Tripathy A, Resch W, Xu L, Valdivia H H, Meissner G
Department of Biochemistry and Biophysics, and Department of Physiology, University of North Carolina, Chapel Hill, North Carolina 27599, USA.
J Gen Physiol. 1998 May;111(5):679-90. doi: 10.1085/jgp.111.5.679.
Single-channel and [3H]ryanodine binding experiments were carried out to examine the effects of imperatoxin activator (IpTxa), a 33 amino acid peptide isolated from the venom of the African scorpion Pandinus imperator, on rabbit skeletal and canine cardiac muscle Ca2+ release channels (CRCs). Single channel currents from purified CRCs incorporated into planar lipid bilayers were recorded in 250 mM KCl media. Addition of IpTxa in nanomolar concentration to the cytosolic (cis) side, but not to the lumenal (trans) side, induced substates in both ryanodine receptor isoforms. The substates displayed a slightly rectifying current-voltage relationship. The chord conductance at -40 mV was approximately 43% of the full conductance, whereas it was approximately 28% at a holding potential of +40 mV. The substate formation by IpTxa was voltage and concentration dependent. Analysis of voltage and concentration dependence and kinetics of substate formation suggested that IpTxa reversibly binds to the CRC at a single site in the voltage drop across the channel. The rate constant for IpTxa binding to the skeletal muscle CRC increased e-fold per +53 mV and the rate constant of dissociation decreased e-fold per +25 mV applied holding potential. The effective valence of the reaction leading to the substate was approximately 1.5. The IpTxa binding site was calculated to be located at approximately 23% of the voltage drop from the cytosolic side. IpTxa induced substates in the ryanodine-modified skeletal CRC and increased or reduced [3H]ryanodine binding to sarcoplasmic reticulum vesicles depending on the level of channel activation. These results suggest that IpTxa induces subconductance states in skeletal and cardiac muscle Ca2+ release channels by binding to a single, cytosolically accessible site different from the ryanodine binding site.
进行了单通道和[3H]ryanodine结合实验，以研究imperatoxin激活剂（IpTxa）对兔骨骼肌和犬心肌Ca2+释放通道（CRC）的影响。IpTxa是一种从非洲帝王蝎毒液中分离出的33个氨基酸的肽。将纯化的CRC整合到平面脂质双分子层中，在250 mM KCl培养基中记录单通道电流。在纳摩尔浓度下，将IpTxa添加到胞质（顺式）侧而非腔（反式）侧，可在两种ryanodine受体同工型中诱导亚状态。这些亚状态呈现出轻微的整流电流-电压关系。在-40 mV时的弦电导约为全电导的43%，而在+40 mV的保持电位下约为28%。IpTxa诱导的亚状态形成与电压和浓度有关。对电压和浓度依赖性以及亚状态形成动力学的分析表明，IpTxa在通道电压降的单个位点上可逆地结合到CRC。IpTxa与骨骼肌CRC结合的速率常数每增加+53 mV增加e倍，解离速率常数每增加+25 mV的施加保持电位降低e倍。导致亚状态的反应的有效价约为1.5。计算得出IpTxa结合位点位于从胞质侧起电压降的约23%处。IpTxa在ryanodine修饰的骨骼肌CRC中诱导亚状态，并根据通道激活水平增加或减少[3H]ryanodine与肌浆网囊泡的结合。这些结果表明，IpTxa通过结合到一个与ryanodine结合位点不同的、可从胞质侧接近的单个位点，在骨骼肌和心肌Ca2+释放通道中诱导亚电导状态。
