Hyperpolarization-activated Channels Research Articles

Conserved Voltage Sensor

The voltage sensor for the depolarization-activated, Shaker K + channel (Kv) is well characterized as the transmembrane segment S4. In Kv channels, depolarization stimulates the outward movement (more residues of S4 exposed to the extracellular surface), and the channel opens. Männikkö et al. report that hyperpolarization-activated, cyclic nucleotide-gated, cation nonselective channels (HCN) also use movement of the S4 domain to control channel gating. Cysteine accessibility studies on channels expressed in Xenopus oocytes were used to show that outward movement of the S4 domain was associated with closure of HCN channels at depolarized membrane potentials. Thus, depolarization- and hyperpolarization-activated channels use a similar voltage-sensing mechanism. However, outward movement of S4 results in channel closure for the hyperpolarization-activated HCN channel and channel opening for the depolarization-activated Kv channels. In this way, opposite changes in membrane potential can act through the same domain to produce channel activation. R. Männikkö, F. Elinder, H. P. Larsson, Voltage-sensing mechanism is conserved among ion channels gated by opposite voltages. Nature 419 , 837-841 (2002). [Online Journal]

Keeping Pace with Pacemaker Channels.

Developmental Febrile Seizures Modulate Hippocampal Gene Expression of Hyperpolarization-activated Channels in an Isoform and Cell-specific Manner Brewter A, Bender RA, Chen Y, Dube C, Eghbal-Ahmadi M, and Baram TZ J Neurosci 2002;22:4591–4599 Febrile seizures, in addition to being the most common seizure type of the developing human, may contribute to the generation of subsequent limbic epilepsy. Our previous work demonstrated that prolonged experimental febrile seizures in the immature rat model increased hippocampal excitability in the long term, enhancing susceptibility to future seizures. The mechanisms for these profound proepileptogenic changes did not require cell death and were associated with long-term slowed kinetics of the hyperpolarization activated depolarizing current (Ih). Here we show that these seizures modulate the expression of genes encoding this current, the hyperpolarization-activated, cyclic nucleotide–gated channels (HCNs): In CA1 neurons expressing multiple HCN isoforms, the seizures induced a coordinated reduction of HCN1 mRNA and enhancement of HCN2 expression, thus altering the neuronal HCN phenotype. The seizure-induced augmentation of HCN2 expression involved CA3 in addition to CA1, whereas for HCN4, mRNA expression was not changed by the seizures in either hippocampal region. This isoform- and region-specific transcriptional regulation of HCNs required neuronal activity rather than hyperthermia alone, correlated with seizure duration, and favored the formation of slow-kinetics HCN2-encoded channels. In summary, these data demonstrate a novel, activity-dependent transcriptional regulation of HCN molecules by developmental seizures. These changes result in long-lasting alteration of the HCN phenotype of specific hippocampal neuronal populations, with profound consequences on the excitability of the hippocampal network.

Development of membrane conductance improves coincidence detection in the nucleus laminaris of the chicken.

Coincidence detection at the nucleus laminaris (NL) of a chicken was improved between embryos (embryonic days (E) 16 and 17) and chicks (post-hatch days (P) 2-7) in slice preparations. Electrical stimuli were applied bilaterally to the projection fibres to the NL at various intervals. The response window corresponding to the temporal separation of electrical stimuli that resulted in half-maximal firing probability was adopted as the measure of coincidence detection, and was narrower in chicks (1.4 ms) than in embryos (3.9 ms). Between these two ages, the membrane time constant of NL neurons was reduced from 18.4 to 3.2 ms and the membrane conductance was increased 5-fold, while no difference was measured in the input capacitance. Evoked EPSCs decayed slightly faster in chicks, while the size and the time course of miniature EPSCs were unchanged. Action potentials had lower thresholds and larger after-hyperpolarization in chicks than in embryos. Dendrotoxin-I depolarized cells and increased their input resistance significantly at both ages, eliminated the after-hyperpolarization, and delayed the decay phase of action potentials, indicative of the expression of low-threshold K(+) channels. Cs(+) hyperpolarized the cells, increased the input resistance and eliminated sags during hyperpolarization at both ages, while the hyperpolarization sag was affected by neither Ba(2+) nor TEA. These data indicate the expression of hyperpolarization-activated cation channels. Between these two ages, the maximum conductance of low-threshold K(+) channels increased 4-fold to about 16 nS, and hyperpolarization-activated channels increased 6-fold to about 10 nS. Improvement of coincidence detection correlated with the acceleration of the EPSP time course as a result of the increase of these conductances.

Characteristics of hyperpolarization-activated cation currents in portal vein smooth muscle cells.

Voltage-clamp studies of freshly isolated smooth muscle cells from rabbit portal vein revealed the existence of a time-dependent cation current evoked by membrane hyperpolarization (termed I(h)). Both the rate of activation and the amplitude of I(h) were enhanced by membrane hyperpolarization. Half-maximal activation of I(h) was about -105 mV with conventional whole cell and -80 mV when the perforated patch technique was used. In current clamp, injection of hyperpolarizing current produced a marked depolarizing "sag" followed by rebound depolarization. Activation of I(h) was augmented by an increase in the extracellular K(+) concentration and was blocked rapidly by externally applied Cs(+) (1-5 mM). The bradycardic agent ZD-7288 (10 microM), a selective inhibitor of I(h), produced a characteristically slow inhibition of the portal vein I(h). The depolarizing sag recorded in current clamp was also abolished by application of 5 mM Cs(+). Cs(+) significantly decreased the frequency of spontaneous contractions in both whole rat portal vein and rabbit portal vein segments. Multiplex RT-PCR of rabbit portal vein myocytes using primers derived from existing genes for hyperpolarization-activated cation channels (HCN1-4) revealed the existence of cDNA clones corresponding to HCN2, 3, and 4. The present study shows that portal vein myocytes contain genes shown to encode for hyperpolarization-activated channels and exhibit an endogenous current with characteristics similar to I(h) in other cell types. This conductance appears to determine, in part, the rhythmicity of this vessel.

Blockade of hyperpolarization-activated channels modifies the effect of beta-adrenoceptor stimulation.

Experiments on rats showed that blockade of hyperpolarization-activated currents moderates tachycardia induced by beta-adrenoceptor agonist isoproterenol and potentiates the increase in stroke volume produced by this agonist. Electrical stimulation of the vagus nerve against the background of isoproterenol treatment augmented bradycardia and increased stroke volume. Blockade of hyperpolarization-activated currents followed by application of isoproterenol moderated vagus-induced bradycardia and had no effect on the dynamics of stroke volume.

Parasympathetic nervous system modulates effects of hyperpolarization-activated channel blockade.

Bilateral vagotomy eliminating extracardiac parasympathetic influences on the heart modulates the changes in variational pulsogram during blockade of hyperpolarization-activated currents, but has no significant effect on stroke volume after the blockade.

Hyperpolarization-activated channels HCN1 and HCN4 mediate responses to sour stimuli.

Sour taste is initiated by protons acting at receptor proteins or channels. In vertebrates, transduction of this taste quality involves several parallel pathways. Here we examine the effects of sour stimuli on taste cells in slices of vallate papilla from rat. From a subset of cells, we identified a hyperpolarization-activated current that was enhanced by sour stimulation at the taste pore. This current resembled Ih found in neurons and cardio-myocytes, a current carried by members of the family of hyperpolarization-activated and cyclic-nucleotide-gated (HCN) channels. We show by in situ hybridization and immunohistochemistry that HCN1 and HCN4 are expressed in a subset of taste cells. By contrast, gustducin, the G-protein involved in bitter and sweet taste, is not expressed in these cells. Lowering extracellular pH causes a dose-dependent flattening of the activation curve of HCN channels and a shift in the voltage of half-maximal activation to more positive voltages. Our results indicate that HCN channels are gated by extracellular protons and may act as receptors for sour taste.

Integrated allosteric model of voltage gating of HCN channels.

Hyperpolarization-activated (pacemaker) channels are dually gated by negative voltage and intracellular cAMP. Kinetics of native cardiac f-channels are not compatible with HH gating, and require closed/open multistate models. We verified that members of the HCN channel family (mHCN1, hHCN2, hHCN4) also have properties not complying with HH gating, such as sigmoidal activation and deactivation, activation deviating from fixed power of an exponential, removal of activation "delay" by preconditioning hyperpolarization. Previous work on native channels has indicated that the shifting action of cAMP on the open probability (Po) curve can be accounted for by an allosteric model, whereby cAMP binds more favorably to open than closed channels. We therefore asked whether not only cAMP-dependent, but also voltage-dependent gating of hyperpolarization-activated channels could be explained by an allosteric model. We hypothesized that HCN channels are tetramers and that each subunit comprises a voltage sensor moving between "reluctant" and "willing" states, whereas voltage sensors are independently gated by voltage, channel closed/open transitions occur allosterically. These hypotheses led to a multistate scheme comprising five open and five closed channel states. We estimated model rate constants by fitting first activation delay curves and single exponential time constant curves, and then individual activation/deactivation traces. By simply using different sets of rate constants, the model accounts for qualitative and quantitative aspects of voltage gating of all three HCN isoforms investigated, and allows an interpretation of the different kinetic properties of different isoforms. For example, faster kinetics of HCN1 relative to HCN2/HCN4 are attributable to higher HCN1 voltage sensors' rates and looser voltage-independent interactions between subunits in closed/open transitions. It also accounts for experimental evidence that reduction of sensors' positive charge leads to negative voltage shifts of Po curve, with little change of curve slope. HCN voltage gating thus involves two processes: voltage sensor gating and allosteric opening/closing.

Cellular expression and functional characterization of four hyperpolarization-activated pacemaker channels in cardiac and neuronal tissues

Cellular expression and functional characterization of four hyperpolarization-activated pacemaker channels in cardiac and neuronal tissues

Molecular characterization of a slowly gating human hyperpolarization-activated channel predominantly expressed in thalamus, heart, and testis.

Rhythmic activity of neurons and heart cells is endowed by pacemaker channels that are activated by hyperpolarization and directly regulated by cyclic nucleotides (termed HCN channels). These channels constitute a multigene family, and it is assumed that the properties of each member are adjusted to fit its particular function in the cell in which it resides. Here we report the molecular and functional characterization of a human subtype hHCN4. hHCN4 transcripts are expressed in heart, brain, and testis. Within the brain, the thalamus is the predominant area of hHCN4 expression. Heterologous expression of hHCN4 produces channels of unusually slow kinetics of activation and inactivation. The mean potential of half-maximal activation (V(1/2)) was -75.2 mV. cAMP shifted V(1/2) by 11 mV to more positive values. The hHCN4 gene was mapped to chromosome band 15q24-q25. The characteristic expression pattern and the sluggish gating suggest that hHCN4 controls the rhythmic activity in both thalamocortical neurons and pacemaker cells of the heart.

Molecular cloning of a putative voltage- and cyclic nucleotide-gated ion channel present in the antennae and eyes of Drosophila melanogaster.

The amino acid sequence BCNG-1 (brain cyclic nucleotide gated 1, of the mouse), the first member of mamalian I(h) channels, was used to construct a set of polymerase chain reaction (PCR) primers from possibly conserved regions. Reverse transcription-PCR with Drosophila melanogaster mRNA yielded in a PCR product, which exhibited a high homology to BCNG-1. Using these PCR products to screen a D. melanogaster head cDNA library we isolated a cDNA encoding a member of a new class of putative voltage- and cyclic nucleotide-gated potassium channels from D. melanogaster. The most important features of the amino acid sequence predicted from the cDNA were a C-terminal cyclic nucleotide-binding region, an S4-voltage sensor and a putative potassium-selective pore-forming motif. The high homology of 51% to the sea urchin I(h) channel, which belongs to the same class of ion channels as BCNG-1, leads us to suggest that the Drosophila cDNA is the first insect member of a new class of hyperpolarization-activated and cyclic nucleotide-gated channels. As shown by in situ hybridization, a pronounced mRNA expression was detected in neuronal tissue, including sensory tissue like the compound eyes, and the olfactory and the auditory organs.

Molecular identification of a hyperpolarization-activated channel in sea urchin sperm.

Sea urchin eggs attract sperm through chemotactic peptides, which evoke complex changes in membrane voltage and in the concentrations of cyclic AMP, cyclic GMP and Ca2+ ions The intracellular signalling pathways and their cellular targets are largely unknown. We have now cloned, from sea urchin testis, the complementary DNA encoding a channel polypeptide, SPIH. Functional expression of SPIH gives rise to weakly K+-selective hyperpolarization-activated channels, whose activity is enhanced by the direct action of cAMP. Thus, SPIH is under the dual control of voltage and cAMP. The SPIH channel, which is confined to the sperm flagellum, may be involved in the control of flagellar beating. SPIH currents exhibit all the hallmarks of hyperpolarization-activated currents (Ih), which participate in the rhythmic firing of central neurons, control pacemaking in the heart, and curtail saturation by bright light in retinal photoreceptors. Because of their sequence and functional properties, Ih channels form a class of their own within the superfamily of voltage-gated and cyclic-nucleotide-gated channels.

Membrane properties and monosynaptic retinal excitation of neurons in the turtle accessory optic system.

Using an eye-attached isolated brain stem preparation of a turtle, Pseudemys scripta elegans, in conjunction with whole cell patch techniques, we recorded intracellular activity of accessory optic system neurons in the basal optic nucleus (BON). This technique offered long-lasting stable recordings of individual synaptic events. In the reduced preparation (most of the dorsal structures were removed), large spontaneous excitatory synaptic inputs [excitatory postsynaptic potentials (EPSPs)] were frequently recorded. Spontaneous inhibitory postsynaptic potentials were rarely observed except in few cases. Most EPSPs disappeared after injection of lidocaine into the retina. A few EPSPs of small size remained, suggesting that these EPSPs either were from intracranial sources or may have been miniature spontaneous synaptic potentials from retinal ganglion cell axon terminals. Population EPSPs were synchronously evoked by electrical stimulation of the contralateral optic nerve. Their constant onset latency and their ability to follow short-interval paired stimulation indicated that much of the population EPSP's response was monosynaptic. Visually evoked BON spikes and EPSP inputs to BON showed direction sensitivity when a moving pattern was projected onto the entire contralateral retina. With the use of smaller moving patterns, the receptive field of an individual BON cell was identified. A small spot of light, projected within the receptive field, guided the placement of a bipolar stimulation electrode to activate retinal ganglion cells that provided input to that BON cell. EPSPs evoked by this retinal microstimulation showed features of unitary EPSPs. Those EPSPs had distinct low current thresholds. Recruitment of other inputs was only evident when the stimulation level was increased substantially above threshold. The average size of evoked unitary EPSPs was 7.8 mV, confirming the large size of synaptic inputs of this system relative to nonsynaptic noise. EPSP shape was plotted (rise time vs. amplitude), with the use of either evoked unitary EPSPs or spontaneous EPSPs. Unlike samples of spontaneous EPSPs, data from many unitary EPSPs formed distinct clusters in these scatterplots, indicating that these EPSPs had a unique shape among the whole population of EPSPs. In most BON cells studied, hyperpolarization-activated channels caused a slow depolarization sag that reached a plateau within 0.5-1 s. This property suggests that BON cells may be more complicated than a simple site for convergence of direction-sensitive retinal ganglion cells to form a central retinal slip signal for control of oculomotor reflexes.

Molecular dissection of gating in the ClC-2 chloride channel.

The ClC-2 chloride channel is probably involved in the regulation of cell volume and of neuronal excitability. Site-directed mutagenesis was used to understand ClC-2 activation in response to cell swelling, hyperpolarization and acidic extracellular pH. Similar to equivalent mutations in ClC-0, neutralizing Lys566 at the end of the transmembrane domains results in outward rectification and a shift in voltage dependence, but leaves the basic gating mechanism, including swelling activation, intact. In contrast, mutations in the cytoplasmic loop between transmembrane domains D7 and D8 abolish all three modes of activation by constitutively opening the channel without changing its pore properties. These effects resemble those observed with deletions of an amino-terminal inactivation domain, and suggest that it may act as its receptor. Such a 'ball-and-chain' type mechanism may act as a final pathway in the activation of ClC-2 elicited by several stimuli.

Simulation of mechanisms responsible for initiation and modulation of bursting activity in RPa1 neuron of theHelix snail

A mathematical model of bursting activity in the RPa1 neuron of theHelix snail has been developed. The model allowed us to describe the processes of initiation and augmentation of the bursting activity related to transient secretion of a modulatory factor. Based on the analysis of computer simulations of various mechanisms underlying the effect of a modulating factor on the ionic membrane conductances in the bursting neuron, we suggested that modulating factor evokes a transition of non-voltage-dependent sodium channels and hyperpolarization-activated outward current channels to an active state and influences the gating of voltage-dependent sodium channels.

The Role of Potassium Channels in the Temperature Control of Stomatal Aperture.

We used the patch-clamp technique to examine the effect of temperature (13-36[deg]C) on the depolarization-activated K channels (KD channels) and on the hyperpolarization-activated channels (KH channels) in the plasma membrane of Vicia faba guard-cell protoplasts. The steady-state whole-cell conductance of both K channel types increased with temperature up to 20[deg]C. However, whereas the whole-cell conductance of the KH channels increased further and saturated at 28[deg]C, that of KD channels decreased at higher temperatures. The unitary conductance of both channel types increased with temperature like the rate of diffusion in water (temperature quotient of approximately 1.5), constituting the major contribution to the conductance increase in the whole cells. The mean number of available KH channels was not affected significantly by temperature, but the mean number of available KD channels increased significantly between 13 and 20[deg]C and declined drastically above 20[deg]C. This decrease and the reduced steady-state voltage-dependent probability of opening of the KD channels above 28[deg]C (because of a shift of voltage dependence by +21 mV) account for the depression of the whole-cell KD conductance at the higher temperatures. This may be a basic mechanism by which leaves of well-watered plants keep their stomata open during heat stress to promote cooling by transpiration.

Transmembrane Segments Critical for Potassium Channel Function

The relative contribution of the transmembrane segments in the α-subunit of Shaker-type potassium channels was investigated in relation to potassium channel function. Starting from a wild-type Kv1.1 channel, four different deletion mutants were made, missing respectively transmembrane segments S1 and S2, S2 and S3, S1 to S3, and S1 to S4. To ensure the assembly of the different subunits, the hydrophylic N-terminal domain was always conserved. The lack of transmembrane segments S1 to S4 converts a depolarization-activated WT Kv1.1 channel with outward rectification into a hyperpolarization-activated channel with inward rectification. In contrast, mutant channels missing transmembrane segments S1 and S2, S2 and S3, or S1 to S3 did not reveal functional expression.

Reversal of rectification and alteration of selectivity and pharmacology in a mammalian Kv1.1 potassium channel by deletion of domains S1 to S4.

1. A possible relation between the family of inwardly rectifying K+ channels and the Shaker superfamily of K+ channels was investigated using a deletion mutant (DelS1-S4) of a delayed rectifier Kv1.1 (RCK1) K+ channel. 2. The mutant DelS1-S4 was made by eliminating the sequence coding for transmembrane domains S1 to S4 of the Kv1.1 K+ channel, and re-ligating the sequence coding for the cytoplasmic amino terminus to transmembrane domain S5. Microelectrode voltage-clamp and patch-clamp experiments were performed on Xenopus laevis oocytes after injection of in vitro transcribed mRNA coding for mutant and wild-type channels. 3. The lack of transmembrane domains S1 to S4 converts a depolarization-activated wild-type Kv1.1 K+ channel with outward rectification into a hyperpolarization-activated channel with inward rectification. Although the pore region of the deletion mutant is identical to the wild-type channel, the mutant channel is a non-selective cation channel and is characterized by an altered pharmacology profile.

Voltage, calcium, and stretch activated ionic channels and intracellular calcium in bone cells.

Embryonic chick bone cells express various types of ionic channels in their plasma membranes for as yet unresolved functions. Chick osteoclasts (OCL) have the richest spectrum of channel types. Specific for OCL is a K+ channel, which activates (opens) when the inside negative membrane potential (Vm) becomes more negative (hyperpolarization). This is consistent with findings of others on rat OCL. The membrane conductance constituted by these channels is called the inward rectifying K+ conductance (GKi), or inward rectifier, because the hyperpolarization-activated channels cause cell-inward K+ current to pass more easily through the membrane than outward K+ current. Besides GKi channels, OCL may express two other types of voltage-activated K+ channels. One constitutes the transient outward rectifying K+ conductance (GKto), which is activated upon making the membrane potential less negative (depolarization) but has a transient nature. This conductance favors transient K+ conduction in the cell-outward direction. The GKto also occurs in a small percentage of cells in osteoblast (OBL) and periosteal fibroblast (PFB) cultures. The other OCL K+ conductance, the GKCa, is activated by both membrane depolarization and a rise in [Ca2+]i. GKCa channels are also present in the other chick bone cell types, that is, OBL, osteocytes (OCY), and PFB. Furthermore, in excised patches of all bone cell types, channels have been found that conduct anions, including Cl- and phosphate ions. These channels are only active around Vm = 0 mV. While searching for a membrane mechanism for adaptation of bone to mechanical loading, we found stretch-activated channels in chick osteoclasts; other investigators have found stretch-activated cation channels (K+ or aselective) in rat and human osteogenic cell lines. In contrast to other studies on cell lines or OBL from other species, we have not found any of the classic macroscopic voltage-activated calcium conductances (GCa) in any of the chick bone cells under our experimental conditions. However, our fluorescence measurements of [Ca2+]i in single cells indicate the presence of Ca2+ conductive pathways through the plasma membrane of osteoblastic cells and osteoclasts, consistent with other studies. We discuss possible roles for GKi, GKCa, and anion channels in acid secretion by OCL and for stretch-activated channels in OCL locomotion.

Hyperpolarization-activated cationic channels in smooth muscle cells are stretch sensitive

The properties of hyperpolarization-activated channels were studied in single smooth muscle cells from the stomach of the toad, Bufo marinus, using the patch-clamp technique. In cell-attached patches, inward channel currents were activated by hyperpolarizing pulses from a holding potential of -20 mV to potentials more negative than -60 mV. The activity of the channels increased and their latency of activation decreased as the hyperpolarization was increased. The slope conductance of the channels with standard high sodium concentration pipette solution was 64.2 +/- 9.1 pS (SD, n = 17). Stretching the patch, by suction applied to the back of the patch pipette, also increased the activity and shortened the latency of activation. We designate these channels as HA-SACs (hyperpolarization- and stretch-activated channels). HA-SACs were observed in 83% (175/210) of the patches studied. HA-SAC currents were carried by sodium and potassium ions, but their amplitude was increased by replacing extracellular sodium with potassium. Extracellular magnesium and calcium ions significantly reduced the single-channel conductance of HA-SACs. These permeation characteristics and the single-channel conductance of HA-SACs were indistinguishable from those of stretch-activated channels (SACs) previously described in these cells. The following observations are consistent with HA-SACs being a subset of SACs. First, SACs were at times found in cell-attached patches which lacked HA-SACs. Second, the number of channels in a cell-attached patch simultaneously activated by stretch (usually 5-10 and often more) exceeded by far the number simultaneously activated by hyperpolarization (usually one or two).(ABSTRACT TRUNCATED AT 250 WORDS)