An evolutionarily conserved pacemaker role for HCN ion channels in smooth muscle

Abstract

Although HCN ion channels are well established to underlie cardiac pacemaker activity, their role in smooth muscle organs remains controversial. HCN expressing cells are localized to renal pelvic smooth muscle (RPSM) pacemaker tissues of the murine upper urinary tract and HCN channel conductance is required for peristalsis. To date, however, the Ih pacemaker current conducted by HCN channels has never been detected in these cells, raising questions on the identity of RPSM pacemakers. Indeed, the RPSM pacemaker mechanisms of the unique multicalyceal upper urinary tract exhibited by humans remains unknown. Here, we developed immunopanning purification protocols and demonstrate that 96% of isolated HCN+ cells exhibit Ih. Single molecule STORM to whole-tissue imaging showed HCN+ cells express single HCN channels on their plasma membrane and integrate into the muscular syncytium. By contrast, PDGFR-+ cells exhibiting the morphology of ICC gut pacemakers were shown to be vascular mural cells. Translational studies in the homologous human and porcine multicalyceal upper urinary tracts showed that contractions and pacemaker depolarizations originate in proximal calyceal RPSM. Critically, HCN+ cells were shown to integrate into calyceal RPSM pacemaker tissues, and HCN channel block abolished electrical pacemaker activity and peristalsis of the multicalyceal upper urinary tract. Cumulatively, these studies demonstrate that HCN ion channels play a broad, evolutionarily conserved pacemaker role in both cardiac and smooth muscle organs and have implications for channelopathies as putative etiologies of smooth muscle disorders.