Hyperpolarization-activated cyclic nucleotide-gated (HCN) channels and pain.

Hyperpolarization-activated cyclic nucleotide-gated (HCN) channels underlie the pacemaker currents in neurons and cardiac cells designated as I(h) and I(f), respectively. HCN channels are activated at negative membrane potentials and specifically upon repolarization following action potential firing resulting in a depolarizing current influencing the threshold for subsequent action potential generation. Consequently, HCN channels and I(h)/I(f) play a critical role in regulating excitability and rhythmic activity in excitable cells. The distribution of the four HCN channel subtypes has been studied in some detail in sensory neurons demonstrating a diverse and widespread distribution and raising the question as to their potential involvement in pain pathophysiology, frequently ascribed to aberrant neuronal hyperexcitability. This review discusses the evidence implicating a role for HCN channels in pain.

Characterization of compounds on nicotinic acetylcholine receptor alpha7 channels using higher throughput electrophysiology.

Alpha7 nicotinic acetylcholine receptor channels are important ligand-gated ion channels that are fast desensitizing, cation selective and have been implicated in the pathophysiology of schizophrenia and Alzheimer's disease. We report here high quality alpha7 parallel patch clamp recordings using the QPatch automated patch clamp system. The QPatch patch clamps up to 48 cells in parallel with the same high fidelity as conventional patch clamp. EC(50) and IC(50) values were comparable to values obtained with conventional patch clamp. The EC(50) value for acetylcholine (ACh) on the QPatch with area under the curve (AUC) analysis was 26microM compared to a value of 29microM determined from conventional patch clamp experiments. Sequential additions of ACh can be made with minimal decay of the peak amplitude. The competitive alpha7 antagonist methyllycaconitine (MLA) blocked currents with an IC(50) value of 0.25nM which is similar to published IC(50) values for MLA. Finally, two different classes of positive allosteric modulators represented by PNU-120596 and NS-1738 elicited characteristic responses, thus allowing accurate characterization of modulation and measurements of potency. These results demonstrate that alpha7 nicotinic acetylcholine receptor channels can be studied reliably in a higher throughput, parallel manner with the QPatch automated patch clamp system.

Hyperpolarization-activated cyclic nucleotide-gated channel mRNA and protein expression in large versus small diameter dorsal root ganglion neurons: correlation with hyperpolarization-activated current gating.

Hyperpolarization-activated cyclic nucleotide-gated channels (HCN) are responsible for the functional hyperpolarization-activated current (I(h)) in dorsal root ganglion (DRG) neurons. We studied HCN1-4 channel mRNA and protein expression and correlated these findings with I(h) functional properties in rat DRG neurons of different size. Quantitative RT-PCR (TaqMan) analysis demonstrated that HCN2 and HCN1 mRNAs were more abundantly expressed in large diameter (55-80 microm) neurons, while HCN3 mRNA was preferentially expressed in small diameter (20-30 microm) neurons. HCN4 mRNA expression was very low in neurons of all sizes. At the protein level, subunit-selective polyclonal antibodies and immunofluorescence indicated that HCN1 and HCN3 are present in large diameter neurons and small diameter neurons. Staining in small diameter neurons was in IB4-positive (non-peptidergic) and IB4-negative (peptidergic) cells. HCN2 immunofluorescent staining was heterogeneous and predominantly in large diameter neurons and in small diameter IB4-negative neurons. HCN4 was poorly expressed in all neurons. Functionally, I(h) amplitude and density were significantly larger, and activation kinetics faster, in large diameter neurons when compared with small neurons. I(h) activation rates in small and large diameter DRG neurons were consistent with the relative abundance of HCN subunits in the respective cell type, considering the reported HCN channel activation rates in heterologous systems (HCN1>HCN2 approximately HCN3>HCN4), suggesting exclusivity of roles of different HCN subunits contributing to the excitability of DRG neurons of different size. Additionally, a functional role of I(h) in small DRG neuron excitability was evaluated using a computational model.

Modulation of A-type potassium channels by a family of calcium sensors.

In the brain and heart, rapidly inactivating (A-type) voltage-gated potassium (Kv) currents operate at subthreshold membrane potentials to control the excitability of neurons and cardiac myocytes. Although pore-forming alpha-subunits of the Kv4, or Shal-related, channel family form A-type currents in heterologous cells, these differ significantly from native A-type currents. Here we describe three Kv channel-interacting proteins (KChIPs) that bind to the cytoplasmic amino termini of Kv4 alpha-subunits. We find that expression of KChIP and Kv4 together reconstitutes several features of native A-type currents by modulating the density, inactivation kinetics and rate of recovery from inactivation of Kv4 channels in heterologous cells. All three KChIPs co-localize and co-immunoprecipitate with brain Kv4 alpha-subunits, and are thus integral components of native Kv4 channel complexes. The KChIPs have four EF-hand-like domains and bind calcium ions. As the activity and density of neuronal A-type currents tightly control responses to excitatory synaptic inputs, these KChIPs may regulate A-type currents, and hence neuronal excitability, in response to changes in intracellular calcium.

Neuroprotective interaction effects of NMDA and AMPA receptor antagonists in an in vitro model of cerebral ischemia.

An in vitro model of ischemia was developed and characterized using the acute rat hippocampal slice preparation. Neuroprotective concentrations of several competitive and noncompetitive glutamate subtype-selective antagonists (CGS-19755, MK-801, YM90K and GYKI-52466) were initially determined in anoxia-enhanced agonist-induced excitotoxicity experiments. Concentrations which proved to be effective in these studies were subsequently tested for their effectiveness against an ischemic episode. Ischemia was defined as a 30-min exposure to aglycemic media ending in 5 min of concurrent anoxia, a protocol which was arrived at by empirically determining the effect of various hypoglycemic and anoxic insults on the ability of hippocampal slices to retain their electrophysiological viability. Exposure to such an ischemic episode resulted in a loss of viability by most slices, an effect which was strongly dependent on extracellular calcium. AMPA antagonists applied alone produced no neuroprotective effect in the present model of in vitro ischemia, while NMDA antagonists applied alone had a modest neuroprotective effect. In contrast, the coapplication of 10 microM MK-801 and 300 microM GYKI-52466, noncompetitive NMDA and AMPA receptor antagonists, respectively, resulted in almost complete neuroprotection. This protection was comparable to that obtained by withholding extracellular calcium, indicating that the toxic effects of glutamate receptor overstimulation can be accounted for solely by calcium influx. The effect of this combination treatment on the survival rate of hippocampal slices was synergistic, that is greater than the sum of the effects of the individual compounds. The results indicate that neuroprotection against acute ischemic insults may require a combination therapy approach.

Modulation of the Kv1.3 potassium channel by receptor tyrosine kinases.

The voltage-dependent potassium channel, Kv1.3, is modulated by the epidermal growth factor receptor (EGFr) and the insulin receptor tyrosine kinases. When the EGFr and Kv1.3 are coexpressed in HEK 293 cells, acute treatment of the cells with EGF during a patch recording can suppress the Kv1.3 current within tens of minutes. This effect appears to be due to tyrosine phosphorylation of the channel, as it is blocked by treatment with the tyrosine kinase inhibitor erbstatin, or by mutation of the tyrosine at channel amino acid position 479 to phenylalanine. Previous work has shown that there is a large increase in the tyrosine phosphorylation of Kv1.3 when it is coexpressed with the EGFr. Pretreatment of EGFr and Kv1.3 cotransfected cells with EGF before patch recording also results in a decrease in peak Kv1.3 current. Furthermore, pretreatment of cotransfected cells with an antibody to the EGFr ligand binding domain (alpha-EGFr), which blocks receptor dimerization and tyrosine kinase activation, blocks the EGFr-mediated suppression of Kv1.3 current. Insulin treatment during patch recording also causes an inhibition of Kv1.3 current after tens of minutes, while pretreatment for 18 h produces almost total suppression of current. In addition to depressing peak Kv1.3 current, EGF treatment produces a speeding of C-type inactivation, while pretreatment with the alpha-EGFr slows C-type inactivation. In contrast, insulin does not influence C-type inactivation kinetics. Mutational analysis indicates that the EGF-induced modulation of the inactivation rate occurs by a mechanism different from that of the EGF-induced decrease in peak current. Thus, receptor tyrosine kinases differentially modulate the current magnitude and kinetics of a voltage-dependent potassium channel.

Kinetic variability and modulation of dSlo, a cloned calcium-dependent potassium channel.

We have examined, using patch recording, the modulation by ATP gamma S of the cloned Drosophila slopoke calcium-dependent potassium channel (dSlo) expressed in Xenopus oocytes. There is a large variation in the gating kinetics, open probability, and conductance level of the channel in this expression system, which complicates the analysis of modulatory events. Addition of ATP gamma S to the intracellular face of the patch does not consistently alter the overall open probability of dSlo, but it does increase the frequency of appearance of an exceptionally long-lived closed state of the channel. This modulation is not blocked by an inhibitor of several serine/threonine protein kinases, nor by mutation of a serine residue that is a target for phosphorylation by protein kinase A. Thus, ATP gamma S may alter dSlo kinetic properties by some mechanism other than serine/threonine phosphorylation.

Block of cloned voltage-gated potassium channels by the second messenger diacylglycerol independent of protein kinase C.

1. Diacylglycerols (DAGs) are common intracellular second messengers produced as a result of activation of phospholipase C. We have examined the direct effects of DAG on currents from cloned voltage-dependent potassium channels. Potassium channels were studied by overexpression of their cRNAs in Xenopus oocytes or of their cDNAs in HEK 293 cells, and macroscopic currents were recorded from inside-out membrane patches. 2. When applied to the intracellular side of the patch, 1,2-dioctanoyl-sn-glycerol (C8:0) (DOG) blocks Shaker IR, Kv1.3, and Kv1.6 channels. This block appears macroscopically as a large speeding of the inactivation rate. Longer carbon chain length DAGs (10 and 12 carbons) are less effective in producing this response. 3. DOG is effective at low concentrations, doubling the apparent inactivation rate at 162 nM, and has a fast time course, with a wash-in and reversal to control each within approximately 30 s. 4. Voltage steps delivered with a two pulse protocol in the presence of DOG indicate that recovery from DOG block is voltage dependent. Recovery occurs quickly (tau = 507 ms) when channels are closed quickly by hyperpolarized (-90 mV) potentials, and occurs slowly (tau = 1.3 s) when channels are closed incompletely by depolarized (-60 mV) potentials. 5. The action of DOG is independent of protein kinase C (PKC) activation, because it does not require ATP, nor is it blocked by staurosporin or by the PKC inhibitor peptide 19-36.(ABSTRACT TRUNCATED AT 250 WORDS)

Intracellular and extracellular amino acids that influence C-type inactivation and its modulation in a voltage-dependent potassium channel.

The rate of C-type inactivation of the cloned voltage-gated potassium channel, Kv1.3, measured in membrane patches from Xenopus oocytes, increases when the patch is detached from the cell; the structural basis for this on-cell/off-cell change was examined. First, four serine and threonine residues, that are putative sites for phosphorylation by protein kinases A and C, were mutated to alanines. Mutating any one of these residues, or two or three of them simultaneously, does not eliminate the change in C-type inactivation. However, the basal rate of C-type inactivation in the cell-attached patch is markedly slower in the triple phosphorylation site mutant. Second, a homologous potassium channel, Kv 1.6, does not exhibit the on-cell/off-cell change. When an extracellular histidine at position 401 of Kv1.3 is replaced with tyrosine, the residue at the equivalent position (430) in Kv1.6, the resulting Kv1.3 H401Y mutant channel does not undergo the on-cell/off-cell change. The results indicate that several potentially phosphorylatable intracellular amino acids influence the basal rate of C-type inactivation, but are not essential for the on-cell/off-cell change in inactivation kinetics. In contrast, an extracellular amino acid is critical for this on-cell/off-cell change.

Pregnenolone sulfate potentiation of N-methyl-D-aspartate receptor channels in hippocampal neurons.

Many actions of the classical gonadal and adrenal steroid hormones are at the level of transcriptional regulation. Recent studies have shown, however, that endogenous brain metabolites of steroids exert important nongenomic modulatory effects on neuronal mechanisms. Potentiation of the N-methyl-D-aspartate (NMDA) subtype of glutamate receptor by the neurosteroid pregnenolone sulfate (PS) was studied using cultured hippocampal neurons and patch-clamp techniques. The magnitude of NMDA-activated whole-cell currents was approximately doubled in the presence of 100 microM PS. The dose-response curve of PS action showed significant potentiation above 250 nM and a half-maximal effect at approximately 29 microM. Maximum potentiation was reached within 25 sec, and the potentiation was completely reversed with 60 sec of washout. The enhancement of the NMDA current is probably not due to activation of a new ionic conductance, because the reversal potential of the I-V curve did not shift in the presence of PS. Potentiation is specific for the NMDA subtype of glutamate receptor; non-NMDA currents showed only a slight inhibition (approximately 6%) in the presence of 50 microM PS. Potentiation of the NMDA current by PS occurred in the presence of saturating concentrations of NMDA and glycine, indicating that at saturating concentrations of the coagonists PS does not change the affinity between the coagonists and the NMDA receptor. The dose-response relations for NMDA and glycine were shifted slightly to the left, and the percent potentiation was significantly higher for lower concentrations of coagonists, suggesting that at low concentrations of the coagonists PS may slightly increase their affinity for the NMDA receptor. The fractional open time (nPo) of single NMDA-activated channels was potentiated by PS in patch-clamp recordings using both the outside-out and cell-attached configurations. The potentiation of nPo resulted from increases in the frequency of opening and in the mean channel open time. No effect was seen on single-channel conductances.

Ultrastructure and Neuronal Control of Luminous Cells in the Copepod Gaussia princeps.

The physiology of light production in copepods is largely unknown. The mesopelagic copepod Gaussia princeps possesses luminous glands, each consisting of a single large cell discharging through a cuticular pore. Slow flashes external to the cuticle are triggered from excised abdomens by electrical stimulation of the ventral nerve cord. Each luminous cell contains UV fluorescent secretory vesicles distally, which are secreted through a valved cuticular pore. Each luminous cell, except for the most proximal portion, is surrounded by a cellular sheath, which appears to form the distal valve. Luminous cells have a stem containing small, electron-lucent precursors to secretory vesicles proximal to the fluorescent vesicles. Nerve terminals, filled with large synaptic vesicles, are associated with the unsheathed proximal cell membrane. Gap junctions interconnect the nerve terminals, and possibly serve to accelerate conduction to the luminous cell to achieve a synchronous effector output.

Patterns of Stimulated Bioluminescence in Two Pyrosomes (Tunicata: Pyrosomatidae).

Pyrosomes are colonial tunicates that, in contrast with typical luminescent plankton, generate brilliant, sustained bioluminescence. They are unusual in numbering among the few marine organisms reported to luminesce in response to light. Each zooid within a colony detects light and emits bioluminescence in response. To investigate the luminescence responsivity of Pyrosoma atlanticum and Pyrosomella verticillata, photic, electrical, and mechanical stimuli were used. Photic stimulation of 1.5 x 109 photons{middot}s-1{middot}cm-2, at wavelengths between 350 and 600 nm, induced bioluminescence, with the maximum response induced at 475 nm. The photic-excitation half-response constant was 1.1 x 107 photons{middot}s-1{middot}cm-2 at 475 nm for P. atlanticum; P. verticillata had a significantly higher half-response constant of 9.3 x 107 photons{middot}s-1{middot}cm-2. Individual zooids within a colony, however, appeared to have different half-response constants. Stimulus strength influenced recruitment of zooids and, in turn, luminescent duration and quantum emission. Image intensification revealed saltatory propagation of luminescence across the colony, owing to photic triggering among zooids. Repetitive, regular mechanical or electrical stimulation elicited rhythmic flashing characterized by alternating periods of high and low light intensities.