Yuill K, Ashmole I, Stanfield P R
Molecular Physiology Group, Department of Biological Sciences, University of Warwick, Gibbet Hill Road, Coventry, CV4 7AL, UK.
Pflugers Arch. 2004 Apr;448(1):63-9. doi: 10.1007/s00424-003-1218-5. Epub 2004 Jan 14.
We have studied pH sensitivity and ionic selectivity of the tandem pore K(+) channel TASK-1 heterologously expressed in Xenopus oocytes. We fit pH sensitivity assuming that only one of the two residues H98 need be protonated for channels to be shut. The effect of protons was weakly voltage dependent with a p K(a) of 6.02 at +40 mV. Replacement of His (H98D, H98N) reduced pH sensitivity but did not abolish it. Use of a concatameric channel permitted replacement of one His residue only; this concatamer was fully pH-sensitive. Increasing the number of His residues to 4 (mutant D204H) abolished pH sensitivity over the physiological range. The implication that D204 plays a role in pH-sensitivity was confirmed by the finding that pH sensitivity over the physiological range was also abolished in the mutant D204N. Ionic selectivity was also altered in D204H, D204N and H98D mutants. P(Rb)/ P(K) was increased from 0.80+/-0.04 (n=19) in wild type to 1.06+/-0.04 (n=19) in D204H. H98D, D204H and D204N were permeable to Na(+) with P(Na)/ P(K)=0.39+/-0.03 (n=14) in H98D, 0.64+/-0.04 (n=18) in D204H and 0.33+/-0.07 (n=3) in D204N. Thus, the arrangement of ring of residues HDHD appears to optimise both pH sensitivity and ionic selectivity.
我们研究了在非洲爪蟾卵母细胞中异源表达的串联孔钾通道TASK-1的pH敏感性和离子选择性。我们通过假设通道关闭仅需两个残基H98中的一个被质子化来拟合pH敏感性。质子的作用对电压的依赖性较弱，在 +40 mV时的pK(a)为6.02。His的替换（H98D、H98N）降低了pH敏感性，但并未消除。使用串联通道仅允许替换一个His残基；该串联通道对pH完全敏感。将His残基数量增加到4个（突变体D204H）在生理范围内消除了pH敏感性。突变体D204N在生理范围内的pH敏感性也被消除，这一发现证实了D204在pH敏感性中起作用。D204H、D204N和H98D突变体的离子选择性也发生了改变。P(Rb)/P(K)从野生型的0.80±0.04（n = 19）增加到D204H中的1.06±0.04（n = 19）。H98D、D204H和D204N对Na(+)具有通透性，H98D中的P(Na)/P(K)=0.39±0.03（n = 14），D204H中的为0.64±0.04（n = 18），D204N中的为0.33±0.07（n = 3）。因此，HDHD残基环的排列似乎优化了pH敏感性和离子选择性。
