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Anionic lipids unlock the gates of select ion channels in the pacemaker family.

ABSTRACT: Lipids play important roles in regulating membrane protein function, but the molecular mechanisms used are elusive. Here we investigated how anionic lipids modulate SthK, a bacterial pacemaker channel homolog, and HCN2, whose activity contributes to pacemaking in the heart and brain. Using SthK allowed the reconstitution of purified channels in controlled lipid compositions for functional and structural assays that are not available for the eukaryotic channels. We identified anionic lipids bound tightly to SthK and their exact binding locations and determined that they potentiate channel activity. Cryo-EM structures in the most potentiating lipids revealed an open state and identified a nonannular lipid bound with its headgroup near an intersubunit salt bridge that clamps the intracellular channel gate shut. Breaking this conserved salt bridge abolished lipid modulation in SthK and eukaryotic HCN2 channels, indicating that anionic membrane lipids facilitate channel opening by destabilizing these interactions. Our findings underline the importance of state-dependent protein-lipid interactions.

SUBMITTER: Schmidpeter PAM

PROVIDER: S-EPMC10022520 | biostudies-literature | 2022 Nov

REPOSITORIES: biostudies-literature

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Anionic lipids unlock the gates of select ion channels in the pacemaker family.

Schmidpeter Philipp A M PAM Wu Di D Rheinberger Jan J Riegelhaupt Paul M PM Tang Haiping H Robinson Carol V CV Nimigean Crina M CM

Nature structural & molecular biology 20221109 11

Lipids play important roles in regulating membrane protein function, but the molecular mechanisms used are elusive. Here we investigated how anionic lipids modulate SthK, a bacterial pacemaker channel homolog, and HCN2, whose activity contributes to pacemaking in the heart and brain. Using SthK allowed the reconstitution of purified channels in controlled lipid compositions for functional and structural assays that are not available for the eukaryotic channels. We identified anionic lipids bound ...[more]

PMID: 36352139

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Project description:Although hyperpolarization-activated cation (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-α+ 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 aetiologies of smooth muscle disorders. KEY POINTS: Pacemakers trigger contractions of involuntary muscles. Hyperpolarization-activated cation (HCN) ion channels underpin cardiac pacemaker activity, but their role in smooth muscle organs remains controversial. Renal pelvic smooth muscle (RPSM) pacemakers trigger contractions that propel waste away from the kidney. HCN+ cells localize to murine RPSM pacemaker tissue and HCN channel conductance is required for peristalsis. The HCN (Ih ) current has never been detected in RPSM cells, raising doubt whether HCN+ cells are bona fide pacemakers. Moreover, the pacemaker mechanisms of the unique multicalyceal RPSM of higher order mammals remains unknown. In total, 97% of purified HCN+ RPSM cells exhibit Ih . HCN+ cells integrate into the RPSM musculature, and pacemaker tissue peristalsis is dependent on HCN channels. Translational studies in human and swine demonstrate HCN channels are conserved in the multicalyceal RPSM and that HCN channels underlie pacemaker activity that drives peristalsis. These studies provide insight into putative channelopathies that can underlie smooth muscle dysfunction.

Project description:Effects of polyvalent ions on the lateral packing of phospholipids have been known for decades, but the physiological consequences have not been systematically studied. Gd(3+) is a relatively nonspecific agent that blocks mechano-gated channels with a variable affinity. In this study, we show that the large mechanosensitive channel MscL of Escherichia coli is effectively blocked by Gd(3+) only when reconstituted with negatively charged phospholipids (e.g., PS). Taking this lead, we studied effects of Gd(3+) on monolayers and unilamellar vesicles made of natural brain PS, DMPS, and its mixtures with DMPC. In monolayer experiments, we found that muM Gd(3+) present in the subphase leads to approximately 8% lateral compaction of brain PS (at 35 mN/m). Gd(3+) more strongly shrinks and rigidifies DMPS films causing a spontaneous liquid expanded-to-compact transition to the limiting 40 A(2)/mol. Pressure-area isotherms of uncharged DMPC were unaffected by Gd(3+), and neutralization of DMPS surface by low pH did not produce strong compaction. Upshifts of surface potential isotherms of DMPS monolayers reflected changes in the diffuse double layer due to neutralization of headgroup charges by Gd(3+), whereas the increased packing density produced up to a 200 mV change in the interfacial dipole potential. The slopes of surface potential versus reciprocal area predicted that Gd(3+) induced a modest ( approximately 18%) increase in the magnitude of the individual lipid dipoles in DMPS. Isothermal titration calorimetry indicated that binding of Gd(3+) to DMPS liposomes in the gel state is endothermic, whereas binding to liquid crystalline liposomes produces heat consistent with the isothermal liquid-to-gel phase transition induced by the ion. Both titration curves suggested a K(b) of approximately 10(6) M(-1). We conclude that anionic phospholipids serve as high-affinity receptors for Gd(3+) ions, and the ion-induced compaction generates a lateral pressure increase estimated as tens of mN/m. This pressure can "squeeze" the channel and shift the equilibrium toward the closed state.

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Anionic lipids unlock the gates of select ion channels in the pacemaker family.

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Anionic lipids unlock the gates of select ion channels in the pacemaker family.

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