Blocker state dependence and trapping in hyperpolarization-activated cation channels: Evidence for an intracellular activation gate

Hyperpolarization-activated cation currents (I-h) are key determinants of r epetitive electrical activity in heart and nerve cells. The bradycardic age nt ZD7288 is a selective blocker of these currents. We studied the mechanis m for ZD7288 blockade of cloned I-h channels in excised inside-out patches. ZD7288 blockade of the mammalian mHCN1 channel appeared to require opening of the channel, but strong hyperpolarization disfavored blockade. The stee pness of this voltage-dependent effect (an apparent valence of similar to4) makes it unlikely to arise solely from a direct effect of voltage on block er binding. Instead, it probably indicates a differential affinity of the b locker for different channel conformations. Similar properties were seen fo r ZD7288 blockade of the sea urchin homologue of I-h channels (SPIH), but s ome of the blockade Was irreversible. To explore the molecular basis for th e difference in reversibility we constructed chimeric channels from mHCN1 a nd SPIH and localized the structural determinant for the reversibility to t hree residues in the S6 region likely to line the pore. Using a triple poin t mutant in S6, ive also revealed the trapping of ZD7288 by the closing of the channel. Overall, the observations led us to hypothesize that the resid ues responsible for ZD7288 block of I-h channels are located in the pore li ning, and are guarded by an intracellular activation gate of the channel.