Deactivation Time Research Articles

Desensitization Masks Nanomolar Potency of ATP for the P2X1 Receptor

ATP-gated P2X1 receptors feature fast activation and fast desensitization combined with slow recovery from desensitized states. Here, we exploited a non-desensitizing P2X2/P2X1 chimera that includes the entire P2X1 ectodomain (Werner, P., Seward, E. P., Buell, G. N., and North, R. A. (1996) Proc. Natl. Acad. Sci. U. S. A. 93, 15485-15490) to obtain a macroscopic representation of intrinsic receptor kinetics without bias arising from the overlap of channel activation and desensitization. From the stationary currents made amenable to analysis by this chimera, an EC50 for ATP of 3.3 nM was derived, representing a >200- and >7000-fold higher ATP potency than observed for the parental P2X1 and P2X2A receptors, respectively. Also, other agonists activated the P2X2/P2X1 chimera with nanomolar EC50 values ranging from 3.5 to 73 nM in the following rank order: 2-methylthio-ATP, 2',3'-O-(4-benzoylbenzoyl)-ATP, alpha,beta-methylene-ATP, adenosine 5'-O-(3-thiotriphosphate). Upon washout, the P2X2/P2X1 chimera deactivated slowly with a time constant (ranging from 63 to 2.5 s) that is inversely related to the EC50 value for the corresponding agonist. This suggests that deactivation time courses reflect unbinding rates, which by themselves define agonist potencies. The P2X2/P2X1 chimera and the P2X1 receptor possess virtually identical sensitivity to inhibition by the P2X1 receptor-selective antagonist NF279, a suramin analog. These results suggest that the P2X1 ectodomain confers nanomolar ATP sensitivity, which, within the wild-type P2X1 receptor, is obscured by desensitization such that only a micromolar ATP potency can be deduced from peak current measurements, representing an amalgam of activation and desensitization.

Kinetics of Pyruvate Decarboxylase Deactivation by Benzaldehyde

Based on experimental data, a kinetic model for the deactivation of partially purified pyruvate decarboxylase (PDC) by benzaldehyde (0–200 mM) in MOPS buffer (2.5 M) has been developed. An initial lag period prior to deactivation was found to occur. With first order dependencies of PDC deactivation on exposure time and on benzaldehyde concentration, a reaction time deactivation constant of 2.64×10−3 h−1 and a benzaldehyde deactivation coefficient of 1.98×10−4 mM−1 h−1 were determined for benzaldehyde concentrations up to 200 mM. The PDC deactivation kinetic equations established in this study are an essential component in an overall model being developed to describe the enzymatic biotransformation of benzaldehyde and pyruvate to produce the pharmaceutical intermediate (R)-phenylacetylcarbinol (R-PAC).

Tityustoxin-K(alpha) blockade of the voltage-gated potassium channel Kv1.3.

1. We investigated the action of TsTX-Kalpha on cloned Kv1.3 channels of the Shaker subfamily of voltage-gated potassium channels, using the voltage-clamp technique. Highly purified TsTX-Kalpha was obtained from the venom of the Brazilian scorpion Tityus serrulatus using a new purification protocol. Our results show that TsTX-Kalpha blocks Kv1.3 with high affinity in two expression systems. 2. TsTX-Kalpha blockade of Kv1.3 channels expressed in Xenopus oocytes was found to be completely reversible and to exhibit a pH dependence. The K(D) was 3.9 nM at pH 7.5, 9.5 nM at pH 7.0 and 94.5 nM at pH 6.5. 3. The blocking properties of TsTX-Kalpha in a mammalian cell line (L929), stably transfected to express Kv1.3, were studied using the patch-clamp technique. In this preparation, the toxin had a K(D) of 19.8 nM at pH 7.4. 4. TsTX-Kalpha was found to affect neither the voltage-dependence of activation, nor the activation and deactivation time constants. The block appeared to be independent of the transmembrane voltage and the toxin did not interfere with the C-type inactivation process. 5. Taken as a whole, our findings indicate that TsTX-Kalpha acts as a simple blocker of Kv1.3 channels. It is concluded that this toxin is a useful tool for probing not only the physiological roles of Kv1.2, but also those mediated by Kv1.3 channels.

NR2B and NR2D Subunits Coassemble in Cerebellar Golgi Cells to Form a Distinct NMDA Receptor Subtype Restricted to Extrasynaptic Sites

NMDA receptors (NMDARs) are thought to be tetrameric assemblies composed of NR1 and at least one type of NR2 subunit. The identity of the NR2 subunit (NR2A, -B, -C, -D) is critical in determining many of the functional properties of the receptor, such as channel conductance and deactivation time. Further diversity may arise from coassembly of more than one type of NR2 subunit, if the resulting triheteromeric assembly (NR1 plus two types of NR2) displays distinct functional properties. We have used gene-ablated mice (NR2D -/-) to examine the effects of the NR2D subunit on NMDAR channels and NMDAR EPSCs in cerebellar Golgi cells. These cells are thought to express both NR2B and NR2D subunits, a combination that occurs widely in the developing nervous system. Our experiments provide direct evidence that the low conductance NMDAR channels in Golgi cells arise from diheteromeric NR1/NR2D assemblies. To investigate whether a functionally distinct triheteromeric assembly was also expressed, we analyzed the kinetic and pharmacological properties of single-channel currents in isolated extrasynaptic patches. We found that after the loss of the NR2D subunit, the properties of the 50 pS NMDAR channels were altered. This result is consistent with the presence of a triheteromeric assembly (NR1/NR2B/NR2D) in cells from wild-type mice. However, we could find no difference in the properties of NMDAR-mediated EPSCs between wild-type and NR2D subunit ablated mice. Our experiments suggest that although both diheteromeric and triheteromeric NR2D-containing receptors are expressed in cerebellar Golgi cells, neither receptor type participates in parallel fiber to Golgi cell synaptic transmission. The presence of the NR2D subunit within an assembly may therefore result in its restriction to extrasynaptic sites.

Stimulatory Action of Internal Protons on Slo1 BK Channels

We investigated the internal pH-sensitivity of heterologously expressed hSlo1 BK channels. In the virtual absence of Ca(2+) and Mg(2+) to isolate the voltage-dependent gating transitions, low internal pH enhanced macroscopic hSlo1 currents by shifting the voltage-dependence of activation to more negative voltages. The activation time course was faster and the deactivation time course was slower with low pH. The estimated K(d) value of the stimulatory effect was approximately pH = 6.5 or 0.35 micro M. The stimulatory effect was maintained when the auxiliary subunit mouse beta1 was coexpressed. Treatment of the hSlo1 channel with the histidine modifying agent diethyl pyrocarbonate also enhanced the hSlo1 currents and greatly diminished the internal pH sensitivity, suggesting that diethyl pyrocarbonate and low pH may work on the same effector mechanism. High concentrations of Ca(2+) or Mg(2+) also masked the stimulatory effect of low internal pH. These results indicate that the acid-sensitivity of the Slo BK channel may involve the channel domain implicated in the divalent-dependent activation.

Selective knockout of mouse ERG1 B potassium channel eliminates I(Kr) in adult ventricular myocytes and elicits episodes of abrupt sinus bradycardia.

The ERG1 gene encodes a family of potassium channels. Mutations in human ERG1 lead to defects in cardiac repolarization, referred to as the long QT syndrome. Through homologous recombination in mouse embryonic stem cells the ERG1 B potassium channel transcript was eliminated while the ERG1 A transcript was maintained. Heterologous expression of ERG1 isoforms had previously indicated that the deactivation time course of ERG1 B is 10-fold more rapid than that of ERG1 A. In day-18 fetal +/+ myocytes, I(Kr) exhibited two time constants of deactivation (3,933 +/- 404 and 350 +/- 19 ms at -50 mV), whereas in age-matched ERG1 B(-/-) mice the rapid component was absent. Biexponential deactivation rates (2,039 +/- 268 and 163 +/- 43 ms at -50 mV) were also observed in adult +/+ myocytes. In adult ERG1 B(-/-) myocytes no I(Kr) was detected. Electrocardiogram intervals were similar in +/+ and -/- mice. However, adult -/- mice manifested abrupt spontaneous episodes of sinus bradycardia (>100 ms of slowing) in 6 out of 21 mice. This phenomenon was never observed in +/+ mice (0 out of 16). We conclude that ERG1 B is necessary for I(Kr) expression in the surface membrane of adult myocytes. Knockout of ERG1 B predisposes mice to episodic sinus bradycardia.

Activation of muscarinic m5 receptors inhibits recombinant KCNQ2/KCNQ3 K + channels expressed in HEK293T cells

A variety of G-protein-coupled receptors regulate membrane excitability via M-type K + current (M-current) modulation. Muscarinic m1 and m3 acetylcholine receptors have both been implicated in the modulation of M-current. The muscarinic m5 receptor, like muscarinic m1 and m3 receptors, couples to phospholipase C via a pertussis toxin-insensitive G protein. Since a number of other receptors which activate phospholipase C also modulate M-current, we investigated if muscarinic m5 receptors could modulate recombinant M-type (KCNQ2/KCNQ3) K + channels after heterologous expression in human embryonic kidney (HEK) 293T cells. Application of Oxo-tremorine M to HEK293T cells expressing muscarinic m1, m3, or m5 receptors produced a similar robust inhibition of M-current, whereas muscarinic m2 and m4 receptor stimulation was without effect. Muscarinic m1, m3, or m5 receptor stimulation decreased the deactivation time constants of M-current at −50 mV. The inhibition of M-current by stimulation of muscarinic m1, m3, or m5 receptors was insensitive to overnight treatment with pertussis toxin or cholera toxin, which interfere with G i/o and G s G-protein signaling. These data suggest that muscarinic m1, m3, and m5 receptors inhibit M-channels via the activation of a common G protein.

Slow Deactivation and U-Shaped Inactivation Properties in Cloned Ca v1.2b Channels in Chinese Hamster Ovary Cells

Whole-cell patch-clamp techniques were applied to Chinese hamster ovary cells stably expressing cloned smooth muscle Ca 2+ channel α 1-subunits. In the presence of Ba 2+ as a charge carrier, U-shaped inactivation was observed in the presence and absence of Ca 2+ agonists. Also, tail currents deactivated slowly when conditioning steps of positive potential were applied. The deactivation time constant was decreased by hyperpolarizing the repolarization step. Application of ATP- γ-S or H-7 had little effect on the conditions necessary to induce slow tail, suggesting involvement of physical processes in the channel protein. In the presence of Bay K 8644, additional application of nifedipine decreased the amplitudes of the test and tail currents induced by a test step preceded by a conditioning step to +80 mV, but did not affect the decay time constant of the tail current. From these results and assumptions we have drawn up a kinetic scheme with one closed state, two open states ( O 1, O 2) and two inactivated states linked to the closed state and open state O 1, respectively, i.e., open state O 2 protected from inactivation. Computer calculation reconstructed slow deactivation and U-shaped inactivation properties. A similar kinetic scheme with Ca 2+-agonist-binding states accounted for the results in the presence of Ca 2+ agonists.

GDNF Acutely Potentiates Ca2+ Channels and Excitatory Synaptic Transmission in Midbrain Dopaminergic Neurons

Glial cell line-derived neurotrophic factor (GDNF) is best known for its long-term survival effect on dopaminergic neurons in the ventral midbrain. A recent study showed that acute application of GDNF to these neurons suppresses A-type potassium channels and potentiates neuronal excitability. Here we have characterized the acute effects of GDNF on Ca²⁺ channels and synaptic transmission. GDNF rapidly and reversibly potentiated the high voltage-activated (HVA) Ca²⁺ channel currents in cultured dopaminergic neurons. Analyses of channel kinetics indicate that GDNF decreased the activation time constant, increased the inactivation and deactivation time constants of HVA Ca²⁺ channel currents. Ca²⁺ imaging experiments demonstrate that GDNF facilitated Ca²⁺ influx induced by membrane depolarization. To investigate the physiological consequences of the Ca²⁺ channel modulation, we examined the acute effects of GDNF on excitatory synaptic transmission at synapses made by these dopaminergic neurons, which co-release the transmitter glutamate. Within 3 min of application, GDNF increased the amplitude of spontaneous and evoked excitatory autaptic- or multiple-postsynaptic currents. The frequency as well as the amplitude of miniature excitatory postsynaptic currents was also increased. These results reveal, for the first time, an acute effect of GDNF on synaptic transmission and its potential mechanisms, and suggest that an important function of GDNF for midbrain dopaminergic neurons is the acute modulation of transmission and ion channels.

Reduction in the Sodium Currents in Isolated Ventricular Myocytes of Guinea Pigs Treated by Chronic L‐Thyroxin Medication

Objective: Cardiac remodeling induced by chronic medication of L-thyroxin is manifested by a much more severe cardiac arrhythmias on the occlusion/reperfusion of the coronary artery in rats. A pattern of changes in ion currents in a diseased heart (L-thyroxin induced cardiac remodeling) is possibly provided as a basis of promoting malignant cardiac arrhythmias. An enhanced delayed outward rectifier potassium currents the rapid (IKr) and slow (IKS) component was found in the remodeled heart by L-thyroxin chronic medication. It is interested to investigate the changes in the sodium currents in the L-thyroxin remodeled guinea pig ventricle. Method: The remodeling model in guinea pig was developed by L-thyroxin 4 mg po for 10 days. On d 11, the heart was removed and perfused to isolate ventricular myocytes with medium of Ca2+ free medium containing collagen. The whole cell holding technique was applied. Results: The INa density in the L-thyroxin caused hypertrophied myocytes was reduced significantly at holding potential −30 mV, −53.20 +/−10.78pA/pF against −73.78+/−14.66pA/pF in the normal. (n = 45, p < 0.001). No difference in the steady-state inactivation and recovery kinetics between the remodeled and the normal was found. The recovery constant 37.54+/−3.63 ms in the remodeled vs 36.57+/−2.81 ms in the normal (n = 18, p > 0.05). The accelerated deactivation time constant 3.67+/−0.14 of the remodeled (n = 39) against the normal 4.14+/−0.15 ms (n = 43) (p < 0.05). Conclusion: There is a reduced INa in the L-thyroxin remodeled ventricular myocytes and the deactivation of the current is accelerated. A changed depolarization of the affected myocardium is likely involved in the mechanism of arrhythmogenesis of the remodeled ventricle.

A-type potassium currents active at subthreshold potentials in mouse cerebellar Purkinje cells.

Voltage-dependent and calcium-independent K+ currents were whole-cell recorded from cerebellar Purkinje cells in slices. Tetraethylammonium (TEA, 4 mM) application isolated an A-type K+ current (I(K(A))) with a peak amplitude, at +20 mV, of about one third of the total voltage-dependent and calcium-independent K+ current. The I(K(A)) activated at about -60 mV, had a V(0.5) of activation of -24.9 mV and a V(0.5) of inactivation of -69.2 mV. The deactivation time constant at -70 mV was 3.4 +/- 0.4 ms, while the activation time constant at +20 mV was 0.9 +/- 0.2 ms. The inactivation kinetics was weakly voltage dependent, with two time constants; those at +20 mV were 19.3 +/- 3.1 and 97.6 +/- 9.8 ms. The recovery from inactivation had two time constants of 60.8 ms (78.4 %) and 962.3 ms (21.6 %). The I(K(A)) was blocked by 4-aminopyridine with an IC50 of 67.6 microM. Agitoxin-2 (2 nM) blocked 17.4 +/- 2.1 % of the I(K(A)). Flecainide completely blocked the I(K(A)) with a biphasic effect with IC50 values of 4.4 and 183.2 microM. In current-clamp recordings the duration of evoked action potentials was affected neither by agitoxin-2 (2 nM) nor by flecainide (3 microM), but action potentials that were already broadened by TEA were further prolonged by 4-aminopyridine (100 microM). The amplitude of the hyperpolarisation at the end of depolarising steps was reduced by all these blockers.

Direct inhibition of the cloned Kv1.5 channel by AG-1478, a tyrosine kinase inhibitor.

The action of tyrphostin AG-1478, a potent protein tyrosine kinase (PTK) inhibitor, on rat brain Kv1.5 channels (Kv1.5) stably expressed in Chinese hamster ovary cells was investigated using the whole cell patch-clamp technique. AG-1478 rapidly and reversibly inhibited Kv1.5 currents at 50 mV in a concentration-dependent manner with an IC50 of 9.82 microM. AG-1478 accelerated the decay rate of inactivation of Kv1.5 currents without modifying the kinetics of current activation. Pretreatment with the structurally dissimilar PTK inhibitors (genistein and lavendustin A) had no effect on the AG-1478-induced inhibition of Kv1.5 and did not modify the AG-1478-induced current kinetics. The rate constants for binding and unbinding of AG-1478 were 1.46 microM(-1) x s(-1) and 10.19 s(-1), respectively. The AG-1478-induced inhibition of Kv1.5 channels was voltage dependent, with a steep increase over the voltage range of channel opening. However, the inhibition exhibited voltage independence over the voltage range in which channels are fully activated. AG-1478 produced no significant effect on the steady-state activation or inactivation curves. AG-1478 slowed the deactivation time course, resulting in a tail crossover phenomenon. Inhibition of Kv1.5 by AG-1478 was use dependent. The present results suggest that AG-1478 acts directly on Kv1.5 currents as an open-channel blocker and independently of the effects of AG-1478 on PTK activity.

An M-like outward current regulates the excitability of spinal motoneurones in the adult turtle.

The excitatory action of muscarine on spinal motoneurones was investigated with intracellular recordings in a slice preparation from adult turtles. In these cells muscarine is known to facilitate a persistent inward current mediated by L-type Ca(2+) channels. When this effect was blocked by nifedipine, muscarine still increased the excitability. In voltage clamp, a slowly activating outward current, generated during depolarizing voltage commands and deactivating as a tail current on return to the holding voltage, was reduced by muscarine. This outward current was activated when the voltage was stepped to potentials positive to -60 mV, was voltage sensitive and had a deactivation time constant of approximately 80 ms. These findings are compatible with an M-current. This possibility was also supported by the finding that the current was reduced by XE-991 - a selective blocker of the KCNQ potassium channels underlying M-currents in other cell types. Our findings suggest that an M-like current, mediated by a KCNQ channel, contributes to the intrinsic response properties of motoneurones in the adult spinal cord by increasing adaptation of repetitive firing and decreasing the slope of the frequency-current relation.

Roscovitine: a novel regulator of P/Q-type calcium channels and transmitter release in central neurons.

Roscovitine is widely used for inhibition of cdk5, a cyclin-dependent kinase expressed predominantly in the brain. A novel function of roscovitine, i.e. an effect on Ca(2+) channels and transmitter release in central neurons, was studied by whole-cell voltage-clamp recordings and time-lapse fluorescence imaging techniques. Extracellular application of roscovitine markedly enhanced the tail calcium current following repolarization from depolarized voltages. This effect was rapid, reversible and dose dependent. Roscovitine dramatically slowed the deactivation kinetics of calcium channels. The deactivation time constant was increased 3- to 6-fold, suggesting that roscovitine could prolong the channel open state and increase the calcium influx. The potentiation of tail calcium currents caused by roscovitine and by the L-channel activator Bay K 8644 was not occluded but additive. Roscovitine-induced potentiation of tail calcium currents was significantly blocked by the P/Q-channel blocker CgTx-MVIIC, indicating that the major target of roscovitine is the P/Q-type calcium channel. In mutant mice with targeted deletion of p35, a neuronal specific activator of cdk5, roscovitine regulated calcium currents in a manner similar to that observed in wild-type mice. Moreover, intracellular perfusion of roscovitine failed to modulate calcium currents. These results suggest that roscovitine acts on extracellular site(s) of calcium channels via a cdk5-independent mechanism. Roscovitine potentiated glutamate release at presynaptic terminals of cultured hippocampal neurons detected with the vesicle trafficking dye FM1-43, consistent with the positive effect of roscovitine on the P/Q-type calcium channel, the major mediator of action potential-evoked transmitter release in the mammalian CNS.

Differing Effects of Inotropic Agents on Length Change Deactivation of Isolated Rat Myocardium

BackgroundA rapid change in length of cardiac muscle during isometric contraction is followed by developed force that is less than appropriate for the new length because of deactivation of the...

Roscovitine: a novel regulator of P/Q-type calcium channels and transmitter release in central neurons

Roscovitine is widely used for inhibition of cdk5, a cyclin-dependent kinase expressed predominantly in the brain. A novel function of roscovitine, i.e. an effect on Ca2+ channels and transmitter release in central neurons, was studied by whole-cell voltage-clamp recordings and time-lapse fluorescence imaging techniques. Extracellular application of roscovitine markedly enhanced the tail calcium current following repolarization from depolarized voltages. This effect was rapid, reversible and dose dependent. Roscovitine dramatically slowed the deactivation kinetics of calcium channels. The deactivation time constant was increased 3- to 6-fold, suggesting that roscovitine could prolong the channel open state and increase the calcium influx. The potentiation of tail calcium currents caused by roscovitine and by the L-channel activator Bay K 8644 was not occluded but additive. Roscovitine-induced potentiation of tail calcium currents was significantly blocked by the P/Q-channel blocker CgTx-MVIIC, indicating that the major target of roscovitine is the P/Q-type calcium channel. In mutant mice with targeted deletion of p35, a neuronal specific activator of cdk5, roscovitine regulated calcium currents in a manner similar to that observed in wild-type mice. Moreover, intracellular perfusion of roscovitine failed to modulate calcium currents. These results suggest that roscovitine acts on extracellular site(s) of calcium channels via a cdk5-independent mechanism. Roscovitine potentiated glutamate release at presynaptic terminals of cultured hippocampal neurons detected with the vesicle trafficking dye FM1–43, consistent with the positive effect of roscovitine on the P/Q-type calcium channel, the major mediator of action potential-evoked transmitter release in the mammalian CNS.

Agonist Trapping by GABAAReceptor Channels

GABAergic IPSCs have a relatively slow decay (deactivation) that appears to result from GABA(A) receptor channel openings that occur well beyond the predicted duration of free GABA at central synapses. Open and desensitized states have been suggested to prevent dissociation of agonist from the receptor, thus prolonging deactivation. However, simultaneous assessment of GABA binding and channel gating has not been possible. We developed a functional assay for occupancy of the GABA binding site or sites to test the GABA "trapping" hypothesis. Deactivation currents were compared in the absence and presence of bicuculline, a competitive antagonist that also allosterically inhibits GABA(A) receptors. This provided a model-independent, functional test of the hypothesis that GABA is trapped on the receptor during gating: bicuculline could only inhibit the channel if it was open but unbound by GABA. Although bicuculline inhibited spontaneous and neurosteroid-activated GABA(A) receptor currents, it failed to alter the deactivation time course of GABA-activated GABA(A) receptor currents. Protection of deactivation current from bicuculline block indicated that GABA remained bound to the receptors while the channel was open, thus suggesting that all open states, as well as all closed and desensitized states from which channel opening can occur, must be GABA liganded states. Trapping may be specific to agonists, because the positive allosteric modulator diazepam unbound from GABA(A) receptors independent of GABA binding and channel activity.

Low threshold T-type calcium current in rat embryonic chromaffin cells.

1. The gating kinetics and functions of low threshold T-type current in cultured chromaffin cells from rats of 19-20 days gestation (E19-E20) were studied using the patch clamp technique. Exocytosis induced by calcium currents was monitored by the measurement of membrane capacitance and amperometry with a carbon fibre sensor. 2. In cells cultured for 1-4 days, the embryonic chromaffin cells were immunohistochemically identified by using polyclonal antibodies against dopamine beta-hydroxylase (DBH) and syntaxin. The immuno-positive cells could be separated into three types, based on the recorded calcium current properties. Type I cells showed exclusively large low threshold T-type current, Type II cells showed only high voltage activated (HVA) calcium channel current and Type III cells showed both T-type and HVA currents. These cells represented 44 %, 46 % and 10 % of the total, respectively. 3. T-type current recorded in Type I cells became detectable at -50 mV, reached its maximum amplitude of 6.8 +/- 1.2 pA pF(-1) (n = 5) at -10 mV and reversed around +50 mV. The current was characterized by criss-crossing kinetics within the -50 to -30 mV voltage range and a slow deactivation (deactivation time constant, tau(d) = 2 ms at -80 mV). The channel closing and inactivation process included both voltage-dependent and voltage-independent steps. The antihypertensive drug mibefradil (200 nM) reduced the current amplitude to about 65 % of control values. Ni(2+) also blocked the current in a dose-dependent manner with an IC(50) of 25 microM. 4. T-type current in Type I cells did not induce exocytosis, while catecholamine secretion by exocytosis could be induced by HVA calcium current in both Type II and Type III cells. The failure to induce exocytosis by T-type current in Type I cells was not due to insufficient Ca(2+) influx through the T-type calcium channel. 5. We suggest that T-type current is expressed in developing immature chromaffin cells. The T-type current is replaced progressively by HVA calcium current during pre- and post-natal development accompanying the functional maturation of the exocytosis mechanism.

Hidden Markov model analysis of intermediate gating steps associated with the pore gate of shaker potassium channels.

Cooperativity among the four subunits helps give rise to the remarkable voltage sensitivity of Shaker potassium channels, whose open probability changes tenfold for a 5-mV change in membrane potential. The cooperativity in these channels is thought to arise from a concerted structural transition as the final step in opening the channel. Recordings of single-channel ionic currents from certain other channel types, as well as our previous recordings from T442S mutant Shaker channels, however, display intermediate conductance levels in addition to the fully open and closed states. These sublevels might represent stepwise, rather than concerted, transitions in the final steps of channel activation. Here, we report a similar fine structure in the closing transitions of Shaker channels lacking the mutation. Describing the deactivation time course with hidden Markov models, we find that two subconductance levels are rapidly traversed during most closing transitions of chimeric, high conductance Shaker channels. The lifetimes of these levels are voltage-dependent, with maximal values of 52 and 22 micros at -100 mV, and the voltage dependences of transitions among these states suggest that they arise from equivalent conformational changes occurring in individual subunits. At least one subconductance level is found to be traversed in normal conductance Shaker channels. We speculate that voltage-dependent conformational changes in the subunits give rise to changes in a "pore gate" associated with the selectivity filter region of the channel, producing the subconductance states. As a control for the hidden Markov analysis, we applied the same procedures to recordings of the recovery from N-type inactivation in Shaker channels. These transitions are found to be instantaneous in comparison.

Functional diversity of recombinant human AMPA type glutamate receptors: possible implications for selective vulnerability of motor neurons

Lower motor neurons are known to be susceptible to glutamate-mediated cell damage via overstimulation of AMPA type glutamate receptors (GluR). The molecular basis of an important hypothesis in investigating amyotrophic lateral sclerosis (ALS) is glutamate-excitotoxicity. The aim of this study was to define desensitization and deactivation kinetics of recombinant human GluR1 and GluR2 receptor channels and their splice variants by means of patch-clamp experiments employing ultrafast solution exchange techniques. By this approach, the desensitization time constants of homooligomeric channels could be measured as τ Des=2.95±0.22 ms ( n=10) for GluR1flip, τ Des=3.17±0.19 ms ( n=10) for GluR1flop, τ Des=9.86±0.79 ms ( n=10) for GluR2flip, and τ Des=1.87±0.26 ms ( n=10) for GluR2flop, respectively. In the case of GluR1flip/flop and GluR2flop, a nondesensitising steady state current of less than 1% of peak current amplitude was observed, while GluR2flip channel currents showed a marked steady state component of about 10% of the maximum current. No significant differences were detected comparing the deactivation time course of GluR1 and GluR2 splice variants. These results suggest that the human GluR subtypes tested comprise no fundamental difference to their rodent analogous. Therefore, we describe a preparation that will be useful for further investigation of motor neuron physiological properties and a methodological approach allowing to study functional recombinant human GluR channels under reliable conditions.