsevier Sci ence B.V. All rights reserved.