cAMP binding to closed pacemaker ion channels is non-cooperative.

Electrical activity in the brain and heart depends on rhythmic generation of action potentials by pacemaker ion channels (HCN) whose activity is regulated by cAMP binding1. Previous work has uncovered evidence for both positive and negative cooperativity in cAMP binding2,3, but such bulk measurements suffer from limited parameter resolution. Efforts to eliminate this ambiguity using single-molecule techniques have been hampered by the inability to directly monitor binding of individual ligand molecules to membrane receptors at physiological concentrations. Here we overcome these challenges using nanophotonic zero-mode waveguides4 to directly resolve binding dynamics of individual ligands to multimeric HCN1 and HCN2 ion channels. We show that cAMP binds independently to all four subunits when the pore is closed, despite a subsequent conformational isomerization to a flip state at each site. The different dynamics in binding and isomerization are likely to underlie physiologically distinct responses of each isoform to cAMP5 and provide direct validation of the ligand-induced flip-state model6-9. This approach for observing stepwise binding in multimeric proteins at physiologically relevant concentrations can directly probe binding allostery at single-molecule resolution in other intact membrane proteins and receptors.

Preparation of Giant Escherichia coli spheroplasts for Electrophysiological Recordings.

Prokaryotic ion channels have been instrumental in furthering our understanding of many fundamental aspects of ion channels' structure and function. However, characterizing the biophysical properties of a prokaryotic ion channel in a native membrane system using patch-clamp electrophysiology is technically challenging. Patch-clamp is regarded as a gold standard technique to study ion channel properties in both native and heterologous expression systems. The presence of a cell wall and the small size of bacterial cells makes it impossible to directly patch clamp using microelectrodes. Here, we describe a method for the preparation of giant E. coli spheroplasts in order to investigate the electrophysiological properties of bacterial cell membranes. Spheroplasts are formed by first inhibiting bacterial cell wall synthesis, followed by enzymatic digestion of the outer cell wall in the presence of a permeabilizing agent. This protocol can be used to characterize the function of any heterologous ion channels or ion transporters expressed in E. coli membranes.

Activation of the archaeal ion channel MthK is exquisitely regulated by temperature.

Physiological response to thermal stimuli in mammals is mediated by a structurally diverse class of ion channels, many of which exhibit polymodal behavior. To probe the diversity of biophysical mechanisms of temperature-sensitivity, we characterized the temperature-dependent activation of MthK, a two transmembrane calcium-activated potassium channel from thermophilic archaebacteria. Our functional complementation studies show that these channels are more efficient at rescuing K+ transport at 37°C than at 24°C. Electrophysiological activity of the purified MthK is extremely sensitive (Q10 >100) to heating particularly at low-calcium concentrations whereas channels lacking the calcium-sensing RCK domain are practically insensitive. By analyzing single-channel activities at limiting calcium concentrations, we find that temperature alters the coupling between the cytoplasmic RCK domains and the pore domain. These findings reveal a hitherto unexplored mechanism of temperature-dependent regulation of ion channel gating and shed light on ancient origins of temperature-sensitivity.

cAMP binds to closed, inactivated, and open sea urchin HCN channels in a state-dependent manner.

Hyperpolarization-activated cyclic-nucleotide-modulated (HCN) channels are nonselective cation channels that regulate electrical activity in the heart and brain. Previous studies of mouse HCN2 (mHCN2) channels have shown that cAMP binds preferentially to and stabilizes these channels in the open state-a simple but elegant implementation of ligand-dependent gating. Distinct from mammalian isoforms, the sea urchin (spHCN) channel exhibits strong voltage-dependent inactivation in the absence of cAMP. Here, using fluorescently labeled cAMP molecules as a marker for cAMP binding, we report that the inactivated spHCN channel displays reduced cAMP binding compared with the closed channel. The reduction in cAMP binding is a voltage-dependent process but proceeds at a much slower rate than the movement of the voltage sensor. A single point mutation in the last transmembrane domain near the channel's gate, F459L, abolishes inactivation and concurrently reverses the response to hyperpolarizing voltage steps from a decrease to an increase in cAMP binding. ZD7288, an open channel blocker that interacts with a region close to the activation/inactivation gate, dampens the reduction of cAMP binding to inactivated spHCN channels. In addition, compared with closed and "locked" closed channels, increased cAMP binding is observed in channels purposely locked in the open state upon hyperpolarization. Thus, the order of cAMP-binding affinity, measured by the fluorescence signal from labeled cAMP, ranges from high in the open state to intermediate in the closed state to low in the inactivated state. Our work on spHCN channels demonstrates intricate state-dependent communications between the gate and ligand-binding domain and provides new mechanistic insight into channel inactivation/desensitization.

Singlet oxygen modification abolishes voltage-dependent inactivation of the sea urchin spHCN channel.

Photochemically or metabolically generated singlet oxygen (1O2) reacts broadly with macromolecules in the cell. Because of its short lifetime and working distance, 1O2 holds potential as an effective and precise nanoscale tool for basic research and clinical practice. Here we investigate the modification of the spHCN channel that results from photochemically and chemically generated 1O2 The spHCN channel shows strong voltage-dependent inactivation in the absence of cAMP. In the presence of photosensitizers, short laser pulses transform the gating properties of spHCN by abolishing inactivation and increasing the macroscopic current amplitude. Alanine replacement of a histidine residue near the activation gate within the channel's pore abolishes key modification effects. Application of a variety of chemicals including 1O2 scavengers and 1O2 generators supports the involvement of 1O2 and excludes other reactive oxygen species. This study provides new understanding about the photodynamic modification of ion channels by 1O2 at the molecular level.

Shank3-deficient thalamocortical neurons show HCN channelopathy and alterations in intrinsic electrical properties.

KEY POINTS: Shank3 increases the HCN channel surface expression in heterologous expression systems. Shank3Δ13-16 deficiency causes significant reduction in HCN2 expression and Ih current amplitude in thalamocortical (TC) neurons. Shank3Δ13-16 - but not Shank3Δ4-9 -deficient TC neurons share changes in basic electrical properties which are comparable to those of HCN2-/- TC neurons. HCN channelopathy may critically mediate events downstream from Shank3 deficiency. ABSTRACT: SHANK3 is a scaffolding protein that is highly enriched in excitatory synapses. Mutations in the SHANK3 gene have been linked to neuropsychiatric disorders especially the autism spectrum disorders. SHANK3 deficiency is known to cause impairments in synaptic transmission, but its effects on basic neuronal electrical properties that are more localized to the soma and proximal dendrites remain unclear. Here we confirmed that in heterologous expression systems two different mouse Shank3 isoforms, Shank3A and Shank3C, significantly increase the surface expression of the mouse hyperpolarization-activated, cyclic-nucleotide-gated (HCN) channel. In Shank3Δ13-16 knockout mice, which lack exons 13-16 in the Shank3 gene (both Shank3A and Shank3C are removed) and display a severe behavioural phenotype, the expression of HCN2 is reduced to an undetectable level. The thalamocortical (TC) neurons from the ventrobasal (VB) complex of Shank3Δ13-16 mice demonstrate reduced Ih current amplitude and correspondingly increased input resistance, negatively shifted resting membrane potential, and abnormal spike firing in both tonic and burst modes. Impressively, these changes closely resemble those of HCN2-/- TC neurons but not of the TC neurons from Shank3Δ4-9 mice, which lack exons 4-9 in the Shank3 gene (Shank3C still exists) and demonstrate moderate behavioural phenotypes. Additionally, Shank3 deficiency increases the ratio of excitatory/inhibitory balance in VB neurons but has a limited impact on the electrical properties of connected thalamic reticular (RTN) neurons. These results provide new understanding about the role of HCN channelopathy in mediating detrimental effects downstream from Shank3 deficiency.