I f , encoded by the hyperpolarization-activated cyclic nucleotide-modulated (HCN) channel family, is a key player in cardiac and neuronal pacing. Although HCN channels structurally resemble voltage-gated K ؉ (Kv) channels, their structure-function correlation is much less clear. Here we probed the functional importance of the HCN1 S3-S4 linker by multiple substitutions of its residues. Neutralizing Glu 235 , an acidic S3-S4 linker residue conserved in all hyperpolarization-activated channels, by Ala substitution produced a depolarizing activation shift (V1 ⁄2 ‫؍‬ ؊65.0 ؎ 0.7 versus ؊70.6 ؎ 0.7 mV for wild-type HCN1); the charge-reversed mutation E235R shifted activation even more positively (؊56.2 ؎ 0.5 mV). Increasing external Mg 2؉ mimicked the progressive rightward shifts of E235A and E235R by gradually shifting activation (V1 ⁄2 ‫؍‬ 1 < 3 < 10 < 30 mM); ⌬V1 ⁄2 induced by 30 mM Mg 2؉ was significantly attenuated for E235A (؉7.9 ؎ 1.2 versus ؉11.3 ؎ 0.9 mV for wild-type HCN1) and E235R (؉3.3 ؎ 1.4 mV) channels, as if surface charges were already shielded. Consistent with an electrostatic role, the energetic changes associated with ⌬V1 ⁄2 resulting from various Glu 235 substitutions (i.e. Asp, Ala, Pro, His, Lys, and Arg) displayed a strong correlation with their charges (⌬⌬G ‫؍‬ ؊2.1 ؎ 0.3 kcal/mol/charge; r ‫؍‬ 0.94). In contrast, D233E, D233A, D233G, and D233R did not alter activation gating. D233C (in C318S background) was also not externally accessible when probed with methanethiosulfonate ethylammonium (MTSEA). We conclude that the S3-S4 linker residue Glu 235 influences activation gating, probably by acting as a surface charge.I f or I h , encoded by the hyperpolarization-activated cyclic nucleotide-modulated (HCN) 1 or the so-called pacemaker channel gene family, is a key contributor to spontaneous rhythmic activity in cardiac and neuronal cells (1-9). Although HCN channels structurally resemble voltage-gated K ϩ (Kv) channels (for instance, both are tetramers made up of monomeric subunits consisting of six membrane-spanning segments) (7-10), a distinguishing functional feature that discriminates pacemaker channels from the Kv counterparts is their signature "backward" gating (i.e. activation upon hyperpolarization rather than depolarization). The molecular basis of this unique gating phenotype is unknown. Recently, it has been suggested that the voltage-sensing mechanism of the sea urchin sperm HCN (i.e. SPIH or spHCN) and Kv channels may be conserved (11). In any case, the structure-function correlation of HCN channels is much less defined compared with the well studied Kv channels. Comparison of these related yet functionally distinct ion channels should provide important insights into the unique behavior of HCN channels.Previous studies of Kv channels have demonstrated that the S3-S4 linker influences activation gating (12-15). By analogy to Kv channels, it is possible that the S3-S4 linker (defined as residues 229 -237 here, HCN1 numbering) of HCN channels also contributes to activation gating. Ho...

African trypanosomiasis (sleeping sickness), caused by protozoan Trypanosoma brucei species, is a debilitating disease that is lethal if untreated. Available drugs are antiquated, toxic, and compromised by emerging resistance. The indenoisoquinolines are a class of noncamptothecin topoisomerase IB poisons that are under development as anticancer agents. We tested a variety of indenoisoquinolines for their ability to kill T. brucei. Indenoisoquinolines proved trypanocidal at submicromolar concentrations in vitro. Structure-activity analysis yielded motifs that enhanced potency, including alkylamino substitutions on N-6, methoxy groups on C-2 and C-3, and a methylenedioxy bridge between C-8 and C-9. Detailed analysis of eight water-soluble indenoisoquinolines demonstrated that in trypanosomes the compounds inhibited DNA synthesis and acted as topoisomerase poisons. Testing these compounds on L1210 mouse leukemia cells revealed that all eight were more effective against trypanosomes than against mammalian cells. In preliminary in vivo experiments one compound delayed parasitemia and extended survival in mice subjected to a lethal trypanosome challenge. The indenoisoquinolines provide a promising lead for the development of drugs against sleeping sickness.

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