Peterson Blaise Z, Catterall William A
Cellular and Molecular Physiology, H166, Penn State Milton S. Hershey Medical Center, College of Medicine, 500 University Dr., Room C6603, P.O. Box 850, Hershey, PA 17033-0850, USA.
Mol Pharmacol. 2006 Aug;70(2):667-75. doi: 10.1124/mol.105.020644. Epub 2006 May 4.
Dihydropyridines (DHPs) are an important class of drugs, used extensively in the treatment of angina pectoris, hypertension, and arrhythmia. The molecular mechanism by which DHPs modulate Ca(2+) channel function is not known in detail. We have found that DHP binding is allosterically coupled to Ca(2+) binding to the selectivity filter of the skeletal muscle Ca(2+) channel Ca(V)1.1, which initiates excitation-contraction coupling and conducts L-type Ca(2+) currents. Increasing Ca(2+) concentrations from approximately 10 nM to 1 mM causes the DHP receptor site to shift from a low-affinity state to a high-affinity state with an EC(50) for Ca(2+) of 300 nM. Substituting each of the four negatively charged glutamate residues that form the ion selectivity filter with neutral glutamine or positively charged lysine residues results in mutant channels whose DHP binding affinities are decreased up to 10-fold and are up to 150-fold less sensitive to Ca(2+) than wild-type channels. Analysis of mutations of amino acid residues adjacent to the selectivity filter led to identification of Phe-1013 and Tyr-1021, whose mutation causes substantial changes in DHP binding. Thermo-dynamic mutant cycle analysis of these mutants demonstrates that Phe-1013 and Tyr-1021 are energetically coupled when a single Ca(2+) ion is bound to the channel pore. We propose that DHP binding stabilizes a nonconducting state containing a single Ca(2+) ion in the pore through which Phe-1013 and Tyr-1021 are energetically coupled. The selectivity filter in this energetically coupled high-affinity state is blocked by bound Ca(2+), which is responsible for the high-affinity inhibition of Ca(2+) channels by DHP antagonists.
二氢吡啶类（DHPs）是一类重要的药物，广泛用于治疗心绞痛、高血压和心律失常。DHPs调节Ca(2+)通道功能的分子机制尚不清楚。我们发现，DHP结合与Ca(2+)结合到骨骼肌Ca(2+)通道Ca(V)1.1的选择性过滤器上存在变构偶联，Ca(V)1.1启动兴奋-收缩偶联并传导L型Ca(2+)电流。将Ca(2+)浓度从约10 nM增加到1 mM会导致DHP受体位点从低亲和力状态转变为高亲和力状态，Ca(2+)的EC(50)为300 nM。用中性谷氨酰胺或带正电荷的赖氨酸残基取代形成离子选择性过滤器的四个带负电荷的谷氨酸残基中的每一个，会产生突变通道，其DHP结合亲和力降低高达10倍，对Ca(2+)的敏感性比野生型通道低多达150倍。对与选择性过滤器相邻的氨基酸残基突变的分析导致鉴定出Phe-1013和Tyr-1021，它们的突变会导致DHP结合发生重大变化。对这些突变体的热力学突变循环分析表明，当单个Ca(2+)离子结合到通道孔时，Phe-1013和Tyr-1021在能量上是偶联的。我们提出，DHP结合通过Phe-1013和Tyr-1021在能量上偶联的方式稳定了孔中含有单个Ca(2+)离子的非传导状态。在这种能量偶联的高亲和力状态下，选择性过滤器被结合的Ca(2+)阻断，这就是DHP拮抗剂对Ca(2+)通道产生高亲和力抑制的原因。
