Novel blockers of hyperpolarization-activated current with isoform selectivity in recombinant cells and native tissue.

Background and purpose.  Selective HCN blockers represent an important therapeutic goal due to the wide distribution and multiple functions of these proteins, representing the molecular correlate of f- and h-current (I(f) or I(h) ). Recently, new compounds able to block differentially the homomeric HCN isoforms expressed in HEK293 were disclosed. In the present work, the electrophysiological and pharmacological properties of these new HCN blockers were characterized and their activity evaluated on native channels. Experimental approach.  HEK293 expressing mHCN1, mHCN2 and hHCN4 isoforms were used to verify channel blockade. Selected compounds were tested on native guinea-pig sino-atrial node cells and neurons from mouse dorsal root ganglia by patch-clamp recordings, and on dog Purkinje fibres by intracellular recordings. Key results.  In HEK293 cells, EC18 resulted to be significantly selective for HCN4 and MEL57A for HCN1 at physiological membrane potential. When tested on guinea-pig sino-atrial node cells, EC18 (10 µM) maintained its activity reducing I(f) by 67\% at -120mV, while MEL57A (3 µM) reduced I(f) by 18\%. On the contrary, in mouse DRG neurons, only MEL57A (30 and 100 µM) significantly reduced I(h) by 60\% at -80 mV. In dog cardiac Purkinje fibres EC18, but not MEL57A, reduced the amplitude and slowed the slope of the spontaneous diastolic depolarization. Conclusions.  Our results identified novel and highly selective HCN isoform blockers, EC18 and MEL57A; the selectivity found in recombinant system was maintained in various tissues expressing different HCN isoforms.