DNA binding by the eukaryotic transcription factor Ets-1 is negatively regulated by an intramolecular mechanism. Quantitative binding assays compared the DNA-binding activities of native Ets-1, three deletion mutants, and three tryptic fragments. Ets-1 and activated Ets-1 polypeptides differed in DNA-binding affinity as much as 23-fold. Inhibition was mediated by two regions flanking the minimal DNA-binding domain. Both regions regulated affinity by enhancing dissociation of the protein-DNA complex. Three lines of evidence indicated that inhibition requires cooperative interaction between the two regions: first, the two inhibitory regions acted through a common mechanism; second, neither region functioned independently of the other; finally, mutation of the C-terminal inhibitory region altered the conformation of the N-terminal inhibitory region. In addition, partial proteolysis detected an identical altered conformation in the N-terminal inhibitory region of Ets-1 bound to DNA. This finding suggested that repression is transiently disrupted during DNA binding. These results provide evidence that the two inhibitory regions of Ets-1 are structurally, as well as functionally, coupled. In addition, conformational change is shown to be a critical component of the inhibition mechanism. A cooperative, allosteric model of autoinhibition is described. Autoinhibition of Ets-1 could be relieved by either protein partner(s) or posttranslational modifications.

The structural basis for the activation gate of voltage‐dependent K+ channels is not known, but indirect evidence has implicated the S4‐S5 linker, the cytoplasmic region between the fourth and fifth transmembrane domains of the channel subunit. We have studied the effects of mutations in the S4‐S5 linker of HERG (human ether‐á‐go‐go‐related gene), a human delayed rectifier K+ channel, in Xenopus oocytes. Mutation of acidic residues (D540, E544) in the S4‐S5 linker of HERG channels to neutral (Ala) or basic (Lys) residues accelerated the rate of channel deactivation. Most mutations greatly accelerated the rate of activation. However, E544K HERG channels activated more slowly than wild‐type HERG channels. Mutation of residues in the S4‐S5 linker had little or no effect on fast inactivation, consistent with independence of HERG channel activation and inactivation In response to large hyperpolarizations, D540K HERG channels can reopen into a state that is distinct from the normal depolarization‐induced open state. It is proposed that substitution of a negatively charged Asp with the positively charged Lys disrupts a subunit interaction that normally stabilizes the channel in a closed state at negative transmembrane potentials. The results indicate that the S4‐S5 linker is a crucial component of the activation gate of HERG channels.

The effects of a mutation in the human ether‐a‐go‐go‐related gene (HERG) (Ser631 to Ala, S631A) on the voltage‐ and extracellular [K+] dependence of inactivation were studied in Xenopus oocytes using two microelectrode and single channel voltage‐clamp techniques. The voltage required for half‐inactivation of S631A HERG was 102 mV more positive than for wild‐type (WT)‐HERG, resulting in reduced rectification of the steady‐state current‐voltage relationship. In contrast, the voltage dependence of channel activation was not altered by the S631A mutation. These findings indicate that inactivation of HERG channels is not linked to activation. Rectification of whole‐cell S631A HERG current was caused by a voltage‐dependent reduction in open probability, and inward rectification of the current‐voltage relationship of single channels. Elevation of extracellular [K+] from 2 to 20 mm shifted the half‐point for inactivation by +20 mV for WT‐HERG, and +25 mV for S631A HERG. Thus, elevated [K+]o and the S631A mutation affect HERG inactivation by different mechanisms. The S631A mutation altered the ion translocation rate of HERG channels. The single channel conductance (γ) of S631A HERG was 20 pS between ‐40 and‐100 mV, and 6.0 pS between +40 and +100 mV (120 mm extracellular K+). This compares to a γ of 12.1 and 5.1 pS for WT‐HERG channels under the same conditions.

LQT1-associated mutations in KVLQT1 caused a spectrum of dysfunction in I(Ks) and KvLQT1 channels. The degree of I(Ks) dysfunction did not correlate with the QTc interval or the presence of symptoms in the respective gene carriers. In contrast to previous reports, we found that loss of function mutations are not exclusive to recessively inherited LQT.