Mistrík Pavel, Torre Vincent
Scuola Internazionale Superiore di Studi Avanzati, via Beirut 2-4, 34014 Trieste, Italy.
Pflugers Arch. 2004 Apr;448(1):76-84. doi: 10.1007/s00424-003-1228-3. Epub 2004 Feb 6.
Sperm motility is a tightly regulated process. One of the crucial factors determining the swimming of the sea-urchin sperm is an elevation of intracellular pH (pH(i)). The possibility that its hyperpolarisation-activated cyclic nucleotide-gated channel (SpHCN) is modulated directly by pH is addressed here. Site-directed mutagenesis showed that histidine 518 from the linker connecting the S6 helix with the cyclic nucleotide binding domain is responsible for the pH modulation of current kinetics and voltage dependence of activation. The effect of mutating histidine 518 to serine (H518S) on the time constant of activation was maximal at pH 6.4: 180+/-20 ms in the wild-type (wt) but only 56+/-10 ms in the H518S mutant channel. Furthermore, histidine 518 accounted for 31% of the shift in the voltage of half activation ( V(1/2)) in wt following a pH change from 6.4 to 8.4. The mutation H518S also shifted V(1/2) by 19 mV at pH 7.4 (-50.2+/-0.2 and -69+/-2 mV for H518S and wt, respectively). This indicates that histidine 518 couples voltage sensing to gating. The wt and H518S channels had a different affinity for cyclic adenosine monophosphate (cAMP) (IC(50) 1.0+/-0.02 and 2.5+/-0.06 microM, respectively). Changes in pH(i) also modulated channel selectivity.
精子活力是一个受到严格调控的过程。决定海胆精子游动的关键因素之一是细胞内pH值（pH(i)）的升高。本文探讨了其超极化激活的环核苷酸门控通道（SpHCN）是否直接受pH值调节的可能性。定点诱变表明，连接S6螺旋与环核苷酸结合域的连接子中的组氨酸518负责电流动力学的pH调节和激活的电压依赖性。将组氨酸518突变为丝氨酸（H518S）对激活时间常数的影响在pH 6.4时最大：野生型（wt）为180±20毫秒，而H518S突变通道仅为56±10毫秒。此外，在pH值从6.4变为8.4后，组氨酸518占野生型半数激活电压（V(1/2)）变化的31%。H518S突变在pH 7.4时也使V(1/2)偏移了19 mV（H518S和野生型分别为-50.2±0.2和-69±2 mV）。这表明组氨酸518将电压传感与门控联系起来。野生型和H518S通道对环磷酸腺苷（cAMP）具有不同的亲和力（IC(50)分别为1.0±0.02和2.5±0.06 microM）。pH(i)的变化也调节通道选择性。
