Hyperpolarized-activated and cyclic nucleotide-gated (HCN) channels are the only members of the voltage-gated ion channel superfamily in mammals that open upon hyperpolarization, conferring them pacemaker properties that are instrumental for rhythmic firing of cardiac and neuronal cells. Activation of their voltage-sensor domains (VSD) upon hyperpolarization occurs through a downward movement of the S4 helix bearing the gating charges, which triggers a break in the alpha-helical hydrogen bonding pattern at the level of a conserved Serine residue. Previous structural and molecular simulation studies had however failed to capture pore opening that should be triggered by VSD activation, presumably because of a low VSD/pore electromechanical coupling efficiency and the limited timescales accessible to such techniques. Here, we have used advanced modeling strategies, including enhanced sampling molecular dynamics simulations exploiting comparisons between non-domain swapped voltage-gated ion channel structures trapped in closed and open states to trigger pore gating and characterize electromechanical coupling in HCN1. We propose that the coupling mechanism involves the reorganization of the interfaces between the VSD helices, in particular S4, and the pore-forming helices S5 and S6, subtly shifting the balance between hydrophobic and hydrophilic interactions in a ‘domino effect’ during activation and gating in this region. Remarkably, our simulations reveal state-dependent occupancy of lipid molecules at this emergent coupling interface, suggesting a key role of lipids in hyperpolarization-dependent gating. Our model provides a rationale for previous observations and a possible mechanism for regulation of HCN channels by the lipidic components of the membrane.

Kinetic models have been employed to understand the logic of substrate transport through transporters of the Solute Carrier (SLC) family. All SLC transporters operate according to the alternate access model, which posits that substrate transport occurs in a closed loop of partial reactions (i.e., a transport cycle). Kinetic models can help to find realistic estimates for conformational transitions between individual states of the transport cycle. When constrained by experimental results, kinetic models can faithfully describe the function of a candidate transporter at a pre-steady state. In addition, we show that kinetic models can accurately predict the intra- and extracellular substrate concentrations maintained by the transporter at a steady state, even under the premise of loose coupling between the electrochemical gradient of the driving ion and of the substrate. We define the criteria for the design of a credible kinetic model of the SLC transporter. Parsimony is the guiding principle of kinetic modeling. We argue, however, that the level of acceptable parsimony is limited by the need to account for the substrate gradient established by a secondary active transporter, and for random order binding of co-substrates and substrate. Random order binding has consistently been observed in transporters of the SLC group.

Plasmalemmal solute carriers (SLCs) gauge and control solute abundance across cellular membranes. By virtue of this action, they play an important role in numerous physiological processes. Mutations in genes encoding the SLCs alter amino acid sequence that often leads to impaired protein function and onset of monogenic disorders. To understand how these altered proteins cause disease, it is necessary to undertake relevant functional assays. These experiments reveal descriptors of SLC function such as the maximal transport velocity (V_max), the Michaelis constant for solute uptake (K_M), potencies for inhibition of transporter function (IC₅₀/EC₅₀), and many more. In several instances, the mutated versions of different SLC transporters differ from their wild-type counterparts in the value of these descriptors. While determination of these experimental parameters can provide conjecture as to how the mutation gives rise to disease, they seldom provide any definitive insights on how a variant differ from the wild-type transporter in its operation. This is because the experimental determination of association between values of the descriptors and several partial reactions a transporter undergoes is casual, but not causal, at best. In the present study, we employ kinetic models that allow us to derive explicit mathematical terms and provide experimental descriptors as a function of the rate constants used to parameterize the kinetic model of the transport cycle. We show that it is possible to utilize these mathematical expressions to deduce, from experimental outcomes, how the mutation has impinged on partial reactions in the transport cycle.

Hyperpolarization-activated and cyclic nucleotide–gated (HCN) ion channels are members of the cyclic nucleotide–binding family and are crucial for regulating cellular automaticity in many excitable cells. HCN channel activation contributes to pain perception, and propofol, a widely used anesthetic, acts as an analgesic by inhibiting the voltage-dependent activity of HCN channels. However, the molecular determinants of propofol action on HCN channels remain unknown. Here, we use a propofol-analog photoaffinity labeling reagent to identify propofol binding sites in the human HCN1 isoform. Mass spectrometry analyses combined with molecular dynamics simulations show that a binding pocket is formed by extracellularly facing residues in the S3 and S4 transmembrane segments in the resting voltage-sensor conformation. Mutations of residues within the putative binding pocket mitigate or eliminate voltage-dependent modulation of HCN1 currents by propofol. Together, these findings reveal a conformation-specific propofol binding site that underlies voltage-dependent inhibition of HCN currents and provides a framework for identifying highly specific modulators of HCN channel gating.

Hyperpolarization and cyclic-nucleotide (HCN) activated ion channels play a critical role in generating self-propagating action potentials in pacemaking and rhythmic electrical circuits in the human body. Unlike most voltage-gated ion channels, the HCN channels activate upon membrane hyperpolarization, but the structural mechanisms underlying this gating behavior remain unclear. Here, we present cryo-electron microscopy structures of human HCN1 in Closed, Intermediate, and Open states. Our structures reveal that the inward motion of two gating charges past the charge transfer center (CTC) and concomitant tilting of the S5 helix drives the opening of the central pore. In the intermediate state structure, a single gating charge is positioned below the CTC and the pore appears closed, whereas in the open state structure, both charges move past CTC and the pore is fully open. Remarkably, the downward motion of the voltage sensor is accompanied by progressive unwinding of the inner end of S4 and S5 helices disrupting the tight gating interface that stabilizes the Closed state structure. This “melting” transition at the intracellular gating interface leads to a concerted iris-like displacement of S5 and S6 helices, resulting in pore opening. These findings reveal key structural features that are likely to underlie reversed voltage-dependence of HCN channels.

Hyperpolarized-activated and Cyclic Nucleotide-gated (HCN) channels are the only members of the voltage-gated ion channel superfamily in mammals that open upon hyperpolarization, conferring them pacemaker properties that are instrumental for rhythmic firing of cardiac and neuronal cells. Activation of their voltage-sensor domains (VSD) upon hyperpolarization occurs through a downward movement of the S4 helix bearing the gating charges, which triggers a break in the alpha-helical hydrogen bonding pattern at the level of a conserved Serine residue. Previous structural and molecular simulation studies had however failed to capture pore opening that should be triggered by VSD activation, presumably because of a low VSD/pore electromechanical coupling efficiency and the limited timescales accessible to such techniques. Here, we have used advanced modeling strategies, including enhanced sampling molecular dynamics simulations exploiting comparisons between non-domain swapped voltage-gated ion channel structures trapped in closed and open states to trigger pore gating and characterize electromechanical coupling in HCN1. We propose that the coupling mechanism involves the reorganization of the interfaces between the VSD helices, in particular S4, and the pore-forming helices S5 and S6, subtly shifting the balance between hydrophobic and hydrophilic interactions in a “domino effect” during activation and gating in this region. Remarkably, our simulations reveal state-dependent occupancy of lipid molecules at this emergent coupling interface, suggesting a key role of lipids in hyperpolarization-dependent gating. Our model provides a rationale for previous observations and a possible mechanism for regulation of HCN channels by the lipidic components of the membrane.