Yasuda Takahiro, Lewis Richard J, Adams David J
The University of Queensland, Queensland 4072, Australia.
J Gen Physiol. 2004 Apr;123(4):401-16. doi: 10.1085/jgp.200308967. Epub 2004 Mar 15.
It has been shown that beta auxiliary subunits increase current amplitude in voltage-dependent calcium channels. In this study, however, we found a novel inhibitory effect of beta3 subunit on macroscopic Ba(2+) currents through recombinant N- and R-type calcium channels expressed in Xenopus oocytes. Overexpressed beta3 (12.5 ng/cell cRNA) significantly suppressed N- and R-type, but not L-type, calcium channel currents at "physiological" holding potentials (HPs) of -60 and -80 mV. At a HP of -80 mV, coinjection of various concentrations (0-12.5 ng) of the beta3 with Ca(v)2.2alpha(1) and alpha(2)delta enhanced the maximum conductance of expressed channels at lower beta3 concentrations but at higher concentrations (>2.5 ng/cell) caused a marked inhibition. The beta3-induced current suppression was reversed at a HP of -120 mV, suggesting that the inhibition was voltage dependent. A high concentration of Ba(2+) (40 mM) as a charge carrier also largely diminished the effect of beta3 at -80 mV. Therefore, experimental conditions (HP, divalent cation concentration, and beta3 subunit concentration) approaching normal physiological conditions were critical to elucidate the full extent of this novel beta3 effect. Steady-state inactivation curves revealed that N-type channels exhibited "closed-state" inactivation without beta3, and that beta3 caused an approximately 40-mV negative shift of the inactivation, producing a second component with an inactivation midpoint of approximately -85 mV. The inactivation of N-type channels in the presence of a high concentration (12.5 ng/cell) of beta3 developed slowly and the time-dependent inactivation curve was best fit by the sum of two exponential functions with time constants of 14 s and 8.8 min at -80 mV. Similar "ultra-slow" inactivation was observed for N-type channels without beta3. Thus, beta3 can have a profound negative regulatory effect on N-type (and also R-type) calcium channels by causing a hyperpolarizing shift of the inactivation without affecting "ultra-slow" and "closed-state" inactivation properties.
已有研究表明，β辅助亚基可增加电压依赖性钙通道的电流幅度。然而，在本研究中，我们发现β3亚基对非洲爪蟾卵母细胞中表达的重组N型和R型钙通道的宏观Ba(2+)电流具有一种新的抑制作用。在-60和-80 mV的“生理”钳制电位（HP）下，过表达的β3（12.5 ng/细胞cRNA）显著抑制N型和R型钙通道电流，但不抑制L型钙通道电流。在-80 mV的钳制电位下，将不同浓度（0 - 12.5 ng）的β3与Ca(v)2.2α(1)和α(2)δ共注射，在较低β3浓度下可增强表达通道的最大电导，但在较高浓度（>2.5 ng/细胞）时则导致明显抑制。β3诱导的电流抑制在-120 mV的钳制电位下被逆转，表明这种抑制是电压依赖性的。高浓度的Ba(2+)（40 mM）作为电荷载体在-80 mV时也大大减弱了β3的作用。因此，接近正常生理条件的实验条件（钳制电位、二价阳离子浓度和β3亚基浓度）对于阐明这种新的β3效应的全部程度至关重要。稳态失活曲线显示，N型通道在没有β3时表现出“关闭状态”失活，而β3导致失活发生约40 mV的负向移位，产生第二个成分，其失活中点约为-85 mV。在高浓度（12.5 ng/细胞）的β3存在下，N型通道的失活发展缓慢，在-80 mV时，时间依赖性失活曲线最适合用两个指数函数之和拟合，时间常数分别为14 s和8.8 min。在没有β3的N型通道中也观察到类似的“超慢”失活。因此，β3可通过引起失活的超极化移位而对N型（以及R型）钙通道产生深远的负调节作用，而不影响“超慢”和“关闭状态”失活特性。
