Qian Xiang, Nimigean Crina M, Niu Xiaowei, Moss Brenda L, Magleby Karl L
Department of Physiology and Biophysics, University of Miami School of Medicine, Miami, FL 33101-6430, USA.
J Gen Physiol. 2002 Dec;120(6):829-43. doi: 10.1085/jgp.20028692.
Functional large-conductance Ca(2+)- and voltage-activated K(+) (BK) channels can be assembled from four alpha subunits (Slo1) alone, or together with four auxiliary beta1 subunits to greatly increase the apparent Ca(2+) sensitivity of the channel. We examined the structural features involved in this modulation with two types of experiments. In the first, the tail domain of the alpha subunit, which includes the RCK2 (regulator of K(+) conductance) domain and Ca(2+) bowl, was replaced with the tail domain of Slo3, a BK-related channel that lacks both a Ca(2+) bowl and high affinity Ca(2+) sensitivity. In the second, the Ca(2+) bowl was disrupted by mutations that greatly reduce the apparent Ca(2+) sensitivity. We found that the beta1 subunit increased the apparent Ca(2+) sensitivity of Slo1 channels, independently of whether the alpha subunits were expressed as separate cores (S0-S8) and tails (S9-S10) or full length, and this increase was still observed after the Ca(2+) bowl was mutated. In contrast, beta1 subunits no longer increased Ca(2+) sensitivity when Slo1 tails were replaced by Slo3 tails. The beta1 subunits were still functionally coupled to channels with Slo3 tails, as DHS-I and 17 beta-estradiol activated these channels in the presence of beta1 subunits, but not in their absence. These findings indicate that the increase in apparent Ca(2+) sensitivity induced by the beta1 subunit does not require either the Ca(2+) bowl or the linker between the RCK1 and RCK2 domains, and that Slo3 tails cannot substitute for Slo1 tails. The beta1 subunit also induced a decrease in voltage sensitivity that occurred with either Slo1 or Slo3 tails. In contrast, the beta1 subunit-induced increase in apparent Ca(2+) sensitivity required Slo1 tails. This suggests that the allosteric activation pathways for these two types of actions of the beta1 subunit may be different.
功能性大电导钙（2+）和电压激活钾（BK）通道可以仅由四个α亚基（Slo1）组装而成，也可以与四个辅助β1亚基一起组装，以大大提高通道的表观钙（2+）敏感性。我们通过两种类型的实验研究了这种调节所涉及的结构特征。在第一个实验中，α亚基的尾部结构域，包括RCK2（钾离子电导调节剂）结构域和钙（2+）碗状结构，被Slo3的尾部结构域所取代，Slo3是一种与BK相关的通道，既缺乏钙（2+）碗状结构，也缺乏高亲和力的钙（2+）敏感性。在第二个实验中，钙（2+）碗状结构通过突变被破坏，这些突变大大降低了表观钙（2+）敏感性。我们发现，β1亚基增加了Slo1通道的表观钙（2+）敏感性，无论α亚基是作为单独的核心（S0-S8）和尾部（S9-S10）表达还是全长表达，并且在钙（2+）碗状结构发生突变后仍能观察到这种增加。相比之下，当Slo1尾部被Slo3尾部取代时，β1亚基不再增加钙（2+）敏感性。β1亚基在功能上仍然与具有Slo3尾部的通道偶联，因为DHS-I和17β-雌二醇在存在β1亚基的情况下激活这些通道，但在不存在β1亚基的情况下则不能。这些发现表明，β1亚基诱导的表观钙（2+）敏感性增加既不需要钙（2+）碗状结构，也不需要RCK1和RCK2结构域之间的连接子，并且Slo3尾部不能替代Slo1尾部。β1亚基还诱导了电压敏感性的降低，无论Slo1还是Slo3尾部都会出现这种情况。相比之下，β1亚基诱导的表观钙（2+）敏感性增加需要Slo1尾部。这表明β1亚基这两种类型作用的变构激活途径可能不同。
