Depolarizing Voltage Steps Research Articles

Regulation of Cardiac IKs Potassium Current by Membrane Phosphatidylinositol 4,5-Bisphosphate

Regulation of the slowly activating component of delayed rectifier K+ current (IKs) by membrane phospholipid phosphatidylinositol 4,5-bisphosphate (PtdIns-(4,5)P2) was examined in guinea pig atrial myocytes using the whole-cell patch clamp method. IKs was elicited by depolarizing voltage steps given from a holding potential of -50 mV, and the effect of various test reagents on IKs was assessed by measuring the amplitude of tail current elicited upon return to the holding potential following a 2-s depolarization to +30 mV. Intracellular application of 50 microM wortmannin through a recording pipette evoked a progressive increase in IKs over a 10-15-min period to 208.5 +/- 14.6% (n = 9) of initial magnitude obtained shortly after rupture of the patch membrane. Intracellular application of anti-PtdIns(4,5)P2 monoclonal antibody also increased the amplitude of IKs to 198.4 +/- 19.9% (n = 5). In contrast, intracellular loading with exogenous PtdIns(4,5)P2 at 10 and 100 mum produced a marked decrease in the amplitude of IKs to 54.3 +/- 3.8% (n = 5) and 44.8 +/- 8.2% (n = 5), respectively. Intracellular application of neomycin (50 microM) or aluminum (50 microM) evoked an increase in the amplitude of IKs to 161.0 +/- 13.5% (n = 4) and 150.0 +/- 8.2% (n = 4), respectively. These results strongly suggest that IKs channel is inhibited by endogenous membrane PtdIns(4,5)P2 through the electrostatic interaction with the negatively charged head group on PtdIns(4,5)P2. Potentiation of IKs by P2Y receptor stimulation with 50 microM ATP was almost totally abolished when PtdIns(4,5)P2 was included in the pipette solution, suggesting that depletion of membrane PtdIns(4,5)P2 is involved in the potentiation of IKs by P2Y receptor stimulation. Thus, membrane PtdIns(4,5)P2 may act as an important physiological regulator of IKs in guinea pig atrial myocytes.

Effects of Kv1.2 Intracellular Regions on Activation of Kv2.1 Channels

Depolarizing voltage steps activate voltage-dependent K + (Kv) channels by moving the voltage sensor, which triggers a coupling reaction leading to the opening of the pore. We constructed chimeric channels in which intracellular regions of slowly activating Kv2.1 channels were replaced by respective regions of rapidly activating Kv1.2 channels. Substitution of either the N-terminus, S4–S5 linker, or C-terminus generated chimeric Kv2.1/1.2 channels with a paradoxically slow and approximately exponential activation time course consisting of a fast and a slow component. Using combined chimeras, each of these Kv1.2 regions further slowed activation at the voltage of 0 mV, irrespective of the nature of the other two regions, whereas at the voltage of 40 mV both slowing and accelerating effects were observed. These results suggest voltage-dependent interactions of the three intracellular regions. This observation was quantified by double-mutant cycle analysis. It is concluded that interactions between N-terminus, S4–S5 linker, and/or C-terminus modulate the activation time course of Kv2.1 channels and that part of these interactions is voltage dependent.

Octopaminergic modulation of the membrane currents in the central feeding system of the pond snail Lymnaea stagnalis.

Octopamine is released by the intrinsic OC interneurons in the paired buccal ganglia and serves both as a neurotransmitter and a neuromodulator in the central feeding network of the pond snail Lymnaea stagnalis. The identified B1 buccal motoneuron receives excitatory inputs from the OC interneurons and is more excitable in the presence of 10 microM octopamine in the bath. This modulatory effect of octopamine on the B1 motoneuron was studied using the two electrode voltage clamp method. In normal physiological saline depolarising voltage steps from the holding potential of -80 mV evoke a transient inward current, presumably carried by Na(+) ions. The peak values of this inward current are increased in the presence of 10 microM octopamine in the bath. In contrast, both the transient (IA) and delayed (IK) outward currents are unaffected by octopamine application. Replacing the normal saline with a Na(+)-free bathing solution containing K(+) channel blockers (50 mM TEACl, 4 mM 4AP) revealed the presence of an additional inward current of the B1 neurons, carried by Ca(2+). Octopamine (10 microM) in the bath decreased the amplitudes of this current. These results suggest that the membrane mechanisms which underlie the modulatory effect of octopamine on the B1 motoneuron include selective changes of the Na(+)- and Ca(2+)-channels.

Hydrogen-Deuterium Exchange Effects on β-Endorphin Release from AtT20 Murine Pituitary Tumor Cells

Abundant evidences demonstrate that deuterium oxide (D 2O) modulates various secretory activities, but specific mechanisms remain unclear. Using AtT20 cells, we examined effects of D 2O on physiological processes underlying β-endorphin release. Immunofluorescent confocal microscopy demonstrated that 90% D 2O buffer increased the amount of actin filament in cell somas and decreased it in cell processes, whereas β-tubulin was not affected. Ca 2+ imaging demonstrated that high-K +-induced Ca 2+ influx was not affected during D 2O treatment, but was completely inhibited upon D 2O washout. The H 2O/D 2O replacement in internal solutions of patch electrodes reduced Ca 2+ currents evoked by depolarizing voltage steps, whereas additional extracellular H 2O/D 2O replacement recovered the currents, suggesting that D 2O gradient across plasma membrane is critical for Ca 2+ channel kinetics. Radioimmunoassay of high-K +-induced β-endorphin release demonstrated an increase during D 2O treatment and a decrease upon D 2O washout. These results demonstrate that the H 2O-to-D 2O-induced increase in β-endorphin release corresponded with the redistribution of actin, and the D 2O-to-H 2O-induced decrease in β-endorphin release corresponded with the inhibition of voltage-sensitive Ca 2+ channels. The computer modeling suggests that the differences in the zero-point vibrational energy between protonated and deuterated amino acids produce an asymmetric distribution of these amino acids upon D 2O washout and this causes the dysfunction of Ca 2+ channels.

Adenine nucleotides inhibit recombinant N‐type calcium channels via G protein‐coupled mechanisms in HEK 293 cells; involvement of the P2Y13 receptor‐type

1. N-type Ca(2+) channel modulation by an endogenous P2Y receptor was investigated by the whole-cell patch-clamp method in HEK 293 cells transfected with the functional rabbit N-type calcium channel. 2. The current responses (I(Ca(N))) to depolarizing voltage steps were depressed by ATP in a concentration-dependent manner. Inclusion of either guanosine 5'-O-(3-thiodiphosphate) or pertussis toxin into the pipette solution as well as a strongly depolarizing prepulse abolished the inhibitory action of ATP. 3. In order to identify the P2Y receptor subtype responsible for this effect, several preferential agonists and antagonists were studied. Whereas the concentration-response curves of ADP and adenosine 5'-O-(2-thiodiphosphate) indicated a higher potency of these agonists than that of ATP, alpha,beta-methylene ATP, UTP and UDP were considerably less active. The effect of ATP was abolished by the P2Y receptor antagonists suramin and N(6)-(2-methylthioethyl)-2-(3,3,3-trifluoropropylthio)-beta,gamma-dichloromethylene-ATP, but not by pyridoxalphosphate-6-azophenyl-2',4'-disulfonic acid, 2'deoxy-N(6)-methyladenosine-3',5'-diphosphate or 2-methylthio AMP. 4. Using reverse transcription and polymerase chain reaction, mRNA for the P2Y(1), P2Y(4), P2Y(6), P2Y(11) and P2Y(13) receptor subtypes, but not the P2Y(2), and P2Y(12) subtypes, was detected in HEK 293 cells. 5. Immunocytochemistry confirmed the presence of P2Y(1), and to a minor extent that of P2Y(4), but not of P2Y(2) receptors. 6. Hence, it is tempting to speculate that P2Y(13) receptors may inhibit N-type Ca(2+) channels via the betagamma subunits of the activated G(i) protein.

Nitric oxide decreases a calcium-activated potassium current via activation of phosphodiesterase 2 in Helix U-cells

In the present study, we investigated the underlaying mechanism of nitric oxide (NO) and cGMP on the decline of a Ca 2+-activated potassium (K Ca) current in U-cells of the right parietal ganglion of the pulmonate snail, Helix pomatia. Using a two-electrode voltage-clamp technique, we activated a K Ca-current either by opening of endogenous voltage-gated Ca 2+-channels during depolarizing voltage steps or by ionophoretic injection of Ca 2+ via a third electrode containing 100 mM Ca 2+. K Ca-current amplitude in U-cells was sensitive to Ba 2+, TEA, iberiotoxin, kaliotoxin and charybdotoxin (ChTX), but not to 4-aminopyridine (4-AP) (up to 30 mM) and apamin (up to 300 nM). Thus, the biophysical and pharmacological profile of the K Ca-current in U-cells shares similarities with the large-conductance K Ca channel (BK(Ca)). The NO-donor sodium nitroprusside (SNP) or S-nitro- N-acetylpenicillamine (SNAP) as well as NO-gas decreased the K Ca-current amplitude and decreased the rate of K Ca-current activation elicited by Ca 2+-injection. Decline of the current amplitude and decrease of activation of K Ca-current were qualitatively mimicked by the membrane-permeable cGMP analogue dibutyryl-cGMP (db-cGMP). NO-induced decrease of K Ca-current was blocked by methylene blue (50 μM), an inhibitor of the guanylyl-cyclase, and by erytho-9-(2-hydroxyl-3-nonyl) adenine (EHNA) (100 μM), an inhibitor of the cGMP-stimulated phosphodiesterase 2 (PDE2). These experiments suggest that the NO-mediated decrease of K Ca-current in U-cells results from synthesis of cGMP by activation of a guanylyl-cyclase and subsequent activation of PDE2.

Voltage-independent KCNQ4 currents induced by (+/-)BMS-204352.

The compound BMS-204352 has been targeted for use against acute ischemic stroke, due to its activation of the large-conductance Ca2+-activated K-channel (BK). We have previously described that the racemate (+/-)BMS-204352 reversibly modulates KCNQ4 voltage dependency. Here we show that (+/-)BMS-204352 also induces a voltage-independent KCNQ4 current. The channels were stably expressed in human embryonic kidney cells (HEK293), and investigated by use of the whole-cell mode of the patch-clamp technique. (+/-)BMS-204352 was applied extracellularly (10 microM) in order to precipitate the robust appearance of the voltage-independent current. The voltage-independent KCNQ4 currents were recorded as instantaneous increases in currents upon hyperpolarizing or depolarizing voltage steps elicited from holding potentials of -90 or -110 mV. The voltage-independent current reversed at the equilibrium potential for potassium ( E(K)), hence was carried by a K+ conductance, and was blocked by the selective KCNQ channel blockers XE991 and linopirdine. Similar results were obtained with KCNQ4 channels transiently transfected into Chinese hamster ovary cells (CHO). When (+/-)BMS-204352 was applied to stably expressed BK channels, only the voltage dependency was modulated. Retigabine, the classic activator of KCNQ channels, did not induce voltage-independent currents. Our data indicate that KCNQ4 channels may conduct voltage-dependent and voltage-independent currents in the presence of (+/-)BMS-204352.

Adult alveolar epithelial cells express multiple subtypes of voltage-gated K+ channels that are located in apical membrane.

Whole cell perforated patch-clamp experiments were performed with adult rat alveolar epithelial cells. The holding potential was -60 mV, and depolarizing voltage steps activated voltage-gated K(+) (Kv) channels. The voltage-activated currents exhibited a mean reversal potential of -32 mV. Complete activation was achieved at -10 mV. The currents exhibited slow inactivation, with significant variability in the time course between cells. Tail current analysis revealed cell-to-cell variability in K(+) selectivity, suggesting contributions of multiple Kv alpha-subunits to the whole cell current. The Kv channels also displayed steady-state inactivation when the membrane potential was held at depolarized voltages with a window current between -30 and 5 mV. Analysis of RNA isolated from these cells by RT-PCR revealed the presence of eight Kv alpha-subunits (Kv1.1, Kv1.3, Kv1.4, Kv2.2, Kv4.1, Kv4.2, Kv4.3, and Kv9.3), three beta-subunits (Kvbeta1.1, Kvbeta2.1, and Kvbeta3.1), and two K(+) channel interacting protein (KChIP) isoforms (KChIP2 and KChIP3). Western blot analysis with available Kv alpha-subunit antibodies (Kv1.1, Kv1.3, Kv1.4, Kv4.2, and Kv4.3) showed labeling of 50-kDa proteins from alveolar epithelial cells grown in monolayer culture. Immunocytochemical analysis of cells from monolayers showed that Kv1.1, Kv1.3, Kv1.4, Kv4.2, and Kv4.3 were localized to the apical membrane. We conclude that expression of multiple Kv alpha-, beta-, and KChIP subunits explains the variability in inactivation gating and K(+) selectivity observed between cells and that Kv channels in the apical membrane may contribute to basal K(+) secretion across the alveolar epithelium.

Membrane-based gating mechanism for auditory information in the mouse inferior colliculus

In order to analyse the intrinsic membrane-based mechanisms of neurons in the mouse inferior colliculus that are likely to contribute to the processing of acoustic signals, this study use whole cell patch clamp recordings in brain slices to characterize the dependence of depolarization evoked inward and outward currents on different prestimulus membrane potentials. Eighty-seven of one-hundred and one inferior colliculus neurons reacted during depolarizing voltage steps from a holding potential of −60 or −80 mV with a fast inactivating inward current followed by a slow inactivating outward current (type I neurons). Fourteen neurons showed outward currents but no inward currents during depolarizing voltage steps from a holding potential of −60 mV (type II neurons). However, these neurons reacted with TTX-sensitive fast inward currents, if the holding potential was set to −80 mV before the voltage steps occurred. The resting potential was not significantly different between type I (−64.3±3.5 mV) and type II (62.7±2.9 mV) neurons. If the neuronal behavior is the same in vivo, type II neurons must receive an inhibition which hyperpolarizes the membrane potential prior to the arrival of excitatory inputs to be able to generate action potentials. This finding suggests a further function for feedforward inhibition in the IC, namely to open a gate for transmission of excitatory information within a distinct time window. With this membrane based gating mechanism it is possible to detect time related information within an acoustic stimulus (e. g. coincidence) which is an essential task e. g. in the neuronal processing of speech.

A slow fraction of Mg2+ unblock of NMDA receptors limits their contribution to spike generation in cortical pyramidal neurons.

The timing of voltage-dependent removal of Mg(2+) block of N-methyl-d-aspartate receptors (NMDARs) is potentially critical for determining their nonlinear contribution to excitability. Here, we measure the kinetics of NMDAR unblock in nucleated patch and whole cell recordings of rat cortical pyramidal neurons during depolarizing voltage steps. At room temperature, the unblock showed a very fast component (tau < 1 ms) and a slower component (tau = 14-23 ms in nucleated patches). The slow component accounted for half of the current at +40 mV and its amplitude and time constant showed some voltage dependence. Blocking with hyperpolarization was very fast (tau < 200 micros). Voltage-clamp with action potential waveforms, at both room temperature and at 33 degrees C, showed that the rising phase of single fast action potentials unblocks far less NMDAR current than expected from the stationary voltage dependence, while a large amplitude of current is uncovered during the upstroke of slow calcium action potentials. The repolarization of fast sodium action potentials uncovers an NMDAR tail current, much bigger than the stationary level of current.

Voltage-dependent Ca 2+ currents in rat cardiac dorsal root ganglion neurons

This study presents the kinetic and pharmacological properties of voltage-gated Ca 2+ currents in anatomically defined cardiac dorsal root ganglion (DRG) neurons in rats. The neurons were labelled by prior injection of fluorescent tracer Fast Blue into the pericardial sack. There were three distinct groups of neurons with respect to cell size: small (27% of total; cell capacitance <30 pF), medium (65% of total; capacitance 30–80 pF) and large neurons (8% of total; capacitance >80 pF). The properties of Ca 2+ currents were tested in small and medium-sized neurons. In large neurons currents could not be adequately controlled and were not analysed. Ca 2+ currents did not completely inactivate during 100 ms depolarising voltage steps. The activation thresholds in small (−36.9±1.3 mV) and medium (−39.0±1.3 mV) size neurons were similar. Current densities were 105.8 pA/pF in small and 97.4 pA/pF in large neurons and also did not differ. The kinetic properties of activation and inactivation did not differ between small and medium-sized cardiac DRG neurons. At membrane potentials between −50 and −60 mV (the expected resting membrane potential in these neurons) 55 to 70% of Ca 2+ currents in small and medium-sized neurons were available for activation. Both, small and medium-sized neurons expressed similar proportions of L (7.5%), N (25%) and P/Q (36%) type Ca 2+ currents. We conclude that small and medium-sized cardiac DRG neurons are homogeneous with respect to the expression and properties of voltage-gated Ca 2+ currents. Voltage-gated Ca 2+ currents probably play an important role in action potential generation in cardiac DRG neurons due to their availability for activation at resting membrane potential, their high density and voltage threshold close to the threshold for voltage-gated Na + currents.

Mechanisms of dopamine activation of fast-spiking interneurons that exert inhibition in rat prefrontal cortex.

Prefrontal cortical dopamine (DA) modulates pyramidal cell excitability directly and indirectly by way of its actions on local circuit GABAergic interneurons. DA modulation of interneuronal functions is implicated in the computational properties of prefrontal networks during cognitive processes and in schizophrenia. Morphologically and electrophysiologically distinct classes of putative GABAergic interneurons are found in layers II-V of rat prefrontal cortex. Our whole cell patch-clamp study shows that DA induced a direct, TTX-insensitive, reversible membrane depolarization, and increased the excitability of fast-spiking (FS) interneurons. The DA-induced membrane depolarization was reduced significantly by D1/D5 receptor antagonist SCH 23390, but not by the D2 receptor antagonist (-)sulpiride, D4 receptor antagonists U101958 or L-745870, alpha1-adrenoreceptor antagonist prazosin, or serotoninergic receptor antagonist mianserin. The D1/5 agonists SKF81297 or dihydrexidine, but not D2 agonist quinpirole, also induced a prolonged membrane depolarization. Voltage-clamp analyses of the voltage-dependence of DA-sensitive currents, and the effects of changing [K(+)](O) on reversal potentials of DA responses, revealed that DA suppressed a Cs(+)-sensitive inward rectifier K(+) current and a resting leak K(+) current. D1/D5, but not D2 agonists mimicked the suppressive effects of DA on the leak current, but the DA effects on the inward rectifier K(+) current were not mimicked by either agonist. In a subgroup of FS interneurons, the slowly inactivating membrane outward rectification evoked by depolarizing voltage steps was also attenuated by DA. Collectively, these data showed that DA depolarizes FS interneurons by suppressing a voltage-independent 'leak' K(+) current (via D1/D5 receptor mechanism) and an inwardly rectifying K(+) current (via unknown DA mechanisms). Additional suppression of a slowly inactivating K(+) current led to increase in repetitive firing in response to depolarizing inputs. This D1-induced increase in interneuron excitability enhances GABAergic transmission to PFC pyramidal neurons and could represent a mechanism via which DA suppresses persistent firing of pyramidal neurons in vivo.

Regional analysis of whole cell currents from hair cells of the turtle posterior crista.

The turtle posterior crista is made up of two hemicristae, each consisting of a central zone containing type I and type II hair cells and a surrounding peripheral zone containing only type II hair cells and extending from the planum semilunatum to the nonsensory torus. Afferents from various regions of a hemicrista differ in their discharge properties. To see if afferent diversity is related to the basolateral currents of the hair cells innervated, we selectively harvested type I and II hair cells from the central zone and type II hair cells from two parts of the peripheral zone, one near the planum and the other near the torus. Voltage-dependent currents were studied with the whole cell, ruptured-patch method and characterized in voltage-clamp mode. We found regional differences in both outwardly and inwardly rectifying voltage-sensitive currents. As in birds and mammals, type I hair cells have a distinctive outwardly rectifying current (I(K,L)), which begins activating at more hyperpolarized voltages than do the outward currents of type II hair cells. Activation of I(K,L) is slow and sigmoidal. Maximal outward conductances are large. Outward currents in type II cells vary in their activation kinetics. Cells with fast kinetics are associated with small conductances and with partial inactivation during 200-ms depolarizing voltage steps. Almost all type II cells in the peripheral zone and many in the central zone have fast kinetics. Some type II cells in the central zone have large outward currents with slow kinetics and little inactivation. Although these currents resemble I(K,L), they can be distinguished from the latter both electrophysiologically and pharmacologically. There are two varieties of inwardly rectifying currents in type II hair cells: activation of I(K1) is rapid and monoexponential, whereas that of I(h) is slow and sigmoidal. Many type II cells either have both inward currents or only have I(K1); very few cells only have I(h). Inward currents are less conspicuous in type I cells. Type II cells near the torus have smaller outwardly rectifying currents and larger inwardly rectifying currents than those near the planum, but the differences are too small to account for variations in discharge properties of bouton afferents innervating the two regions of the peripheral zone. The large outward conductances seen in central cells, by lowering impedances, may contribute to the low rotational gains of some central-zone afferents.

Blocking action of chromanol 293B on the slow component of delayed rectifier K(+) current in guinea-pig sino-atrial node cells.

1. In guinea-pig sino-atrial (SA) node cells the delayed rectifier K(+) current (I(K)) is composed of rapidly and slowly activating components of I(K) (I(Kr) and I(Ks), respectively). The present study was undertaken to characterize the blocking action of the chromanol derivative 293B on I(Ks) in guinea-pig SA node cells using whole-cell patch-clamp technique. 2. Bath application of 293B blocked I(Ks), elicited by 4-s depolarizing voltage pulses from a holding potential of -50 mV, under conditions in which the L-type Ca(2+) current (I(Ca,L)) and I(Kr) were inhibited; the effect was concentration-dependent with an IC(50) of 5.3 microM, when evaluated by the decrease in the amplitude of I(Ks) tail current following 4-s depolarizing voltage steps to +50 mV. 3. The 293B block of I(Ks) progressed with time during depolarizing voltage steps with a more rapid block at higher concentrations. 4. The block of I(Ks) by 293B was fully reversed within a few minutes after washing off the drug, even when a maximal effect (a nearly full block) was achieved at high drug concentration (50 microM). 5. Bath application of 293B at 50 microM greatly and reversibly reduced the amplitude of I(Ks) which is maximally stimulated by beta-adrenergic agonist isoprenaline (1 microM), while the degree of 293B block of the isoprenaline-stimulated I(Ks) was slightly but significantly smaller than that of non-stimulated I(Ks) (94.0+/-0.98% block, n=6 vs 99.4+/-0.45% block, n=6; P<0.01). 6. We conclude that, in guinea-pig SA node cells (i) 293B is a potent and fully reversible blocker of I(Ks) in control and during beta-adrenergic stimulation and (ii) block with 293B occurs in a time-dependent manner during depolarizing voltage steps.

A population of oligodendrocytes derived from multipotent neural precursor cells expresses a cholinergic phenotype in culture and responds to ciliary neurotrophic factor.

Because oligodendrocytes and their precursors possess receptors for classical transmitters, and neurotransmitters such as glutamate and noradrenaline can mediate oligodendroglial proliferation and differentiation, it is possible that other neurotransmitters can also exert regulatory roles in oligodendrocyte function. We used mitogen-proliferated multipotent neuroepithelial precursors (neurospheres) and identified oligodendroglia that expressed markers traditionally found in cholinergic neurons. Regardless of culture conditions, there existed a large population of cells that resembled oligodendrocytes morphologically and coexpressed the oligodendrocyte-specific marker galactocerebroside (GalC) and the acetylcholine (ACh)-synthesizing enzyme choline acetyltransferase (ChAT). These cells did not express neuronal markers, and whole-cell recordings from cells with similar morphology displayed only outward currents in response to depolarizing voltage steps, further supporting their oligodendroglial identity. Another cholinergic marker, the vesicular ACh transporter, was also detected in GalC(+) oligodendrocytes. Furthermore, neurospheres cultured in the presence of the cholinergic receptor antagonist atropine showed a decrease in the number of GalC(+) spheres, implicating the muscarinic ACh receptor in oligodendrocyte development. The actions of neurotrophins and ciliary neurotrophic factor (CNTF) on these ChAT(+) oligodendrocytes were examined. Among these, CNTF treatment significantly increased oligodendrocytic process outgrowth. These results demonstrate classical cholinergic neuronal markers in oligodendrocytes as well as an effect of muscarinic receptor blockade on oligodendrocyte differentiation.

A-type potassium currents dominate repolarisation of neonatal rat primary auditory neurones in situ

Spiral ganglion neurones provide the afferent innervation to cochlear hair cells. Little is known of the molecular physiological processes associated with the differentiation of these neurones, which occurs up to and beyond hearing onset. We have identified novel A-type (inactivating) potassium currents in neonatal rat spiral ganglion neurones in situ, which have not previously been reported from the mammalian cochlea, presumably as a consequence of altered protein expression associated with other preparations. Under whole-cell voltage clamp, voltage steps activated both A-type and non-inactivating outward currents from around −55 mV. The amplitude of the A-type currents was dependent on the holding potential, with steady-state inactivation relieved at hyperpolarised potentials. At −60 mV (close to the resting potential in situ) the currents were approximately 30% enabled. The inactivation kinetics and the degree of inactivation varied between cells, suggesting heterogeneous expression of multiple inactivating currents. A-type currents provided around 60% of total conductance activated by depolarising voltage steps from the resting potential, and were very sensitive to bath-applied 4-aminopyridine (0.01–1 mM). Tetraethylammonium (0.1–30 mM) also blocked the majority of the A-type currents, and the non-inactivating outward current, but left residual fast inactivating A-type current. Under current clamp, neurones fired single tetrodotoxin-sensitive action potentials. 4-Aminopyridine relieved the A-type current mediated stabilisation of membrane potential, resulting in periodic small amplitude action potentials. This study provides the first electrophysiological evidence for A-type potassium currents in neonatal spiral ganglion neurones and shows that these currents play an integral role in primary auditory neurone firing.

Chemosensing at the carotid body. Involvement of a HERG-like potassium current in glomus cells.

Currently, it is not clear what type of K+ channel(s) is active at the resting membrane potential (RMP) in glomus cells of the carotid body (CB). HERG channels produce currents that are known to contribute to the RMP in other neuronal cells. The goal of the present study was to determine whether CB glomus cells express HERG-like (HL) K+ current, and if so, to determine whether HL currents regulate the RMP. With high [K+]o, depolarizing voltage steps from -85 mV revealed a slowly deactivating inward tail current indicative of HL K+ current in whole-cell, voltage clamped glomus cells. The HL currents were blocked by dofetilide (DOF) in a concentration-dependent manner (IC50 = 13 nM) and high concentrations (1 and 10 mM) of Ba2+. The steady-state activation properties of the HL current (Vh = -45 mV) suggest that it is active at the RMP in glomus cells. Whole-cell, current clamped glomus cells exhibited a RMP of -48 mV. 150 nM DOF caused a significant (14 mV) depolarizing shift in the RMP. In isolated glomus cells, [Ca2+]i increased in response to DOF (1 microM). In an in-vitro CB preparation, DOF increased basal sensory discharge in a concentration-dependent manner and significantly attenuated the sensory response to hypoxia. These results suggest that the HERG-like current is responsible for controlling the RMP in glomus cells of the rabbit CB, and that it is involved in the chemosensory response to hypoxia of the CB.

Properties of reciprocal synapses in the mouse accessory olfactory bulb

Reciprocal dendrodendritic synapses between mitral and granule cells in the accessory olfactory bulb have been implicated in a specialized form of olfactory learning in mice, in which a female forms a memory to the pheromonal signal of the male that mates with her. Relatively little is known, however, about the mechanism of synaptic transmission at the reciprocal synapses. We analyzed synaptic currents generated in accessory olfactory bulb mitral cells in slice preparations with the patch-clamp technique in nystatin-perforated whole-cell configuration. A brief (5–20-ms) depolarizing voltage step from −70 to 0 mV applied to a single mitral cell evoked GABAA receptor-mediated inhibitory postsynaptic currents. The inhibitory postsynaptic currents persisted in the presence of tetrodotoxin, indicating that the inhibitory postsynaptic current in mitral cells can be elicited through purely dendritic interactions. The inhibitory postsynaptic currents were greatly enhanced by washout of extracellular Mg2+. In Mg2+-free solution, the N-methyl-D-aspartate (NMDA) receptor antagonist D,L-2-amino-5-phosphonovaleric acid greatly reduced the inhibitory postsynaptic currents, whereas the non-NMDA receptor antagonist 6-cyano-7-nitroquinoxaline-2,3-(1H,4H)-dione (CNQX) slightly reduced them.These data demonstrate that NMDA receptors play an important role in the generation of dendrodendritic inhibition in mitral cells of the mouse accessory olfactory bulb.

Alterations of single potassium channel activity in CA1 pyramidal neurons after transient forebrain ischemia

Selective neuronal injury in the CA1 zone of hippocampus following transient cerebral ischemia has been well documented ( Pulsinelli et al., 1982; Kirino, 1982). Extracellular potassium concentration markedly increases during ischemia/hypoxia ( Hansen, 1985; Schmidt-Kastener and Freund, 1991). Accumulating evidence has indicated that the outward potassium currents, including delayed rectifier potassium current, not only influence membrane excitability but also mediate apoptosis ( Bortner et al., 1997; Hughes et al., 1997; Yu et al., 1997). It has been shown that the amplitude of delayed rectifier potassium current in CA1 neurons significantly increased after cerebral ischemia ( Chi and Xu, 2000b). To elucidate the mechanisms underlying the changes of potassium currents following ischemia, single potassium channel activities of rat CA1 neurons were compared before and after transient forebrain ischemia. Using cell-attached configuration, depolarizing voltage steps activated outward single channel events. The channel properties, the kinetics and pharmacology of these events resemble the delayed rectifier potassium current. After ischemia, the unitary amplitude of single channels significantly increased, the open probability, mean open time and open time constant also significantly increased while the conductance remained unchanged. These data indicate that the increase of single channel activity is responsible, at least in part, for the increase of delayed rectifier potassium current in CA1 neurons after cerebral ischemia.

Olfactory receptor axons influence the development of glial potassium currents in the antennal lobe of the moth Manduca sexta.

In the olfactory (antennal) lobe of the moth Manduca sexta, olfactory receptor axons strongly influence the distribution and morphology of glial cells. In the present study, we asked whether the development of the electrophysiological properties of the glial cells is influenced by the receptor axons. Whole-cell currents were measured in antennal lobe glial cells in acute brain slices prepared from animals at different stages of metamorphic development (stages 3, 6, and 12). Outward currents were induced by depolarizing voltage steps from a holding potential of -70 mV. At all developmental stages investigated, the outward currents were partly blocked by bath application of the potassium channel blocker 4-aminopyridine (4AP, 10 mM) or by including tetraethylammonium (TEA, 30 mM) in the pipette solution. The relative contribution of the 4AP-sensitive current to the outward current increased from 18% at stages 3 and 6 to 42% at stage 12, while the TEA-sensitive current increased from 18% at stage 3 to 81% at stage 6, and then declined again to 40% at stage 12. In contrast, in the absence of receptor axons, these changes in the contribution of the TEA- and 4AP-sensitive currents to the total outward current did not occur; rather, the current profile remained in the most immature state (stage 3). The results suggest that olfactory receptor axons are essential for development of the mature pattern of glial potassium currents.