Hosoya Y, Yamada M, Ito H, Kurachi Y
Department of Cell Biology and Signaling, Yamagata University School of Medicine, Japan.
J Gen Physiol. 1996 Dec;108(6):485-95. doi: 10.1085/jgp.108.6.485.
To elucidate the functional interaction between the active G protein subunit (GK*) and the cardiac muscarinic K+ (KACh) channel, the effect of intracellular GTP on the channel current fluctuation in the presence of 0.5 microM extracellular acetylcholine was examined in inside-out patches from guinea pig atrial myocytes using spectral analysis technique. The power density spectra of current fluctuations induced at various concentrations of GTP ([GTP]) were well fitted by the sum of two Lorentzian functions. Because the channel has one open state, the open-close transitions of the channel gate represented by the spectra could be described as C2<-->C1<-->O. As [GTP] was raised, the channel activity increased in a positive cooperative manner. The powers of the two Lorentzian components concomitantly increased, while the corner frequencies and the ratio of the powers at 0 Hz remained almost constant. This indicates that G protein activation did not affect the gating of each channel but mainly increased the number of functionally active channels in the patch to enhance the channel activity. Regulation of the number of functionally active channels could be described by a slow transition of the channel states, U (unavailable)<-->A (available), which is independent of the gating. The equilibrium of this slow transition was shifted by GTP from U to A. Monod-Wyman-Changeux's allosteric model for the channel state transition(U<-->A) could well describe the positive cooperative increase in the channel availability by GTP, assuming that, in the presence of saturating concentrations of ACh, [GK*] linearly increased as [GTP] was raised in our experimental range. The model indicates that the cardiac KACh channel could be described as a multimer composed of four or more functionally identical subunits, to each of which one GK* binds.
为阐明活性G蛋白亚基（GK*）与心肌毒蕈碱型钾通道（KACh）之间的功能相互作用，我们使用频谱分析技术，在豚鼠心房肌细胞的内向外膜片中，研究了细胞内鸟苷三磷酸（GTP）对0.5微摩尔/升细胞外乙酰胆碱存在时通道电流波动的影响。在不同浓度的GTP（[GTP]）下诱导的电流波动功率密度谱，能很好地用两个洛伦兹函数之和拟合。由于该通道有一个开放状态，由频谱表示的通道门控的开闭转换可描述为C2<-->C1<-->O。随着[GTP]升高，通道活性以正协同方式增加。两个洛伦兹分量的功率同时增加，而转折频率和0赫兹处的功率比几乎保持不变。这表明G蛋白激活不影响每个通道的门控，而是主要增加了膜片中功能活性通道的数量，以增强通道活性。功能活性通道数量的调节可用通道状态的缓慢转变来描述，即U（不可用）<-->A（可用），这与门控无关。这种缓慢转变的平衡被GTP从U转移到A。假设在乙酰胆碱饱和浓度存在时，在我们的实验范围内，随着[GTP]升高，[GK*]呈线性增加，莫诺德-怀曼-尚热的通道状态转变（U<-->A）变构模型能很好地描述GTP引起的通道可用性的正协同增加。该模型表明，心脏KACh通道可描述为由四个或更多功能相同的亚基组成的多聚体，每个亚基结合一个GK*。
