Substance P-induced inward current in identified auditory efferent neurons in rat brain stem slices

The effects of substance P (SP) on whole cell currents were studied in neurons of the medial olivocochlear efferent system (MOCS) in the ventral nucleus of the trapezoid body (VNTB) of brain stem slices from neonatal rats. Each neuron was identified by retrograde labeling with Fast Blue injected into the cochlea. Bath application of SP (0.1-10 microM) reversibly induced an apparent inward current in 49 of 63 labeled neurons when voltage clamped at near resting voltages. This apparent inward current was consistent with the SP-induced membrane depolarization observed in current-clamp mode. The SP-induced change in current was dose dependent with a half-maximal response dose of 200 nM. It was mimicked by [Cys3,6, Tyr8, Pro9]-SP, a neurokinin (NK1) receptor selective agonist, whereas [Succinyl-Asp6, MePhe8]-SP 6-11 (Senktide), a NK3 receptor agonist, had no detectable effect. The SP effect was not blocked by 10(-6) M tetrodotoxin (TTX) and persisted when the perfusate contained 30 mM tetraethylammonium (TEA) or 100 microM Cd2+ or was in a 0-Ca solution. In a TTX-containing solution, SP caused a voltage-dependent decrease of membrane conductance, and the SP-evoked current reversed at a potential at around -105 mV. The predicted K+ equilibrium potential was -93.8 mV under the experimental conditions. The SP-induced inward current was attenuated by 66% when the perfusate contained 3 mM Cs+. We conclude that the apparent inward current is partly caused by SP decreasing an outward current normally maintained by the inward rectifier K+ channels in these cells. In the presence of Cs solution in the recording pipette and with a perfusate containing 3 mM Cs+, 0.1 mM Cd2+ and 10(-6) M TTX, a residual SP-induced inward current was observed at test voltages ranging from -120 to 40 mV. This subcomponent reversed its polarity at approximately 20 mV. This inward current was reduced substantially (but not abolished) when all NaCl in the external solution was replaced by TEA-Cl. The results indicate that SP also opens an unknown cation channel, which the available data suggests may be relatively nonselective. The results suggest that MOCS neurons are subject to modulation by SP, which depolarizes the cell membrane by decreasing the activity of inward rectifier K+ channels as well as concurrently activating a separate cation conductance. It also was found that in MOCS neurons responsive to both SP and norepinephrine, the norepinephrine effect was abolished by TTX, suggesting that an interneuronal population excited by norepinephrine converges selectively onto SP-sensitive MOCS neurons in the VNTB.     

Synaptic current at the rat ganglionic synapse and its interactions with the neuronal voltage-dependent currents

The membrane current activated by fast nicotinic excitation of intact and mature rat sympathetic neurons was studied at 37 degrees C, by using the two-microelectrode voltage-clamp technique. The excitatory postsynaptic current (EPSC) was modeled as the difference between two exponentials. A fast time constant (tau2; mean value 0.57 ms), which proves to be virtually voltage-independent, governs the current rise phase and a longer time constant (tau1; range 5.2-6.8 ms in 2 mM Ca2+) describes the current decay and shows a small negative voltage dependence. A mean peak synaptic conductance of 0.58 muS per neuron is measured after activation of the whole presynaptic input in 5 mM Ca2+ external solution (0.40 muS in 2 mM Ca2+). The miniature EPSCs also rise and decay with exponential time constants very similar to those of the compound EPSC recorded at the same voltage. A mean peak conductance of 4.04 nS is estimated for the unitary event. Deconvolution procedures were employed to decompose evoked macrocurrents. It is shown that under appropriate conditions the duration of the driving function describing quantal secretion can be reduced to <1 ms. The shape of the EPSC is accurately mimicked by a complete mathematical model of the sympathetic neuron incorporating the kinetic properties of five different voltage-dependent current types, which were characterized in a previous work. We show that IA channels are opened by depolarizing voltage steps or by synaptic potentials in the subthreshold voltage range, provided that the starting holding voltage is sufficiently negative to remove IA steady-state inactivation (less than -50 mV) and the voltage trajectories are sufficiently large to enter the IA activation range (greater than -65 mV). Under current-clamp conditions, this gives rise to an additional fast component in the early phase of membrane repolarization-in response to voltage pulses-and to a consistent distortion of the excitatory postsynaptic potential (EPSP) time course around its peak-in response to the synaptic signal. When the stimulation initiates an action potential, IA is shown to significantly increase the synaptic threshold conductance (up to a factor of 2 when IA is fully deinactivated), compared with that required when IA is omitted. The voltage dependence of this effect is consistent with the IA steady-state inactivation curve. It is concluded that IA, in addition to speeding up the spike repolarization process, also shunts the excitatory drive and delays or prevents the firing of the neuron action potential.     

Potassium current and the effect of cesium on this current during anomalous rectification of the egg cell membrane of a starfish

The kinetics of the membrane current during the anomalous or inward-going rectification of the K current in the egg cell membrane of the starfish Mediaster aequalis were analyzed by voltage clamp. The rectification has instantaneous and time-dependent components. The time-dependent increase in the K conductance for the negative voltage pulse as well as the decrease in the conductance for the positive pulse follows first-order kinetics. The steady-state conductance increases as the membrane potential becomes more negative and reaches the saturation value at about -40 mV more negative than the K equilibrium potential, V(K). The entire K conductance can be expressed by g(K).n; g g(K) represents the component for the time-independent conductance which depends on V-V(K) and [K+]o, and n is a dimensionless number (1 is greater than or equal to n is greater than or equal to 0) and determined by two rate constants which depend only on V-V(K). Cs+ does not carry any significant current through the K channel but blocks the channel at low concentration in the external medium. The blocking effect increases as the membrane potential is made more negative and the potential-dependent blocking by the external Cs+ also has instantaneous and time-dependent components.     

Analysis of voltage-dependent membrane currents in spatially extended neurons from point-clamp data

1. Numerical methods were used to evaluate voltage space-clamp performance in the investigation of a voltage-dependent inward current similar to the noninactivating Ca current. In addition, the cell is equipped with a repolarizing system, represented by leak and outwardly rectifying outward conductances. The electrotonically compact model cell is represented by a cable with an electrotonic length of 1 space constant under control conditions, but that becomes effectively only 0.33 space constants during a 90% reduction of the leak and outward conductance. The cable is perfectly voltage clamped at one end. 2. The apparent voltage dependence, activation, and inactivation of the clamp current depend on the distribution of the membrane slope conductance along the cable; this depends on 1) the distribution of the inward current along the cable and 2) the amplitude of the inward current relative to the amplitudes of the leak and voltage-dependent outward currents. 3. Under control conditions, the membrane voltage decays steeply with distance from the command voltage at the clamp site to almost resting potential for most of the rest of the cable. This is because the leak and outward current are dominant over the inward current. The inward current is activated primarily at the clamped part of the cable. Clamp currents are activated instantaneously. The clamp-current current-voltage (I-V) relation is less steep with depolarization because the membrane potential for locations away from the clamp site lags behind the clamp potential. 4. When the conductances for leak and outward current are reduced by 90%, these conductances lose their dominance. The membrane slope conductance now has a range with negative values.(ABSTRACT TRUNCATED AT 250 WORDS)     

Cortistatin increase of a potassium conductance in rat locus coeruleus in vitro

1. In this study we examined the effects of cortistatin, a putative endogenous ligand for somatostatin (SRIF) receptors, on the membrane properties of rat locus coeruleus (LC) neurones in vitro, by use of intracellular and whole cell patch clamp recording. We have compared the actions of cortistatin with those of SRIF and the SRIF analogue D-Phe-Cys-Tyr-D-Trp-Orn-Thr-Pen-Thr-NH2 (CTOP). 2. When LC neurones were voltage clamped to -60 mV, application of cortistatin caused an outward current in all cells examined (n = 44), with a pEC50 of 6.62. SRIF also caused an outward current in all cells examined (n = 43), with a pEC50 of 6.93. 3. The outward currents caused by cortistatin in 2.5 mM extracellular K+ reversed polarity at -106 mV, very close to the predicted K+ reversal potential of -105 mV. Increasing extracellular K+ to 10.5 mM resulted in a shift of the reversal potential of +38 mV, a shift consistent with a K+ conductance. The conductance activated by cortistatin showed mild inward rectification. 4. Continuous application of a high concentration of SRIF (1 microM) resulted in a decrease of the outward current to a steady level of 49% of the maximum response, with a t1/2 of 131 s. Application of a high concentration of cortistatin (3 microM) during the desensitized portion of the SRIF response did not result in any further outward current. Continuous application of a high concentration of cortistatin (10 microM) resulted in a decrease of the outward current to a steady level of 42% of the maximum response with a t1/2 of 114 s. Application of a high concentration of SRIF (3 microM) during the desensitized portion of the cortistatin response produced only a small outward current. 5. Continuous application of cortistatin (3 microM) also resulted in a decrease of the outward current (by 43%, t1/2 of 136 s) and application of a high concentration of CTOP (10 microM) during the desensitized portion of the cortistatin response did not produce any outward current. Continuous application of a high concentration of CTOP (10 microM) resulted in a decrease of the outward current to a steady level of 70% of the maximum response with a t1/2 of 143 s. Application of a high concentration of cortistatin (3 microM) during the desensitized portion of the CTOP response did not result in any further outward current. 6. The actions of cortistatin (300 nM-10 microM) were not affected by the opioid antagonist naloxone (10 microM). Application of met-enkephalin during the desensitized portion of the response to a high concentration of cortistatin (3 microM) produced an outward current similar to that produced by metenkephalin application alone. 7. Thus cortistatin efficaciously activates an inwardly rectifying K+ conductance in LC neurones. These actions appear to be mediated by a population of SRIF receptors, at which CTOP is also an agonist. Cortistatin does not appear to be a ligand for mu-opioid receptors in rat LC neurons.     

Nitric oxide (NO)-induced activation of large conductance Ca2+-dependent K+ channels (BK(Ca)) in smooth muscle cells isolated from the rat mesenteric artery

1. To assess the action of nitric oxide (NO) and NO-donors on K+ current evoked either by voltage ramps or steps, patch clamp recordings were made from smooth muscle cells freshly isolated from secondary and tertiary branches of the rat mesenteric artery. 2. Inside-out patches contained channels, the open probability of which increased with [Ca2+]i. The channels had a linear slope conductance of 212+/-5 pS (n = 12) in symmetrical (140 mM) K+ solutions which reversed in direction at 4.4 mV. In addition, the channels showed K+ selectivity, in that the reversal potential shifted in a manner similar to that predicted by the Nernst potential for K+. Barium (1 mM) applied to the intracellular face of the channel produced a voltage-dependent block and external tetraethylammonium (TEA; at 1 mM) caused a large reduction in the unitary current amplitude. Taken together, these observations indicate that the channel most closely resembled BK(Ca). 3. In five out of six inside-out patches, NO (45 or 67 microM) produced an increase in BK(Ca) activity. In inside-out patches, BK(Ca) activity was also enhanced in some patches with 100 or 200 microM 3-morpholino-sydnonimine (SIN-1) (4/11) and 100 microM sodium nitroprusside (SNP) (3/8). The variability in channel opening with the NO donors may reflect variability in the release of NO from these compounds. 4. In inside-out patches, 100 microM SIN-1 failed to increase BK(Ca) activity (in all 4 patches tested), while at a higher (500 microM) concentration SIN-1 had a direct blocking effect on the channels (n = 3). NO applied directly to inside-out patches increased (P < 0.05) BK(Ca) activity in two patches. 5. In the majority of cells (6 out of 7), application of NO (45 or 67 microM) evoked an increase in the amplitude of whole-cell currents in perforated patches. This action was not affected by the soluble guanylyl cyclase inhibitor, 1H-[1,2,4] oxadiazolo [4,3-a]quinoxalin-1-one (ODQ). An increase in whole-cell current was also evoked with either of the NO donors, SIN-1 or SNP (each at 100 microM). With SIN-1, the increase in current was blocked with the BK(Ca) channel blocker, iberiotoxin (50 nM). 6. With conventional whole-cell voltage clamp, the increase in the outward K+ current evoked with SIN-1 (50-300 microM) showed considerable variability. Either no effect was obtained (11 out of 18 cells), or in the remaining cells, an average increase in current amplitude of 38.7+/-10.2% was recorded at 40 mV. 7. In cell-attached patches, large conductance voltage-dependent K+ channels were stimulated by SIN-1 (100 microM) applied to the cell (n = 5 patches). 8. These data indicate that NO and its donors can directly stimulate BK(Ca) activity in cells isolated from the rat mesenteric artery. The ability of NO directly to open BK(Ca) channels could play an important functional role in NO-induced relaxation of the vascular smooth muscle cells in this small resistance artery.     

The pharmacology of mesocortical dopamine neurons: a dual-probe microdialysis study in the ventral tegmental area and prefrontal cortex of the rat brain

The development of receptor function at corticothalamic synapses during the first 20 days of postnatal development is described. Whole cell excitatory postsynaptic currents (EPSCs) were evoked in relay neurons of the ventral posterior nucleus (VP) by stimulation of corticothalamic fibers in in vitro slices of mouse brain from postnatal day 1 (P1). During P1-P12, excitatory postsynaptic conductances showed strong voltage dependence at peak current and at 100 ms after the stimulus and were almost completely antagonized by -2-amino-5-phosphonopentoic acid (APV), indicating that N-methyl--aspartate (NMDA) receptor-mediated currents dominate corticothalamic EPSCs at this time. After P12, in 42% of cells, excitatory postsynaptic conductances showed no voltage-dependence at peak current but still showed voltage-dependence 100-ms poststimulus. This voltage-dependent conductance was antagonized by APV. The nonvoltage-dependent component was APV resistant, showed fast decay, and was antagonized by the nonNMDA antagonist, 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX). In the remaining 58% of cells after P12, excitatory postsynaptic conductances showed moderate voltage dependence at peak conductance and strong voltage dependence 100 ms after the stimulus. Analysis of EPSCs before and after APV showed a significant increase in the relative contribution of the non-NMDA conductance after the second postnatal week. From P1 to P16, there was a significant decrease in the time constant of decay of the NMDA EPSC but no change in the voltage dependence of the NMDA response. After P8, slow EPSPs, 1.5-30 s in duration and mediated by metabotropic glutamate receptors (mGluRs), could be evoked by high-frequency stimulation of corticothalamic fibers in the presence of APV and CNQX. Similar slow depolarizations could be evoked by local application of the mGluR agonist (+/-)-1-aminocyclopentane-trans-1,3-dicarboxylic acid (t-ACPD) but from P0. Both conductances were blocked by the mGluR antagonist, (RS)-alpha-methyl-4-carboxyphenylglycine. Hence functional mGluR receptors are present on VP cells from birth, but their synaptic activation at corticothalamic synapses can only be detected after P8. In voltage clamp, the extrapolated reversal potential of the t-ACPD current, with potassium gluconate-based internal solution, was +12 +/- 10 (SE) mV, and the measured reversal potential with cesium gluconate-based internal solution was 1.5 +/- 9.9 mV, suggesting that the mGluR-mediated depolarization was mediated by a nonselective cation current. Replacement of NaCl in the external solution caused the reversal potential of the current to shift to -18 +/- 2 mV, indicating that Na+ is a charge carrier in the current. The current amplitude was not reduced by application of Cs+, Ba2+, and Cd2+, indicating that the t-ACPD current was distinct from the hyperpolarization-activated cation current (IH) and distinct from certain other previously characterized mGluR-activated, nonselective cation conductances.     

Mechanisms of afterhyperpolarization in lobster olfactory receptor neurons

In lobster olfactory receptor neurons (ORNs), depolarizing responses to odorants and current injection are accompanied by the development of an afterhyperpolarization (AHP) that likely contributes to spike-frequency adaptation and that persists for several seconds after termination of the response. A portion of the AHP can be blocked by extracellular application of 5 mM CsCl. At this concentration, CsCl specifically blocks the hyperpolarization-activated cation current (Ih) in lobster ORNs. This current is likely to be active at rest, where it provides a constant, depolarizing influence. Further depolarization deactivates Ih, thus allowing the cell to be briefly hyperpolarized when that depolarizing influence is removed, thus generating an AHP. Reactivation of Ih would terminate the AHP. The component of the AHP that could not be blocked by Cs+ (the Cs(+)-insensitive AHP) was accompanied by decreased input resistance, suggesting that this component is generated by increased conductance to an ion with an equilibrium potential more negative than the resting potential. The Cs(+)-insensitive AHP in current clamp and the underlying current in voltage clamp displayed a reversal potential of approximately -75 mV. Both EK and ECl are predicted to be in this range. Similar results were obtained with the use of a high Cl- pipette solution, although that shifted ECl from -72 mV to -13 mV. However, when EK was shifted to more positive or negative values, the reversal potential also shifted accordingly. A role for the Ca(2+)-mediated K+ current in generating the Cs(+)-independent AHP was explored by testing cells in current and voltage clamp while blocking IK(Ca) with Cs+/Co(2+)-saline. In some cells, the Cs(+)-independent AHP and its underlying current could be completely and reversibly blocked by Cs+/Co2+ saline, whereas in other cells some fraction of it remained. This indicates that the Cs(+)-independent AHP results from two K+ currents, one that requires an influx of extracellular Ca2+ and one that does not. Collectively, these findings indicate that AHPs result from three phenomena that occur when lobster ORNs are depolarized: 1) inactivation of the hyperpolarization-activated cation current, 2) activation of a Ca(2+)-mediated K+ current, and 3) activation of a K+ current that does not require influx of extracellular Ca2+. Roles of these processes in modulating the output of lobster ORNs are discussed.     

Properties of a slow nonselective cation conductance modulated by neurotensin and other neurotransmitters in midbrain dopaminergic neurons

1. A widespread mechanism of slow excitation throughout the nervous system involves overlapping changes in nonselective ion conductance and K+ conductance. We used whole cell patch-clamp recording to characterize such a nonselective conductance induced by neurotensin (NT) and other neurotransmitters in immunocytochemically identified dopaminergic neurons cultured from the rat ventral tegmental area (VTA). 2. The NT-induced inward current consisted of an initial peak and later "hump." The response was blocked reversibly by the nonpeptide NT-receptor antagonist SR48692, suggesting that it resulted from activation of NT receptors. 3. The channel was almost equally permeable to Na+ and K+, as determined from the reversal potential shift upon switching from Na+- to K(+)-containing external solution. The permeability of Cs+ was similar to that of Na+, as determined from the zero-current equation and average reversal potential in the 75 mM Na+ solution. Cl- was not significantly permeable. 4. In Ca(2+)-free external solution, the NT-induced current showed a fourfold increase in amplitude, and in high Mg2+ (20 mM) external solution, the NT-induced current showed an 80% decrease in amplitude, suggesting that external Ca2+ and Mg2+ could block the nonselective conductance. 5. The NT response was unaffected by loading the neurons with either the Ca2+ chelator 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid or with 1 mM ca2+. The nonselective conductance was therefore not Ca2+ activated. 6. Loading the neurons with cyclic GMP or cyclic AMP (each with the phosphodiesterase inhibitor isobutyl-methylxanthine) did not affect the NT response. The NT-induced nonselective conductance was therefore not cyclic nucleotide-activated. 7. The latency of the NT response was long (> or = 185 ms, average 406 ms, 30 degrees C), indicating that NT did not induce the conductance through ligand-gated channels. Thus, NT activated a slow nonselective cation conductance. 8. Neurokinin B, a metabotropic glutamate agonist, and muscarine elicited responses similar to the NT response. The NT response could be elicited after desensitizing the responses to these other neurotransmitters, indicating receptor specificity in the activation of the nonselective conductance.     

Hyperpolarization-activated inward current in neurons of the rat's dorsal nucleus of the lateral lemniscus in vitro

Hyperpolarization-activated inward current in neurons of the rat's dorsal nucleus of the lateral lemniscus in vitro. J. Neurophysiol. 78: 2235-2245, 1997. The hyperpolarization-activated current (Ih) underlying inward rectification in neurons of the rat's dorsal nucleus of the lateral lemniscus (DNLL) was investigated using whole cell patch-clamp techniques. Patch recordings were made from DNLL neurons of young rats (21-30 days old) in 400 micro;m tissue slices. Under current clamp, injection of negative current produced a graded hyperpolarization of the cell membrane, often with a gradual sag in the membrane potential toward the resting value. The rate and magnitude of the sag depended on the amount of hyperpolarizing current. Larger current resulted in a larger and faster decay of the voltage. Under voltage clamp, hyperpolarizing voltage steps elicited a slowly activating inward current that was presumably responsible for the sag observed in the voltage response to a steady hyperpolarizing current recorded under current clamp. Activation of the inward current (Ih) was voltage and time dependent. The current just was seen at a membrane potential of -70 mV and was activated fully at -140 mV. The voltage value of half-maximal activation of Ih was -78.0 +/- 6.0 (SE) mV. The rate of Ih activation was best approximated by a single exponential function with a time constant that was voltage dependent, ranging from 276 +/- 27 ms at -100 mV to 186 +/- 11 ms at -140 mV. Reversal potential (Eh) of Ih current was more positive than the resting potential. Raising the extracellular potassium concentration shifted Eh to a more depolarized value, whereas lowering the extracellular sodium concentration shifted Eh in a more negative direction. Ih was sensitive to extracellular cesium but relatively insensitive to extracellular barium. The current amplitude near maximal-activation (about -140 mV) was reduced to 40% of control by 1 mM cesium but was reduced to only 71% of control by 2 mM barium. When the membrane potential was near the resting potential (about -60 mV), cesium had no effect on the membrane potential, current-evoked firing rate and input resistance but reduced the spontaneous firing. When the membrane potential was more negative than -70 mV, cesium hyperpolarized the cell, decreased current-evoked firing and increased the input resistance. Ih in DNLL neurons does not contribute to the normal resting potential but may enhance the extent of excitation, thereby making the DNLL a consistently powerful inhibitory source to upper levels of the auditory system.     

Firing properties of spherical bushy cells in the anteroventral cochlear nucleus of the gerbil

In gerbils, spherical bushy cells (SBCs) encode low frequency sound signals into a temporal firing pattern. To investigate the support for the timing in this temporal code, we characterized the membrane electrical properties of visually identified SBCs in brainstem slices. A brief depolarizing subthreshold transient potential (TP) triggered, with relatively invariant latency, a single spike at the onset of a response to depolarizing current pulses. The activation of a subthreshold Na+-conductance, sensitive to blockade with tetrodotoxin, and a high threshold Ca2+-conductance, sensitive to blockade with Co2+ or Cd2+, accelerated the rising phase and amplified the TP. A K+-conductance, sensitive to blockade by 4-aminopyridine (4-AP, 50 microM), shaped the decay of the TP. Following a single spike, voltage-gated activation of transient and sustained K+-conductances suppressed any tendency to repetitively discharge. A reduction in either K+-conductance due to application of 4-AP or tetraethylammonium (TEA, 10 mM), converted the single spike mode to repetitive firing during the depolarizing pulses. A persistent, tetrodotoxin-sensitive Na+-conductance amplified steady-state depolarizing responses. A hyperpolarization-activated conductance, greatly decreased by extracellular Cs+ (3 mM) but resistant to Ba2+ (up to 1 mM), filtered the responses to hyperpolarizing current inputs. A depolarized membrane potential promoted repetitive firing in SBCs. This state, expected in pathophysiological conditions, would corrupt the temporal code.     

Isolation and characterization of a persistent potassium current in neostriatal neurons

1. Depolarization-activated, calcium-independent potassium (K+) currents were studied with the use of whole cell voltage-clamp recording from neostriatal neurons acutely isolated from adult (> or = 4 wk old) rats. The whole cell K+ current was composed of transient and persistent components. The aims of the experiments were to isolate the persistent component and then to characterize its voltage dependence and kinetics. 2. Application of 10 mM 4-aminopyridine (4-AP) completely blocked the transient currents while reducing the persistent current by approximately 40% [50% inhibitory concentration (IC50), of blockable current = 125 microM]. The persistent K+ current also was reduced by tetraethylammonium (TEA). Two components to the TEA block were present, having IC50s of 125 microM (23% of the blockable current) and 5.9 mM (77% of the blockable current). Collectively, these results suggested that the persistent components of the total K+ current was pharmacologically heterogeneous. The properties of the 4-AP-resistant, persistent K+ current (IKrp) were subsequently studied. 3. The kinetics of activation and deactivation of IKrp were voltage dependent. Examination of the entire activation/deactivation time constant profile showed that it was bell shaped, with time constants being moderately rapid (tau approximately 50 ms) at membrane potentials corresponding to the resting potential of neostriatal cells (approximately -80 mV), becoming considerably longer (tau approximately 100 ms) at potentials near the cells' spike thresholds (approximately -45 mV), and decreasing to a minimum (tau approximately 5 ms) at potentials associated with the peak of the cells' action potentials (approximately +20 mV). The inactivation kinetics of IKrp also were voltage dependent. The time constants of inactivation varied between 1 and 8 s at potentials between -10 and +35 mV. 4. Unlike persistent K+ currents in many other cell types, IKrp activated at relatively hyperpolarized membrane potentials (approximately -70 mV). The Boltzmann function describing activation had a half-activation voltage of -13 mV and a slope factor of 12 mV. In addition, the Boltzmann function describing the voltage dependence of inactivation of IKrp had a relatively depolarized half-inactivation voltage of -55 and a large slope factor of 19 mV, indicating that this current was available over a broad range of membrane potentials (between -100 and -10 mV). 5. Neostriatal neurons recorded in vivo exhibit subthreshold shifts in membrane potential of variable duration (tens of ms to s) from a hyperpolarized resting state to a depolarized state that is limited in amplitude just below spike threshold. The voltage dependence of activation and inactivation of IKrp indicates that it will be available on depolarization from the hyperpolarized state. However, the slow activation rate of this current suggests that it will contribute little either to limiting the amplitude of the initial depolarization associated with entry into the depolarized state or to depolarizing episodes of short duration (e.g., < 50 ms). However, IKrp should limit the amplitude of membrane depolarizations associated with prolonged excursions into the depolarized state.     

Characteristics of a fast transient outward current in guinea pig trigeminal motoneurons

A fast transient voltage dependent outward current (TOC) in trigeminal motoneurons (TMNs) was studied in guinea pig brainstem slices by use of sharp electrodes in combination with single electrode voltage clamp techniques. In solutions containing TTX, low Ca2+/Mn2+ and 20 mM TEA this current activated around -55 to -60 mV from holding potentials negative to resting potential, obtained its peak amplitude within 5 ms and decayed as a single exponential with a time constant of 6-8 ms. Half maximal values for inactivation and activation were -72 and -37 mV, respectively. Bath application of 5 mM 4-AP suppressed this current by approximately 90% and eliminated the early depolarizing transient membrane rectification observed in response to a constant depolarizing current pulse, prolonged the action potential duration, and reduced the threshold voltage and delay to onset of the action potential. It is suggested that this current resembles the typical A-current observed in many CNS neurons and, as a result of its voltage and time dependent properties, could contribute to control of motoneuronal discharge and timing of burst onset during rhythmical jaw movements. Therefore, any cellular models of masticatory activity should include the properties of this current.     

Chromo-domain proteins: linking chromatin structure to epigenetic regulation

Ion environment and ionic fluxes through membrane are thought to be important in the spermatozoa's maturation, capacitation, and the initiating process of gamete interaction. In this work, the membrane proteins isolated from human sperm plasma membrane were reconstituted into planar lipid bilayers via fusion, and the ion channels activities were observed under voltage clamp mode. In cis 200//trans 100 mM KCl solution, a TEA-sensitive cation-selective channel with a unit conductance of 40 pS was recorded. In a gradient of 200//100 mM NaCl solutions, a Na(+)-selective channel with a unit conductance of 26 pS was recorded. In both cases, reversal potential was about-18 mV, which is close to the predicated value of a perfect Nernst K+ or Na+ electrode. In 50//10 mM CaCl2 solution, a cation channel activity with a unit conductance of 40 pS and reversal potential of about -20 mV was usually observed. In 200//100 mM NMDG(N-methyl-D-glucamine)-Cl solution, where the cation ions were substituted with NMDG, a 30-pS anion-selective channel activity was also detected. The variety in the types of ion channels observed in human spermatozoa plasma membrane suggests that ion channels may play a range of different roles in sperm physiology and gamete interaction.     

Calcium-sensitive calcium influx in photoreceptor inner segments

The effect of external calcium concentration ([Ca2+]o) on membrane potential-dependent calcium signals in isolated tiger salamander rod and cone photoreceptor inner segments was investigated with patch-clamp and calcium imaging techniques. Mild depolarizations led to increases in intracellular Ca2+ levels ([Ca2+]i) that were smaller when [Ca2+]o was elevated to 10 mM than when it was 3 mM, even though maximum Ca2+ conductance increased 30% with the increase in [Ca2+]o. When external calcium was lowered to 1 mM [Ca2+]o, maximum Ca2+ conductance was reduced, as expected, but the mild depolarization-induced increase in [Ca2+]i was larger than in 3 mM [Ca2+]o. In contrast, when photoreceptors were strongly depolarized, the increase in [Ca2+]i was less when [Ca2+]o was reduced. An explanation for these observations comes from an assessment of Ca2+ channel gating in voltage-clamped photoreceptors under changing conditions of [Ca2+]o. Although Ca2+ conductance increased with increasing [Ca2+]o, surface charge effects dictated large shifts in the voltage dependence of Ca2+ channel gating. Relative to the control condition (3 mM [Ca2+]o), 10 mM [Ca2+]o shifted Ca2+ channel activation 8 mV positive, reducing channel open probability over a broad range of potentials. Reducing [Ca2+]o to 1 mM reduced Ca2+ conductance but shifted Ca2+ channel activation negative by 6 mV. Thus the intracellular calcium signals reflect a balance between competing changes in gating and permeation of Ca2+ channels mediated by [Ca2+]o. In mildly depolarized cells, the [Ca2+]o-induced changes in Ca2+ channel activation proved stronger than the [Ca2+]o-induced changes in conductance. In response to the larger depolarizations caused by 80 mM [K+]o, the opposite is true, with conductance changes dominating the effects on channel activation.     

Strategy of small rodent populations from Kanev national park under habitat changes caused by technogenic pollutions and an accident at the Chernobyl nuclear power station

Single channel cell-attached patch and whole-cell clamp experiments on the mode of action of the K+ channel opener (KCO), levcromakalim, were performed in guinea pig isolated portal vein cells. At +20 mV (135/23 mM K+ in bath/pipette), 10 microM levcromakalim activated K+ channels with a chord conductance of 23.2 pS (K(KCO)), which were sensitive to the blocker of ATP-dependent K+ channels (K(ATP)), glibenclamide. Voltage steps from -80 mV to +20 mV activated 4-aminopyridine-sensitive K+ channels of 6.5 pS with properties of delayed rectifier K+ channels (Kv). In patches which upon a previous voltage step had revealed the existence of Kv, levcromakalim reduced the open-probability of Kv, but it did not concomitantly activate K(KCO). During the course of the experiments, but unrelated to the presence of levcromakalim, large conductance K+ channels (BK(Ca)) appeared which could be inhibited by iberiotoxin, a selective blocker of BK(Ca), and by the membrane-permeant calcium buffer, BAPTA/AM, but not by glibenclamide. Whole-cell current-voltage (i-V) relations were established in response to voltage ramps from +50 mV to -100 mV; on subtraction of control i-V curves from i-V curves obtained in the presence of 10 microM levcromakalim, the KCO-induced K+ current remained which was proportional to voltage. This is not compatible with the upward-bent curvature predicted by the GHK current equation for purely resistive channels at high [K+]i versus low [K+]o. In conclusion, in the guinea pig portal vein cells, no evidence could be established for the hypotheses that KCOs may act via conversion of Kv to K(ATP) (Beech and Bolton 1989; Edwards et al. 1993) or by activation of BK(Ca) (Balwierczak et al. 1995). In these cells, mild inward rectification of the levcromakalim-induced current was observed which underlines their relationship to K(ATP) in other tissues.     

DGAP1, a homologue of rasGTPase activating proteins that controls growth, cytokinesis, and development in Dictyostelium discoideum

To study K+ channels in the basolateral membrane of chloride-secreting epithelia, rat tracheal epithelial monolayers were cultured on permeable filters and mounted into an Ussing chamber system. The mucosal membrane was permeabilized with nystatin (180 microg/ml) in the symmetrical high K+ (145 mm) Ringer solution. During measurement of the macroscopic K+ conductance properties of the basolateral membrane under a transepithelial voltage clamp, we detected at least two types of K+ currents: one is an inwardly rectifying K+ current and the other is a slowly activating outwardly rectifying K+ current. The inwardly rectifying K+ current is inhibited by Ba2+. The slowly activating K+ current was potentiated by cAMP and inhibited by clofilium, phorbol 12-myristate 13-acetate (PMA) and lowering temperature. This is consistent with the biophysical characteristics of ISK channel. RT-PCR analysis revealed the presence of ISK cDNA in the rat trachea epithelia. Although 0.1 mM Ba2+ only had minimal affect on short-circuit current (Isc) induced by cAMP in intact epithelia, 0.1 mM clofilium strongly inhibited it. These results indicate that ISK might be important for maintaining cAMP-induced chloride secretion in the rat trachea epithelia.     

Nociceptin receptor coupling to a potassium conductance in rat locus coeruleus neurones in vitro

1. In this study we have examined the effects of nociceptin, an endogenous ligand for the opioid-like receptor ORL1 on the membrane properties of rat locus coeruleus (LC) neurones in vitro, using intracellular and whole cell patch clamp recording. 2. When locus coeruleus neurones were voltage clamped to -60 mV, application to nociceptin caused an outward current in all cells examined (n = 49), with an EC50 of 90 nM. Neither the potency nor the maximal effect of nociceptin was altered in the presence of the peptidase inhibitors, bestatin (20 microM) or thiorphan (2 microM). 3. The outward currents caused by nociceptin in 2.5 mM extracellular K+ reversed polarity at -123 mV, more negative than the predicted K+ reversal potential of -105 mV. Increasing extracellular K+ to 6.5 mM resulted in a shift of the reversal potential of +25 mV, a shift consistent with a K+ conductance. The conductance activated by nociceptin showed mild inward rectification. 4. Application of a high concentration of nociceptin (3 microM) occluded the current produced by simultaneous application of high concentrations of Met-enkephalin (10 microM), (3 microM) somatostatin and UK 14304 (3 microM), indicating that nociceptin activated the same conductance as mu-opioid and somatostatin receptors and alpha 2-adrenoceptors. 5. The actions of nociceptin were weakly antagonized by the opioid antagonist, naloxone, with pKb's estimated from 2 cells of -4.23 and -4.33. The mu-opioid antagonist, CTAP (D-Phe-Cys-Tyr-D-Trp-Arg-Pen-Thr-NH2, 1 microM), the opioid antagonist, nalorphine (30 microM) or the somatostatin antagonist, CPP (cyclo(7-aminoheptanoyl-Phe-D-Trp-Lys-Thr[Bz1]) 3 microM) did not affect the nociceptin-induced current. 6. Dynorphin A (microM), another putative endogenous ligand for ORL1, caused a robust outward current in locus coeruleus neurones that was, however, completely antagonized by moderate concentrations of naloxone (300 nM-1 microM). 7. Continuous application of nociceptin (3 microM) resulted in a decrease of the outward current to a steady level of 70% of the maximum response with a t1/2 of 120s. Desensitization was largely homologous because simultaneous application of Met-enkephalin (30 microM) during the desensitized period of the nociceptin response resulted in an outward current that was 92% of control responses to Met-enkephalin in the same cells. Conversely, continuous application of Met-enkephalin (30 microM) resulted in a decrease of Met-enkephalin current to a steady level that was 54% of the initial current. During this desensitized period application of nociceptin (3 microM) resulted in a current that was 78% of the control responses to nociceptin in the same cells. 8. Thus nociceptin potently activates an inwardly rectifying K+ conductance in locus coeruleus neurones, with a pharmacological profile consistent with activation of the ORL1 receptor. Dynorphin A does not appear to be a ligand for ORL1 in rat locus coeruleus neurones.     

Inhibition by taurine of the inwardly rectifying K+ current in guinea pig ventricular cardiomyocytes

Effects of taurine on the inwardly rectifying K+ current (IK1) in isolated guinea pig ventricular cardiomyocytes were examined using patch voltage-clamp methods. All experiments were performed at 36 degrees C. Taurine (10-20 mM) increased the action potential duration, but failed to affect the resting potential. Holding potential was maintained at -30 mV. The current was activated with an inwardly going rectification, and was completely blocked by Ba2+ (2 mM). Taurine inhibited IK1 at - 120 mV by 28.3+/-1.1% (n=6, P < 0.05) at 10 mM and by 36.0+/-2.1% (n=6, P < 0.01) at 20 mM. The reversal potential was shifted in the hyperpolarizing direction by 3.7+/-0.6 mV (n=6) at 20 mM. In inside-out patch-clamp experiments, the amplitude of unitary channels was -2.7+/-0.3 pA (n=21) at -90 mV. Symmetrical high-K+ (150 mM) solutions in both bath and pipette were used. The channel conductance was 32+/-2 pS (n=9). Taurine did not affect channel conductance, but markedly decreased the open probability at - 120 mV of channel by 21.5+/-2.4% (n=8, P < 0.01) at 10 mM, and by 56.7+/-3.8% (n=8, P < 0.001) at 20 mM. These responses were almost reversible. These results suggest that taurine directly modulates the open probability of the inwardly rectifying K+ current, resulting in regulation of the functions of heart cells.     

Multiple channels mediate calcium leakage in the A7r5 smooth muscle-derived cell line

Ca2+ entry under resting conditions may be important for contraction of vascular smooth muscle, but little is known about the mechanisms involved. Ca2+ leakage was studied in the A7r5 smooth muscle-derived cell line by patch-clamp techniques. Two channels that could mediate calcium influx at resting membrane potentials were characterized. In 110 mM Ba2+, one channel had a slope conductance of 6.0 +/- 0.6 pS and an extrapolated reversal potential of +41 +/- 13 mV (mean +/- SD, n = 8). The current rectified strongly, with no detectable outward current, even at +90 mV. Channel gating was voltage independent. A second type of channel had a linear current-voltage relationship, a slope conductance of 17.0 +/- 3.2 pS, and a reversal potential of +7 +/- 4 mV (n = 9). The open probability increased e-fold per 44 +/- 10 mV depolarization (n = 5). Both channels were also observed in 110 mM Ca2+. Noise analysis of whole-cell currents indicates that approximately 100 6-pS channels and 30 17-pS channels are open per cell. These 6-pS and 17-pS channels may contribute to resting calcium entry in vascular smooth muscle cells.