Ligand with Two Modes of Interaction with the Dopamine D2 Receptor-An Induced-Fit Mechanism of Insurmountable Antagonism.

A solid understanding of the mechanisms governing ligand binding is crucial for rational design of therapeutics targeting the dopamine D2 receptor (D2R). Here, we use G protein-coupled inward rectifier potassium (GIRK) channel activation in Xenopus oocytes to measure the kinetics of D2R antagonism by a series of aripiprazole analogues, as well as the recovery of dopamine (DA) responsivity upon washout. The aripiprazole analogues comprise an orthosteric and a secondary pharmacophore and differ by the length of the saturated carbon linker joining these two pharmacophores. Two compounds containing 3- and 5-carbon linkers allowed for a similar extent of recovery from antagonism in the presence of 1 or 100 µM DA (>25 and >90% of control, respectively), whereas recovery was less prominent (∼20%) upon washout of the 4-carbon linker compound, SV-III-130, both with 1 and 100 µM DA. Prolonging the coincubation time with SV-III-130 further diminished recovery. Curve-shift experiments were consistent with competition between SV-III-130 and DA. Two mutations in the secondary binding pocket (V91A and E95A) of D2R decreased antagonistic potency and increased recovery from SV-III-130 antagonism, whereas a third mutation (L94A) only increased recovery. Our results suggest that the secondary binding pocket influences recovery from inhibition by the studied aripiprazole analogues. We propose a mechanism, supported by in silico modeling, whereby SV-III-130 initially binds reversibly to the D2R, after which the drug-receptor complex undergoes a slow transition to a second ligand-bound state, which is dependent on secondary binding pocket integrity and irreversible during the time frame of our experiments.

Closed and open state dependent block of potassium channels cause opposing effects on excitability - a computational approach.

Block of voltage-gated potassium (Kv) channels has been demonstrated to affect neuronal activity described as increasing excitability. The effect has been associated with a closed-state dependent block. However, the block of Kv channels in e.g. local anesthetic and antiarrhythmics, is open state-dependent. Since the reduced excitability in this case mainly is due to sodium channel block, the role of the Kv channel block is concealed. The present investigation aims to analyse the specific role of state-dependent Kv channel block for excitability. Using a computational approach, with introduced blocked states in the Kv channel of the Frankenhaeuser-Huxley axon membrane model, we calculated the effects on threshold, firing and presynaptic Ca influx. The Ca influx was obtained from an N-type Cav channel model linked to the Frankenhaeuser-Huxley membrane. The results suggested that a selective block of open Kv channels decreased the rate of repetitive firing and the consequent Ca influx, thus challenging the traditional view. In contrast, presence of a closed-state block, increased the firing rate and the Ca influx. These findings propose that Kv channel block may either increase or decrease cellular excitability, thus highlighting the importance of further investigating the role of state-specific blocking mechanisms.

The Beta-Arrestin-Biased Dopamine D2 Receptor Ligand, UNC9994, Is a Partial Agonist at G-Protein-Mediated Potassium Channel Activation.

Background: Previous evidence suggests that UNC9994 is a beta-arrestin2-selective agonist at the dopamine D2 receptor, lacking ability both to activate and antagonize G protein-dependent signaling. However, this has only been reported by one laboratory using a single assay. Methods: We used G protein-coupled inward rectifier potassium channel activation in Xenopus oocytes to investigate UNC9994-induced modulation of G protein-dependent signaling at dopamine D2 receptor and dopamine D3 receptor. Results: At dopamine D2 receptor, UNC9994 induced G protein-coupled inward rectifier potassium channel currents that were 15% of the maximal response to dopamine, with an EC50 of 185 nM. At dopamine D3 receptor, the ligand elicited 89% of the maximal dopamine response with an EC50 of 62 nM. Pertussis toxin abolished G protein-coupled inward rectifier potassium channel activation. Furthermore, UNC9994 antagonized dopamine-induced G protein-coupled inward rectifier potassium channel activation at dopamine D2 receptor. Conclusions: UNC9994 modulates G protein-coupled inward rectifier potassium channel channel activation via pertussis toxin-sensitive G proteins at dopamine D2 receptor and dopamine D3 receptor. These findings may have implications for the interpretation of data obtained with this ligand.

The Importance of the Dissociation Rate in Ion Channel Blocking.

Understanding the relationships between the rates and dynamics of current wave forms under voltage clamp conditions is essential for understanding phenomena such as state-dependence and use-dependence, which are fundamental for the action of drugs used as anti-epileptics, anti-arrhythmics, and anesthetics. In the present study, we mathematically analyze models of blocking mechanisms. In previous experimental studies of potassium channels we have shown that the effect of local anesthetics can be explained by binding to channels in the open state. We therefore here examine models that describe the effect of a blocking drug that binds to a non-inactivating channel in its open state. Such binding induces an inactivation-like current decay at higher potential steps. The amplitude of the induced peak depends on voltage and concentration of blocking drug. In the present study, using analytical methods, we (i) derive a criterion for the existence of a peak in the open probability time evolution for a model with an arbitrary number of closed states, (ii) derive formula for the relative height of the peak amplitude, and (iii) determine the voltage dependence of the relative peak height. Two findings are apparent: (1) the dissociation (unbinding) rate constant is important for the existence of a peak in the current waveform, while the association (binding) rate constant is not, and (2) for a peak to exist it suffices that the dissociation rate must be smaller than the absolute value of all eigenvalues to the kinetic matrix describing the model.

Point mutation of a conserved aspartate, D69, in the muscarinic M2 receptor does not modify voltage-sensitive agonist potency.

The muscarinic M2 receptor (M2R) has been shown to display voltage-sensitive agonist binding, based on G protein-activated inward rectifier potassium channel (GIRK) opening and radioligand binding at different membrane voltages. A conserved aspartate in transmembrane segment (TM) II of M2R, D69, has been proposed as the voltage sensor. While a recent paper instead presented evidence of tyrosines in TMs III, VI, and VII acting as voltage sensors, these authors were not able to record GIRK channel activation by a D69N mutant M2R. In the present study, we succeeded in recording ACh-induced GIRK channel activation by this mutant at -80 and 0 mV. The acetylcholine EC50 was about 2.5-fold higher at 0 mV, a potency shift very similar to that observed at wild-type M2R, indicating that voltage sensitivity persists at the D69N mutant. Thus, our present observations corroborate the notion that D69 is not responsible for voltage sensitivity of the M2R.

Extracellular Linkers Completely Transplant the Voltage Dependence from Kv1.2 Ion Channels to Kv2.1.

The transmembrane voltage needed to open different voltage-gated K (Kv) channels differs by up to 50 mV from each other. In this study we test the hypothesis that the channels' voltage dependences to a large extent are set by charged amino-acid residues of the extracellular linkers of the Kv channels, which electrostatically affect the charged amino-acid residues of the voltage sensor S4. Extracellular cations shift the conductance-versus-voltage curve, G(V), by interfering with these extracellular charges. We have explored these issues by analyzing the effects of the divalent strontium ion (Sr2+) on the voltage dependence of the G(V) curves of wild-type and chimeric Kv channels expressed in Xenopus oocytes, using the voltage-clamp technique. Out of seven Kv channels, Kv1.2 was found to be most sensitive to Sr2+ (50 mM shifted G(V) by +21.7 mV), and Kv2.1 to be the least sensitive (+7.8 mV). Experiments on 25 chimeras, constructed from Kv1.2 and Kv2.1, showed that the large Sr2+-induced G(V) shift of Kv1.2 can be transferred to Kv2.1 by exchanging the extracellular linker between S3 and S4 (L3/4) in combination with either the extracellular linker between S5 and the pore (L5/P) or that between the pore and S6 (LP/6). The effects of the linker substitutions were nonadditive, suggesting specific structural interactions. The free energy of these interactions was ∼20 kJ/mol, suggesting involvement of hydrophobic interactions and/or hydrogen bonds. Using principles from double-layer theory we derived an approximate linear equation (relating the voltage shifts to altered ionic strength), which proved to well match experimental data, suggesting that Sr2+ acts on these channels mainly by screening surface charges. Taken together, these results highlight the extracellular surface potential at the voltage sensor as an important determinant of the channels' voltage dependence, making the extracellular linkers essential targets for evolutionary selection.

The fast-off hypothesis revisited: A functional kinetic study of antipsychotic antagonism of the dopamine D2 receptor.

Newer, "atypical" antipsychotics carry a lower risk of motor side-effects than older, "typical" compounds. It has been proposed that a ~100-fold faster dissociation from the dopamine D2 receptor (D2R) distinguishes atypical from typical antipsychotics. Furthermore, differing antipsychotic D2R affinities have been suggested to reflect differences in dissociation rate constants (koff), while association rate constants (kon) were assumed to be similar. However, it was recently demonstrated that lipophilic accumulation of ligand in the cell interior and/or membrane can cause underestimation of koff, and as high-affinity D2R antagonists are frequently lipophilic, this may have been a confounding factor in previous studies. In the present work, a functional electrophysiology assay was used to measure the recovery of dopamine-mediated D2R responsivity from antipsychotic antagonism, using elevated concentrations of dopamine to prevent the potential bias of re-binding of lipophilic ligands. The variability of antipsychotic kon was also reexamined, capitalizing on the temporal resolution of the assay. kon was estimated from the experimental recordings using a simple mathematical model assumed to describe the binding process. The time course of recovery from haloperidol (typical antipsychotic) was only 6.4- to 2.5-fold slower than that of the atypical antipsychotics, amisulpride, clozapine, and quetiapine, while antipsychotic kons were found to vary more widely than previously suggested. Finally, affinities calculated using our kon and koff estimates correlated well with functional potency and with affinities reported from radioligand binding studies. In light of these findings, it appears unlikely that typical and atypical antipsychotics are primarily distinguished by their D2R binding kinetics.

G protein-gated inwardly rectifying potassium channel subunits 1 and 2 are down-regulated in rat dorsal root ganglion neurons and spinal cord after peripheral axotomy.

BACKGROUND: Increased nociceptive neuronal excitability underlies chronic pain conditions. Various ion channels, including sodium, calcium and potassium channels have pivotal roles in the control of neuronal excitability. The members of the family of G protein-gated inwardly rectifying potassium (GIRK) channels, GIRK1-4, have been implicated in modulating excitability. Here, we investigated the expression and distribution of GIRK1 and GIRK2 in normal and injured dorsal root ganglia (DRGs) and spinal cord of rats. RESULTS: We found that ~70% of the DRG neurons expressed GIRK1, while only <10% expressed GIRK2. The neurochemical profiles of GIRK1- and GIRK2-immunoreactive neurons were characterized using the neuronal markers calcitonin gene-related peptide, isolectin-B4 and neurofilament-200, and the calcium-binding proteins calbindin D28k, calretinin, parvalbumin and secretagogin. Both GIRK subunits were expressed in DRG neurons with nociceptive characteristics. However, while GIRK1 was widely expressed in several sensory neuronal subtypes, GIRK2 was detected mainly in a group of small C-fiber neurons. In the spinal dorsal horn, GIRK1- and -2-positive cell bodies and processes were mainly observed in lamina II, but also in superficial and deeper layers. Abundant GIRK1-, but not GIRK2-like immunoreactivity, was found in the ventral horn (laminae VI-X). Fourteen days after axotomy, GIRK1 and GIRK2 were down-regulated in DRG neurons at the mRNA and protein levels. Both after axotomy and rhizotomy there was a reduction of GIRK1- and -2-positive processes in the dorsal horn, suggesting a presynaptic localization of these potassium channels. Furthermore, nerve ligation caused accumulation of both subunits on both sides of the lesion, providing evidence for anterograde and retrograde fast axonal transport. CONCLUSIONS: Our data support the hypothesis that reduced GIRK function is associated with increased neuronal excitability and causes sensory disturbances in post-injury conditions, including neuropathic pain.

Density of voltage-gated potassium channels is a bifurcation parameter in pyramidal neurons.

Several types of intrinsic dynamics have been identified in brain neurons. Type 1 excitability is characterized by a continuous frequency-stimulus relationship and, thus, an arbitrarily low frequency at threshold current. Conversely, Type 2 excitability is characterized by a discontinuous frequency-stimulus relationship and a nonzero threshold frequency. In previous theoretical work we showed that the density of Kv channels is a bifurcation parameter, such that increasing the Kv channel density in a neuron model transforms Type 1 excitability into Type 2 excitability. Here we test this finding experimentally, using the dynamic clamp technique on Type 1 pyramidal cells in rat cortex. We found that increasing the density of slow Kv channels leads to a shift from Type 1 to Type 2 threshold dynamics, i.e., a distinct onset frequency, subthreshold oscillations, and reduced latency to first spike. In addition, the action potential was resculptured, with a narrower spike width and more pronounced afterhyperpolarization. All changes could be captured with a two-dimensional model. It may seem paradoxical that an increase in slow K channel density can lead to a higher threshold firing frequency; however, this can be explained in terms of bifurcation theory. In contrast to previous work, we argue that an increased outward current leads to a change in dynamics in these neurons without a rectification of the current-voltage curve. These results demonstrate that the behavior of neurons is determined by the global interactions of their dynamical elements and not necessarily simply by individual types of ion channels.

Typical and atypical antipsychotics do not differ markedly in their reversibility of antagonism of the dopamine D2 receptor.

It has been suggested that the favorable side-effect profiles of atypical antipsychotics (e.g. clozapine and amisulpride) are related to their ∼100-fold faster dissociation from dopamine D2 receptors (D2R) compared with typical antipsychotics (e.g. haloperidol and chlorpromazine). Fast dissociation would entail rapidly reversible antagonism; however, this has not been thoroughly studied using functional assays. We compared the reversibilities of D2R antagonism by 17 compounds using an electrophysiological method to measure dopamine-evoked potassium channel activation via D2R. Varying rates and amplitudes of D2R response recovery were observed following antagonist removal. Whereas recovery rates differed 15-fold between atypical drugs, recovery from clozapine and amisulpride antagonism was, unexpectedly, less than twofold faster than from chlorpromazine. The recovery amplitude correlated with calculated water solubility and lipid/water distribution coefficients, suggesting variable drug partitioning into cell membranes. Our data do not support the notion that the rate of reversibility of D2R antagonism is what distinguishes atypical from typical antipsychotics.

Coenzyme Q10 prevents peripheral neuropathy and attenuates neuron loss in the db-/db- mouse, a type 2 diabetes model.

Diabetic peripheral neuropathy (DPN) is the most common complication in both type 1 and type 2 diabetes. Here we studied some phenotypic features of a well-established animal model of type 2 diabetes, the leptin receptor-deficient db(-)/db(-) mouse, and also the effect of long-term (6 mo) treatment with coenzyme Q10 (CoQ10), an endogenous antioxidant. Diabetic mice at 8 mo of age exhibited loss of sensation, hypoalgesia (an increase in mechanical threshold), and decreases in mechanical hyperalgesia, cold allodynia, and sciatic nerve conduction velocity. All these changes were virtually completely absent after the 6-mo, daily CoQ10 treatment in db(-)/db(-) mice when started at 7 wk of age. There was a 33% neuronal loss in the lumbar 5 dorsal root ganglia (DRGs) of the db(-)/db(-) mouse versus controls at 8 mo of age, which was significantly attenuated by CoQ10. There was no difference in neuron number in 5/6-wk-old mice between diabetic and control mice. We observed a strong down-regulation of phospholipase C (PLC) ß3 in the DRGs of diabetic mice at 8 mo of age, a key molecule in pain signaling, and this effect was also blocked by the 6-mo CoQ10 treatment. Many of the phenotypic, neurochemical regulations encountered in lumbar DRGs in standard models of peripheral nerve injury were not observed in diabetic mice at 8 mo of age. These results suggest that reactive oxygen species and reduced PLCß3 expression may contribute to the sensory deficits in the late-stage diabetic db(-)/db(-) mouse, and that early long-term administration of the antioxidant CoQ10 may represent a promising therapeutic strategy for type 2 diabetes neuropathy.

Voltage sensitivities and deactivation kinetics of histamine H3 and H4 receptors.

Agonist potency at some neurotransmitter receptors has been shown to be regulated by voltage, a mechanism which has been suggested to play a crucial role in the regulation of neurotransmitter release by inhibitory autoreceptors. Likewise, receptor deactivation rates upon agonist removal have been implicated in autoreceptor function. Using G protein-coupled potassium (GIRK) channel activation in Xenopus oocytes as readout of receptor activity, we have investigated the voltage sensitivities and signaling kinetics of the hH(3)(445) and hH(3)(365) isoforms of the human histamine H3 receptor, which functions as an inhibitory auto- and heteroreceptor in the nervous system. We have also investigated both the human and the mouse homologues of the related histamine H4 receptor, which is expressed mainly on hematopoietic cells. We found that the hH(3)(445) receptor is the most sensitive to voltage, whereas the hH(3)(365) and H(4) receptors are less affected. We further observed a marked difference in response deactivation kinetics between the hH(3)(445) and hH(3)(365) isoforms, with the hH(3)(365) isoform being five to six-fold slower than the hH(3)(445) receptor. Finally, using synthetic agonists, we found evidence for agonist-specific voltage sensitivity at the hH4 receptor. The differences in voltage sensitivities and deactivation kinetics between the hH(3)(445), hH(3)(365), and H4 receptors might be relevant to their respective physiological roles.

Agonist-specific voltage sensitivity at the dopamine D2S receptor--molecular determinants and relevance to therapeutic ligands.

Voltage sensitivity has been demonstrated for some GPCRs. At the dopamine D(2S) receptor, this voltage sensitivity is agonist-specific; some agonists, including dopamine, exhibit decreased potency at depolarized potentials, whereas others are not significantly affected. In the present study, we examined some of the receptor-agonist interactions contributing to these differences, and investigated how dopamine D(2S) receptor voltage sensitivity affects clinically used dopamine agonists. GIRK channel activation in voltage-clamped Xenopus oocytes was used as readout of receptor activation. Structurally distinct agonists and complementary site-directed mutagenesis of the receptor's binding site were used to investigate the role of agonist-receptor interactions. We also confirmed that the depolarization-induced decrease of dopamine potency in GIRK activation is correlated by decreased binding of radiolabeled dopamine, and by decreased potency in G protein activation. In the mutagenesis experiments, a conserved serine residue as well as the conserved aspartate in the receptor's binding site were found to be important for voltage sensitive potency of dopamine. Furthermore, the voltage sensitivity of the receptor had distinct effects on different therapeutic D(2) agonists. Depolarization decreased the potency of several compounds, whereas for others, efficacy was reduced. For some agonists, both potency and efficacy were diminished, whereas for others still, neither parameter was significantly altered. The present work identifies some of the ligand-receptor interactions which determine agonist-specific effects of voltage at the dopamine D(2S) receptor. The observed differences between therapeutic agonists might be clinically relevant, and make them potential tools for investigating the roles of dopamine D(2) receptor voltage sensitivity in native tissue.

Spontaneous ryanodine-receptor-dependent Ca2+-activated K+ currents and hyperpolarizations in rat medial preoptic neurons.

The aim of the present study was to clarify the identity of slow spontaneous currents, the underlying mechanism and possible role for impulse generation in neurons of the rat medial preoptic nucleus (MPN). Acutely dissociated neurons were studied with the perforated patch-clamp technique. Spontaneous outward currents, at a frequency of approximately 0.5 Hz and with a decay time constant of approximately 200 ms, were frequently detected in neurons when voltage-clamped between approximately -70 and -30 mV. The dependence on extracellular K(+) concentration was consistent with K(+) as the main charge carrier. We concluded that the main characteristics were similar to those of spontaneous miniature outward currents (SMOCs), previously reported mainly for muscle fibers and peripheral nerve. From the dependence on voltage and from a pharmacological analysis, we concluded that the currents were carried through small-conductance Ca(2+)-activated (SK) channels, of the SK3 subtype. From experiments with ryanodine, xestospongin C, and caffeine, we concluded that the spontaneous currents were triggered by Ca(2+) release from intracellular stores via ryanodine receptor channels. An apparent voltage dependence was explained by masking of the spontaneous currents as a consequence of steady SK-channel activation at membrane potentials > -30 mV. Under current-clamp conditions, corresponding transient hyperpolarizations occasionally exceeded 10 mV in amplitude and reduced the frequency of spontaneous impulses. In conclusion, MPN neurons display spontaneous hyperpolarizations triggered by Ca(2+) release via ryanodine receptors and SK3-channel activation. Thus such events may affect impulse firing of MPN neurons.

Ion channel density regulates switches between regular and fast spiking in soma but not in axons.

The threshold firing frequency of a neuron is a characterizing feature of its dynamical behaviour, in turn determining its role in the oscillatory activity of the brain. Two main types of dynamics have been identified in brain neurons. Type 1 dynamics (regular spiking) shows a continuous relationship between frequency and stimulation current (f-I(stim)) and, thus, an arbitrarily low frequency at threshold current; Type 2 (fast spiking) shows a discontinuous f-I(stim) relationship and a minimum threshold frequency. In a previous study of a hippocampal neuron model, we demonstrated that its dynamics could be of both Type 1 and Type 2, depending on ion channel density. In the present study we analyse the effect of varying channel density on threshold firing frequency on two well-studied axon membranes, namely the frog myelinated axon and the squid giant axon. Moreover, we analyse the hippocampal neuron model in more detail. The models are all based on voltage-clamp studies, thus comprising experimentally measurable parameters. The choice of analysing effects of channel density modifications is due to their physiological and pharmacological relevance. We show, using bifurcation analysis, that both axon models display exclusively Type 2 dynamics, independently of ion channel density. Nevertheless, both models have a region in the channel-density plane characterized by an N-shaped steady-state current-voltage relationship (a prerequisite for Type 1 dynamics and associated with this type of dynamics in the hippocampal model). In summary, our results suggest that the hippocampal soma and the two axon membranes represent two distinct kinds of membranes; membranes with a channel-density dependent switching between Type 1 and 2 dynamics, and membranes with a channel-density independent dynamics. The difference between the two membrane types suggests functional differences, compatible with a more flexible role of the soma membrane than that of the axon membrane.

Cocaine produces D2R-mediated conformational changes in the adenosine A(2A)R-dopamine D2R heteromer.

Adenosine A(2A) receptors (A(2A)Rs) and dopamine D(2) receptors (D(2)Rs) form constitutive heteromers in living cells and exhibit a strong functional antagonistic interaction. Recent findings give neurochemical evidence that extended cocaine self-administration in the rat give rise to an up-regulation of functional A(2A)Rs in the nucleus accumbens that return to baseline expression levels during cocaine withdrawal. In the present work, the acute in vitro effects of a concentration of cocaine known to fully block the dopamine (DA) transporter without exerting any toxic actions were investigated on A(2A)R and D(2L)R formed heteromers in transiently co-transfected HEK-293T cells. In vitro treatment of cocaine was found to produce changes in D(2)R homodimers and in A(2A)R-D(2)R heterodimers detected through bioluminescent energy transfer (BRET). Cocaine was found to produce a time- and concentration-dependent reduction in the BRET(max) between A(2A)R-D(2L)R heterodimers and D(2L)R homodimers, but not A(2A)R homodimers, indicating its effect on D(2)R. Cocaine was evaluated with regard to D(2)R binding using a human D(2L)R stable expressing CHO cell line and was found to produce an increase in the affinity of hD(2L)R for DA. At the level of G protein-coupling, cocaine produced a small, but significant increase in DA-stimulated binding of GTPgammaS. However, cocaine failed to modulate D(2)R agonist-induced inhibition of cAMP in stable hD(2L)R CHO cells or the gating of GIRK channels in oocytes. Taken together, these results indicate a direct and specific effect of a moderate concentration of cocaine on the DA D(2L)R, that results in enhanced agonist recognition, G protein-coupling and an altered conformational state of D(2)R homodimers and A(2A)R-D(2)R heterodimers.

Voltage-sensitivity at the human dopamine D2S receptor is agonist-specific.

Recently, we and others have shown that agonist potencies at some, but not all, G protein-coupled receptors are voltage-sensitive. Several of those studies employed electrophysiology assays in Xenopus oocytes with G protein-coupled potassium channels as a readout. Using this assay, we have now obtained evidence that voltage-sensitivity at the dopamine D(2S) receptor is agonist-specific. Whereas the potency of dopamine at the D(2S) receptor is decreased by depolarization, the potencies of beta-phenethylamine, p- and m-tyramine are voltage-insensitive. Furthermore, both monohydroxylated and non-hydroxylated N,N-dipropyl-2-aminotetralin compounds are voltage-sensitive. Differential activation of G protein subtypes or differential ratios between effector and active G protein do not underlie this agonist-selective voltage-sensitivity. This is the first demonstration of voltage-sensitive and voltage-insensitive behaviour of different agonists acting via the same receptor.

Bupivacaine blocks N-type inactivating Kv channels in the open state: no allosteric effect on inactivation kinetics.

Local anesthetics bind to ion channels in a state-dependent manner. For noninactivating voltage-gated K channels the binding mainly occurs in the open state, while for voltage-gated inactivating Na channels it is assumed to occur mainly in inactivated states, leading to an allosterically caused increase in the inactivation probability, reflected in a negative shift of the steady-state inactivation curve, prolonged recovery from inactivation, and a frequency-dependent block. How local anesthetics bind to N-type inactivating K channels is less explored. In this study, we have compared bupivacaine effects on inactivating (Shaker and K(v)3.4) and noninactivating (Shaker-IR and K(v)3.2) channels, expressed in Xenopus oocytes. Bupivacaine was found to block these channels time-dependently without shifting the steady-state inactivation curve markedly, without a prolonged recovery from inactivation, and without a frequency-dependent block. An analysis, including computational testing of kinetic models, suggests binding to the channel mainly in the open state, with affinities close to those estimated for corresponding noninactivating channels (300 and 280 microM for Shaker and Shaker-IR, and 60 and 90 microM for K(v)3.4 and K(v)3.2). The similar magnitudes of K(d), as well as of blocking and unblocking rate constants for inactivating and noninactivating Shaker channels, most likely exclude allosteric interactions between the inactivation mechanism and the binding site. The relevance of these results for understanding the action of local anesthetics on Na channels is discussed.

Differential voltage-sensitivity of D2-like dopamine receptors.

Agonist potency at some neurotransmitter receptors has been shown to be regulated by transmembrane voltage, a mechanism which has been suggested to play a crucial role in the regulation of neurotransmitter release by autoreceptors and in synaptic plasticity. We have recently described the voltage-sensitivity of the dopamine D(2L) receptor and we now extend our studies to include the other members of the D(2)-like receptor subfamily; the D(2S), D(3), and D(4) dopamine receptors. Electrophysiological recordings were performed on Xenopus oocytes coexpressing human dopamine D(2S), D(3), or D(4) receptors with G protein-coupled potassium (GIRK) channels. Comparison of concentration-response relationships at -80 mV and at 0 mV for dopamine-mediated GIRK activation revealed significant rightward shifts for both D(2S) and D(4) upon depolarization. In contrast, the concentration-response relationships for D(3)-mediated GIRK activation were not appreciably different at the two voltages. Our findings provide new insight into the functional differences of these closely related receptors.

Electrophysiology-based analysis of human histamine H(4) receptor pharmacology using GIRK channel coupling in Xenopus oocytes.

The recently cloned histamine H(4) receptor is expressed predominantly in haematopoietic cells and has been found to modulate the function of mast cells, eosinophils, dendritic cells and T lymphocytes. It represents an attractive target for pharmacological interventions against a number of inflammatory and autoimmune disorders. In the present work we used two-electrode voltage-clamp to demonstrate histamine H(4) receptor modulation of G protein-coupled inward rectifier potassium (GIRK) channels heterologously expressed in Xenopus oocytes. In accordance with earlier findings in other effector systems, full agonism by histamine and (R)-alpha-methylhistamine, partial agonism by clobenpropit and inverse agonism by thioperamide were observed. Furthermore, in oocytes injected with low amounts of receptor cRNA, clobenpropit apparently acted as a neutral antagonist. We also used the high temporal resolution afforded by this system to study the differential time courses of response deactivation upon ligand washout for clobenpropit and (R)-alpha-methylhistamine. GIRK channels represent a novel effector system for histamine H(4) receptor modulation, which may be of physiological relevance and prove useful in the development of compounds targeting this receptor.