PFXYD序列参与phospholemman对L型钙通道的调节作用

目的探讨PFXYD序列在phospholemman（PLM）调节L型钙通道中的作用。方法用丙氨酸（Ala／，A）替换组成PFXYD序列的5个氨基酸，得到突变型PLM—ALL5。用磷酸钙沉淀法将编码L型钙通道的cDNA分别与编码载体质粒（empUvector，EV）、野生型PLM（wildtype，wT）、突变型PLM（ALL5）的cDNA转染到HEK293细胞中，待其表达相应蛋白后，用全细胞膜片钳方法记录L型钙电流（ICa（L））并分析其动力学特性。结果在除极化电位为一20mV和一10mV时，ALL5较wT更加明显地减慢了ICa（L）激活速度，而在相同电位下ALLJ5使ICa（L）失活速度也明显减慢；与WT相反，ALL5没有减慢ICa（L）灭活速度，反使其加快了；以上ALL5所致的ICa（L）改变与电流大小无关。结论PFXYD序列理化性质的改变可以引起PLM对L型钙通道调节作用的改变，提示PFXYD序列在PLM调节L型钙通道的过程中发挥重要作用。

PFXYD motif plays a role in the modulation of L-type calcium channels by phospholemman

Objective To explore the role of the PFXYD motif in the modulation of L-type calcium channels by phospholemman (PLM), a newly defined phosphoprotein. Methods We used site-directed muta-genesis to replace amino acids comprising the PFXYD motif with alanine (A) to generate the PLM mutant, ALLS. Calcium phosphate precipitation was used to introduce cDNAs encoding L-type calcium channels with either empty vector (EV), or wild type PLM (WT), or mutant PLM (ALL5) into HEK 293 cells. Whole-cell patch clamp was used to measure the L-type calcium currents (Ica(L)) and analyzed the changes in gating ki-netics. Results At voltage steps of -20 mV and - 10 mV,ALL5 slowed activation of Ica(L) to a much greater degree than WT,and also slowed inactivation of Ica(L). In contrast to WT,ALL5 did not appreciably slow deac-tivation of Ica(L). Importantly,the changes in Ica(L) gating kinetics induced by ALL5 was not attributed to the current density. Conclusion The chemical and/or physical properties of the PFXYD motif can be altered to im-pact PLM-dependent modulation of L-type calcium channels, suggesting that the PFXYD motif plays an impor-tant role in the modulation of L-type calcium channels by PLM.