Penheiter A R, Filoteo A G, Croy C L, Penniston J T
Department of Biochemistry and Molecular Biology, Mayo Clinic, 200 First Street SW, Rochester, MN 55905, USA.
Hear Res. 2001 Dec;162(1-2):19-28. doi: 10.1016/s0378-5955(01)00356-2.
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.
小鼠中的聋摇突变体是在质膜钙泵（PMCA）中发现的第一个自发突变体[斯特里特，V.A.等人，1998年，《自然遗传学》第19卷，第390 - 394页]。聋摇突变体中的一个核苷酸替换导致PMCA同工型2（PMCA2）小细胞质环中第283位氨基酸处的甘氨酸向丝氨酸转变。PMCA2在听觉和前庭毛细胞的静纤毛以及螺旋神经节神经元中大量存在，并参与内耳发育。纯合的聋摇小鼠耳聋且平衡能力差。然而，聋摇小鼠的平衡和听力障碍似乎比功能缺失的移码突变体纯合子或PMCA2纯合敲除小鼠要轻，这表明聋摇PMCA2保留了一些生物活性。为了研究聋摇突变体的酶学效应，通过在COS细胞中瞬时表达以及杆状病毒介导在Sf9昆虫细胞中表达产生了PMCA2野生型和聋摇突变体。对膜制剂进行钙转运和ATP酶活性测定。未发现野生型和聋摇PMCA2b在钙调蛋白调节方面有显著差异。对这两种蛋白质的稳态转运测定和前稳态ATP酶测定表明，钙的K(0.5)、钙调蛋白的K(0.5)、钙调蛋白的激活程度以及钙 - 钙调蛋白的激活速率几乎相同。然而，聋摇泵的钙转运降低到了野生型活性的30%。尽管聋摇泵的钙转运活性降低，但聋摇突变体从ATP形成的总磷酸化酶比野生型略高。50微摩尔/升的LaCl3（它阻断E(1)P到E(2)P的构象转变）使野生型的磷酸化酶稳态水平增加了3倍，但对聋摇突变体没有影响。综合来看，动力学数据表明聋摇突变通过减缓E(1)P到E(2)P的转变影响PMCA2，导致PMCA2介导的钙(2+)输出减少约70%。
