Charge movement in gating-locked HCN channels reveals weak coupling of voltage sensors and gate

HCN (hyperpolarization-activated cyclic nucleotide gated) pacemaker channels have an architecture similar to that of voltage-gated K+ channels, but they open with the opposite voltage dependence. HCN channels use essentially the same positively charged voltage sensors and intracellular activation gates as K+ channels, but apparently these two components are coupled differently. In this study, we examine the energetics of coupling between the voltage sensor and the pore by using cysteine mutant channels for which low concentrations of Cd2+ ions freeze the open–closed gating machinery but still allow the sensors to move. We were able to lock mutant channels either into open or into closed states by the application of Cd2+ and measure the effect on voltage sensor movement. Cd2+ did not immobilize the gating charge, as expected for strict coupling, but rather it produced shifts in the voltage dependence of voltage sensor charge movement, consistent with its effect of confining transitions to either closed or open states. From the magnitude of the Cd2+-induced shifts, we estimate that each voltage sensor produces a roughly three- to sevenfold effect on the open–closed equilibrium, corresponding to a coupling energy of ∼1.3–2 kT per sensor. Such coupling is not only opposite in sign to the coupling in K+ channels, but also much weaker

Distinct Populations of HCN Pacemaker Channels Produce Voltage-dependent and Voltage-independent Currents

Hyperpolarization-activated HCN pacemaker channels are critical for the generation of spontaneous activity and the regulation of excitability in the heart and in many types of neurons. These channels produce both a voltage-dependent current (Ih) and a voltage-independent current (Iinst or VIC). In this study, we explored the molecular basis of the voltage-independent current. We found that for the spHCN isoform, VIC averaged ∼4% of the maximum HCN conductance that could be activated by hyperpolarization. Cyclic AMP increased the voltage-independent current in spHCN to ∼8% of maximum. In HCN2, VIC was ∼2% of the maximal current, and was little affected by cAMP. VIC in both spHCN and HCN2 was blocked rapidly both by ZD7288 (an HCN channel blocker that is thought to bind in the conduction pore) and by application of Cd2+ to channels containing an introduced cysteine in the pore (spHCN-464C or HCN2-436C). These results suggest that VIC flows through the main conduction pathway, down the central axis of the protein. We suspected that VIC simply represented a nonzero limiting open probability for HCN channels at positive voltages. Surprisingly, we found instead that the spHCN channels carrying VIC were not in rapid equilibrium with the channels carrying the voltage-dependent current, because they could be blocked independently; a single application of blocker at a depolarized potential essentially eliminated VIC with little change in Ih. Thus, VIC appears to be produced by a distinct population of HCN channels. This voltage-independent current could contribute significantly to the role of HCN channels in neurons and myocytes; VIC flowing through the channels at physiological potentials would tend to promote excitability by accelerating both depolarization and repolarization

Fast and Slow Voltage Sensor Movements in HERG Potassium Channels

HERG encodes an inwardly-rectifying potassium channel that plays an important role in repolarization of the cardiac action potential. Inward rectification of HERG channels results from rapid and voltage-dependent inactivation gating, combined with very slow activation gating. We asked whether the voltage sensor is implicated in the unusual properties of HERG gating: does the voltage sensor move slowly to account for slow activation and deactivation, or could the voltage sensor move rapidly to account for the rapid kinetics and intrinsic voltage dependence of inactivation? To probe voltage sensor movement, we used a fluorescence technique to examine conformational changes near the positively charged S4 region. Fluorescent probes attached to three different residues on the NH2-terminal end of the S4 region (E518C, E519C, and L520C) reported both fast and slow voltage-dependent changes in fluorescence. The slow changes in fluorescence correlated strongly with activation gating, suggesting that the slow activation gating of HERG results from slow voltage sensor movement. The fast changes in fluorescence showed voltage dependence and kinetics similar to inactivation gating, though these fluorescence signals were not affected by external tetraethylammonium blockade or mutations that alter inactivation. A working model with two types of voltage sensor movement is proposed as a framework for understanding HERG channel gating and the fluorescence signals

Cooperative Gating between Single HCN Pacemaker Channels

HCN pacemaker channels (If, Iq, or Ih) play a fundamental role in the physiology of many excitable cell types, including cardiac myocytes and central neurons. While cloned HCN channels have been studied extensively in macroscopic patch clamp experiments, their extremely small conductance has precluded single channel analysis to date. Nevertheless, there remain fundamental questions about HCN gating that can be resolved only at the single channel level. Here we present the first detailed single channel study of cloned mammalian HCN2. Excised patch clamp recordings revealed discrete hyperpolarization-activated, cAMP-sensitive channel openings with amplitudes of 150–230 fA in the activation voltage range. The average conductance of these openings was ∼1.5 pS at −120 mV in symmetrical 160 mM K+. Some traces with multiple channels showed unusual gating behavior, characterized by a variable long delay after a voltage step followed by runs of openings. Noise analysis on macroscopic currents revealed fluctuations whose magnitudes were systematically larger than predicted from the actual single channel current size, consistent with cooperativity between single HCN channels

Reversal of HCN Channel Voltage Dependence via Bridging of the S4–S5 Linker and Post-S6

Voltage-gated ion channels possess charged domains that move in response to changes in transmembrane voltage. How this movement is transduced into gating of the channel pore is largely unknown. Here we show directly that two functionally important regions of the spHCN1 pacemaker channel, the S4–S5 linker and the C-linker, come into close proximity during gating. Cross-linking these regions with high-affinity metal bridges or disulfide bridges dramatically alters channel gating in the absence of cAMP; after modification the polarity of voltage dependence is reversed. Instead of being closed at positive voltage and activating with hyperpolarization, modified channels are closed at negative voltage and activate with depolarization. Mechanistically, this reversal of voltage dependence occurs as a result of selectively eliminating channel deactivation, while retaining an existing inactivation process. Bridging also alters channel activation by cAMP, showing that interaction of these two regions can also affect the efficacy of physiological ligands

Structural changes during HCN channel gating defined by high affinity metal bridges

Hyperpolarization-activated cyclic nucleotide–sensitive nonselective cation (HCN) channels are activated by membrane hyperpolarization, in contrast to the vast majority of other voltage-gated channels that are activated by depolarization. The structural basis for this unique characteristic of HCN channels is unknown. Interactions between the S4–S5 linker and post-S6/C-linker region have been implicated previously in the gating mechanism of HCN channels. We therefore introduced pairs of cysteines into these regions within the sea urchin HCN channel and performed a Cd2+-bridging scan to resolve their spatial relationship. We show that high affinity metal bridges between the S4–S5 linker and post-S6/C-linker region can induce either a lock-open or lock-closed phenotype, depending on the position of the bridged cysteine pair. This suggests that interactions between these regions can occur in both the open and closed states, and that these regions move relative to each other during gating. Concatenated constructs reveal that interactions of the S4–S5 linker and post-S6/C-linker can occur between neighboring subunits. A structural model based on these interactions suggests a mechanism for HCN channel gating. We propose that during voltage-dependent activation the voltage sensors, together with the S4–S5 linkers, drive movement of the lower ends of the S5 helices around the central axis of the channel. This facilitates a movement of the pore-lining S6 helices, which results in opening of the channel. This mechanism may underlie the unique voltage dependence of HCN channel gating

Movements near the Gate of a Hyperpolarization-activated Cation Channel

Hyperpolarization-activated cation (HCN) channels regulate pacemaking activity in cardiac cells and neurons. Like the related depolarization-activated K+ channels (Kv channels), HCN channels use an intracellular activation gate to regulate access to an inner cavity, lined by the S6 transmembrane regions, which leads to the selectivity filter near the extracellular surface. Here we describe two types of metal interactions with substituted cysteines in the S6, which alter the voltage-controlled movements of the gate. At one position (L466), substitution of cysteine in all four subunits allows Cd2+ ions at nanomolar concentration to stabilize the open state (a “lock-open” effect). This effect depends on native histidines at a nearby position (H462); the lock-open effect can be abolished by changing the histidines to tyrosines, or enhanced by changing them to cysteines. Unlike a similar effect in Kv channels, this effect depends on a Cd2+ bridge between 462 and 466 in the same subunit. Cysteine substitution at another position (Q468) produces two effects of Cd2+: both a lock-open effect and a dramatic slowing of channel activation—a “lock-closed” effect. The two effects can be separated, because the lock-open effect depends on the histidine at position 462. The novel lock-closed effect results from stabilization of the closed state by the binding of up to four Cd2+ ions. During the opening conformational change, the S6 apparently moves from one position in which the 468C cysteines can bind four Cd2+ ions, possibly as a cluster of cysteines and cadmium ions near the central axis of the pore, to another position (or flexible range of positions) where either 466C or 468C can bind Cd2+ in association with the histidine at 462

Imaging energy status in live cells with a fluorescent biosensor of the intracellular ATP-to-ADP ratio

The ATP:ADP ratio is a critical parameter of cellular energy status that regulates many metabolic activities. Here we report an optimized genetically-encoded fluorescent biosensor, PercevalHR, that senses the ATP:ADP ratio. PercevalHR is tuned to the range of intracellular ATP:ADP expected in mammalian cells, and it can be used with one- or two-photon microscopy in live samples. We use PercevalHR to visualize activity-dependent changes in ATP:ADP when neurons are exposed to multiple stimuli, demonstrating that it is a sensitive reporter of physiological changes in energy consumption and production. We also use PercevalHR to visualize intracellular ATP:ADP while simultaneously recording currents from ATP-sensitive potassium (KATP) channels in single cells, showing that PercevalHR enables the study of coordinated variation in ATP:ADP and KATP channel open probability in intact cells. With its ability to monitor changes in cellular energetics within seconds, PercevalHR should be a versatile tool for metabolic research

A PKA activity sensor for quantitative analysis of endogenous GPCR signaling via 2-photon FRET-FLIM imaging

Neuromodulators have profound effects on behavior, but the dynamics of their intracellular effectors has remained unclear. Most neuromodulators exert their function via G-protein-coupled receptors (GPCRs). One major challenge for understanding neuromodulator action is the lack of dynamic readouts of the biochemical signals produced by GPCR activation. The adenylate cyclase/cyclic AMP/protein kinase A (PKA) module is a central component of such biochemical signaling. This module is regulated by several behaviorally important neuromodulator receptors. Furthermore, PKA activity is necessary for the induction of many forms of synaptic plasticity as well as for the formation of long-term memory. In order to monitor PKA activity in brain tissue, we have developed a 2-photon fluorescence lifetime imaging microscopy (2pFLIM) compatible PKA sensor termed FLIM-AKAR, which is based on the ratiometric FRET sensor AKAR3. FLIM-AKAR shows a large dynamic range and little pH sensitivity. In addition, it is a rapidly diffusible cytoplasmic protein that specifically reports net PKA activity in situ. FLIM-AKAR expresses robustly in various brain regions with multiple transfection methods, can be targeted to genetically identified cell types, and responds to activation of both endogenous GPCRs and spatial-temporally specific delivery of glutamate. Initial experiments reveal differential regulation of PKA activity across subcellular compartments in response to neuromodulator inputs. Therefore, the reporter FLIM-AKAR, coupled with 2pFLIM, enables the study of PKA activity in response to neuromodulator inputs in genetically identified neurons in the brain, and sheds light on the intracellular dynamics of endogenous GPCR activation