Studies on voltage-gated K channels such as Shaker have shown that positive charges in the voltage-sensor (S4) can form salt bridges with negative charges in the surrounding transmembrane segments in a state-dependent manner, and different charge pairings can stabilize the channels in closed or open states. The goal of this study is to identify such charge interactions in the hERG channel. This knowledge can provide constraints on the spatial relationship among transmembrane segments in the channel's voltage-sensing domain, which are necessary for modeling its structure. We first study the effects of reversing S4's positive charges on channel activation. Reversing positive charges at the outer (K525D) and inner (K538D) ends of S4 markedly accelerates hERG activation, whereas reversing the 4 positive charges in between either has no effect or slows activation. We then use the 'mutant cycle analysis' to test whether D456 (outer end of S2) and D411 (inner end of S1) can pair with K525 and K538, respectively. Other positive charges predicted to be able, or unable, to interact with D456 or D411 are also included in the analysis. The results are consistent with predictions based on the distribution of these charged residues, and confirm that there is functional coupling between D456 and K525 and between D411 and K538.
KO and WT mice. 200 ml of each semen were used for total RNA extraction using the miRNeasy serum/plasma kit (Qiagen). RESULTS: Prmt5-and histone-associated protein Copr5 is highly expressed intestis and its depletion impacted on the maturation of spermatogonia to spermatids, although Copr5 KO animals were fertile. Extinction of Copr5 in mice testis leads to a down-regulation of the level of Miwi protein, as confirmed by western blot analyses and immunohistochemistry of WT and KO mice testis whole cell extracts and paraffin-embedded testis sections, respectively. In addition, the mRNA level of three pre-pachytene piR-NAs, prepiR1, prepiR2 and prepiR3 was decreased in KO Copr5 compared to WTmice, whereas in KO mice the expression level of the LINE1 mRNA, the most abundant retro-transposons that was usedas a read out of deregulation of the piRNA pathway, was increased. Using both human GEO data and patients with teratozoospermia, the present study reports a correlation between a low level of Copr5 mRNA and teratozoospermia, a human pathology characterized by abnormally shaped sperm that can negatively affect fertility. CONCLUSIONS: Copr5 KO in mice perturbed some genome surveillance mechanisms in germ cells by an alteration of the expression level of two major components present in the Piwi-interacting RNA (piRNA) pathway, the Miwi and the LINE1. In addition, low level of Copr5 mRNA correlates with human teratozoospermia sperm. Supported by: This work was supported by grants from the Ligue Contre le Cancer (CS) and the Association pour la Recherche contre le Cancer (EF).The authors declare no competing or financial interests.
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