Duncan R L, Kizer N, Barry E L, Friedman P A, Hruska K A
Renal Division, Jewish Hospital, Washington University Medical Center St. Louis, Missouri 63110, USA.
Proc Natl Acad Sci U S A. 1996 Mar 5;93(5):1864-9. doi: 10.1073/pnas.93.5.1864.
By patch-clamp analysis, we have shown that chronic, intermittent mechanical strain (CMS) increases the activity of stretch-activated cation channels of osteoblast-like UMR-106.01 cells. CMS also produces a swelling-activated whole-cell conductance (Gm) regulated by varying strain levels. We questioned whether the swelling-activated conductance was produced by stretch-activated cation channel activity. We have identified a gene involved in the increase in conductance by using antisense oligodeoxynucleotides (ODN) derived from the alpha 1-subunit genes of calcium channels found in UMR-106.01 cells (alpha1S, alpha1C, and alpha1D). We demonstrate that alpha 1C antisense ODNs abolish the increase in Gm in response to hypotonic swelling following CMS. Antisense ODNs to alpha1S and alpha1D, sense ODNs to alpha1C, and sham permeabilization had no effect on the conductance increase. In addition, during cell-attached patch-clamp studies, antisense ODNs to alpha1c completely blocked the swelling-activated and stretch-activated nonselective cation channel response to strain. Antisense ODNs to alpha1S treatment produced no effect on either swelling-activated or stretch-activated cation channel activity. There were differences in the stretch-activated and swelling-activated cation channel activity, but whether they represent different channels could not be determined from our data. Our data indicate that the alpha1C gene product is involved in the Gm and the activation of the swelling-activated cation channels induced by CMS. The possibility that swelling-activated cation channel genes are members of the calcium channel superfamily exists, but if alpha1c is not the swelling-activated cation channel itself, then its expression is required for induction of swelling-activated cation channel activity by CMS.
通过膜片钳分析，我们已经表明，慢性间歇性机械牵张（CMS）可增加成骨样UMR-106.01细胞的牵张激活阳离子通道活性。CMS还可产生受不同应变水平调节的肿胀激活全细胞电导（Gm）。我们质疑肿胀激活电导是否由牵张激活阳离子通道活性产生。我们通过使用源自UMR-106.01细胞中发现的钙通道α1亚基基因（α1S、α1C和α1D）的反义寡脱氧核苷酸（ODN），鉴定出一个与电导增加相关的基因。我们证明，α1C反义ODN可消除CMS后低渗肿胀引起的Gm增加。针对α1S和α1D的反义ODN、针对α1C的正义ODN以及假通透处理对电导增加均无影响。此外，在细胞贴附式膜片钳研究中，针对α1c的反义ODN完全阻断了对牵张的肿胀激活和牵张激活非选择性阳离子通道反应。针对α1S的反义ODN处理对肿胀激活或牵张激活阳离子通道活性均无影响。牵张激活和肿胀激活阳离子通道活性存在差异，但从我们的数据无法确定它们是否代表不同的通道。我们的数据表明，α1C基因产物参与Gm以及CMS诱导的肿胀激活阳离子通道的激活。肿胀激活阳离子通道基因是钙通道超家族成员的可能性存在，但如果α1c不是肿胀激活阳离子通道本身，那么其表达是CMS诱导肿胀激活阳离子通道活性所必需的。
