Chung-Chin Kuo

Characterization of lamotrigine inhibition of Na+ channels in rat hippocampal neurones

Lamotrigine (LTG), a new antiepileptic drug, requires long depolarizations to inhibit Na+ currents. This suggests either slow binding of LTG to the fast inactivated state or selective binding of LTG to the slow inactivated state of Na+ channels. To differentiate between these possibilities and to characterize further the action of LTG, we studied the affinity and kinetics of LTG binding to the Na+ channels in acutely dissociated hippocampal neurones of the rat. LTG inhibited more Na+ currents at more depolarized holding potentials. The inhibitory effect at various holding potentials could be described by one‐to‐one binding curves, which yielded an apparent dissociation constant of ∼7μm for LTG binding to the inactivated channels (KI), and a dissociation constant more than 200 times larger for LTG binding to the resting channels. A similar value of KI (∼9μm) was also derived from the LTG concentration‐dependent shift of the inactivation curve. The recovery of LTG‐bound inactivated Na+ channels was faster than the recovery of normal (drug‐free) slow inactivated channels. Moreover, the binding kinetics of LTG onto the inactivated channels were faster than the development of the slow inactivated state, and were linearly correlated with LTG concentrations, with a binding rate constant of ∼10,000M−1s−1. These findings suggest that LTG chiefly binds to the fast inactivated state rather than the slow inactivated state. We conclude that LTG, in therapeutic concentrations and at relatively depolarized membrane potentials, may potently inhibit Na+ currents by slow binding to the fast inactivated state of Na+ channels. Like phenytoin, the slow binding rates may explain why LTG effectively inhibits seizure discharges, yet spares most normal neuronal activities.

Ion permeation through the L-type Ca2+ channel in rat phaeochromocytoma cells: two sets of ion binding sites in the pore.

1. Both inward and outward unitary Li+ currents through the L‐type Ca2+ channel and discrete block of such currents by either internal or external Ca2+ are recorded. Detailed kinetic analyses are obtained for all of the four experimental configurations (internal or external Ca2+ block of either inward or outward Li+ currents). 2. No matter from which side the blocking Ca2+ ion comes, the exit (unblocking) rates are always the same at the same potential in the same direction of Li+ current flow. This indicates that the high‐affinity Ca2+ binding site (the blocking site) is in the pore, and internal and external Ca2+ both go to the same site to produce the block. In other words, there can only be one high‐affinity Ca2+ binding site or one set of such sites (sites separated by insignificant barriers) in the pore. Furthermore, the direction of exit of the blocking Ca2+ ion is always with, not against, the Li+ current flow. This suggests ion‐ion interaction (the ‘long pore effect’) in the high‐affinity sites. Therefore there must be more than one high‐affinity site in the pore. Overall it is concluded that the pore must contain a set of high‐affinity Ca2+ binding sites separated by insignificant energy barriers. 3. The voltage dependence of the off‐ (exit) rates is very similar in amplitude for all the four experimental configurations (e‐fold change per approximately 25 mV depolarization or hyperpolarization). This strong voltage dependence in every configuration cannot be explained by any Ca2+ energy profile alone and must include a certain contribution from Li+. The mechanism underlying such a contribution seems to reside in the enhancement effect of Li+ on the exist of Ca2+. 4. The on‐rates (blocking rates) for external Ca2+ are always fast no matter whether the Li+ currents are outward or inward. In certain cases the rates even approach the diffusion‐controlled limit (approximately 10(9) M‐1 S‐1). This suggests that the high‐affinity sites are very easily accessible from the outside, and probably there is no other ionic site located between the external pore mouth and the high‐affinity sites. 5. The on‐rates for internal Ca2+ are fast and voltage independent in outward Li+ currents, but are very slow and strongly voltage dependent in inward Li+ currents.(ABSTRACT TRUNCATED AT 400 WORDS)

Characterization of the high‐affinity Ca2+ binding sites in the L‐type Ca2+ channel pore in rat phaeochromocytoma cells.

1. Two major types of ion‐ion interaction are demonstrated in the pore of the L‐type Ca2+ channel, the ‘lock‐in’ and the ‘enhancement’ effect. The former denotes that the ion at a certain site in the pore cannot move if the neighbouring site is occupied by the other ion. The latter denotes that the ion occupying a certain site may facilitate the exist of the other ion in the neighbouring site. 2. With inward currents carried by 300 mM external Li+, the exit rates of the blocking Ca2+ ion are decreased by approximately 4 times when the internal Li+ concentration is increased from 55 to 300 mM. With outward currents carried by 300 mM internal Li+, the exist rates of the blocking Ca2+ ion are decreased by approximately 2.5 times when the external Li+ concentration is increased from 55 to 300 mM. These findings demonstrate the ‘lock‐in’ effect. 3. When inward currents are carried in Li+ in the cell‐attached configuration, the on‐rates of the external Ca2+ are decreased in a rectangular hyperbolic fashion with increasing external Li+ concentration (apparent dissociation constant (Kd) approximately 75 mM in activity), suggesting competition between Ca2+ and Li+ for a certain site. On the other hand, the off‐rates of the blocking Ca2+ ion are increased linearly with increasing external Li+ concentration between 75 and 850 mM, and the line extrapolates to the zero point, indicating that Ca2+ exist is negligible at zero Li+. This finding demonstrates not only the existence of the enhancement effect in the channel, but also the indispensability of such an effect for Ca2+ to exit the pore. Moreover, a linear relationship up to 850 mM Li+ suggests that the affinity of Li+ to the enhancement sites is very low (apparent Kd very high) when a Ca2+ ion is present in the neighbouring site. 4. The unitary conductances of inward Ba2+ currents in the cell‐attached configuration are increased with increasing Ba2+ concentration. The apparent Kd obtained from a rectangular hyperbolic fit to the data is approximately 6 mM Ba2+ (in activity). When inward Ba2+ currents are blocked by Cd2+, the on‐rates of Cd2+ are decreased with increasing Ba2+ concentration also in a rectangular hyperbolic fashion, and the apparent Kd is approximately 5.5 mM. The similar Kd from these two different experiments suggests the high‐affinity set can accommodate no more than two Ba2+ ions.(ABSTRACT TRUNCATED AT 400 WORDS)

The Activation Gate and Gating Mechanism of the NMDA Receptor

The NMDA receptor opens in response to binding of NMDA and glycine. However, it remains unclear where and how gating of the NMDA receptor pore is accomplished. We show that different point mutations between S645 and I655 (thus including the highly conserved SYTANLAAF motif) of M3c in NR2B lead to constitutively open channels. The current through these constitutively open channels are readily blocked by external Mg2+ and MK-801 [(+)-5-methyl-10,11-dihydro-5H-dibenzo [a,d] cyclohepten-5,10-imine maleate]. Also, the open-channel blocker MK-801 can no longer be trapped in these channels when NMDA and glycine are washed off. Moreover, M3c residues at or below A651(NR2B, A7 in SYTANLAAF) react with external methanethiosulfonate (MTS) reagents ∼500 to 1000-fold faster in the presence than in the absence of agonists NMDA and glycine. In fact, the MTS modification rate shows exactly the same NMDA concentration dependence as channel activation. In contrast, those residues external to A651 are always modified with similar kinetics whether NMDA and glycine are present or not. Interestingly, MTS modification of A651C(NR2B) holds the channel constitutively open. Mutations of A651(NR2B) into arginine, tryptophan, or phenylalanine, and similar mutations of the corresponding A652 in NR1 also lead to constitutively open channels. Double-mutant cycle analysis further shows that the effects of A652(NR1) and A651(NR2B) mutations are evidently non-additive (i.e., cooperative) if mutated into residues with large side chains or with compensatory charges [e.g., A652E(NR1)+A651R(NR2B)]. The side chain of A7 thus plays a determinant role in the intersubunit distance at this level, which is directly responsible for the activation gate and activation–deactivation gating of the NMDA receptor.

Block of the L‐type Ca2+ channel pore by external and internal Mg2+ in rat phaeochromocytoma cells.

1. Three to eight micromolar external Mg2+ produces discrete block of the unitary inward currents through the L‐type Ca2+ channel carried by 300 mM external Li+. Like the Ca2+ block, increasing Li+ concentration decreases the Mg2+ on‐rate and increases the Mg2+ off‐rate. 2. These kinetic changes are saturating and the apparent dissociation constant (Kd) for the on‐rates in 75 mM Li+ (in activity), the same as that in the case of Ca2+ block. This suggests that Mg2+ and Ca2+ produce the discrete block at the same site. The apparent Kd for the off‐rates is 300 mM, much smaller than that in the case of Ca2+ block. This indicates that Mg2+ exerts much less repulsion on the Li+ ion in the neighbouring (enhancement) site than Ca2+, although Mg2+ and Ca2+ both have two charges. The theoretical fits to the off‐rates also suggest that Mg2+ can exit the blocking sites at a rate of several hundred per second in the absence of any enhancement effect. 3. Seventeen to forty‐eight micromolar internal Mg2+ produces discrete block of the outward unitary currents carried by 300 mM internal Li+. The off‐rates are in general approximately 20 times faster as compared to the Mg2+ off‐rates in the inward currents. This finding suggests that Mg2+ in the high‐affinity sites can much more easily exit to the outside than to the inside, implying significantly higher energy barriers on the inner side of the high‐affinity sites for Mg2+. 4. At least 5‐10 mM internal Mg2+ is needed to produce discrete block of the inward unitary currents carried by 215 mM external Na+. The off‐rates in such experiments are generally the same as those in the case of external Mg2+ block of inward currents. This suggests that internal and external Mg2+ both reach the same site, namely the high‐affinity Ca2+ binding sites in the pore, to produce the discrete block. 5. Other than discrete block, 5‐10 mM internal Mg2+ also decreases the size of the inward unitary current. This is most probably due to a fast block at the more internally located low‐affinity sites in the pore. The fractional decrease of the currents is voltage dependent and can be fitted by a rectangular hyperbola to calculate the apparent Kd, which increases e‐fold per 45 mV hyperpolarization, indicating an electrical distance of 0.3 between the low‐affinity sites and the internal pore mouth.(ABSTRACT TRUNCATED AT 400 WORDS)

Modulation of subthalamic T-type Ca(2+) channels remedies locomotor deficits in a rat model of Parkinson disease.

An increase in neuronal burst activities in the subthalamic nucleus (STN) is a well-documented electrophysiological feature of Parkinson disease (PD). However, the causal relationship between subthalamic bursts and PD symptoms and the ionic mechanisms underlying the bursts remain to be established. Here, we have shown that T-type Ca(2+) channels are necessary for subthalamic burst firing and that pharmacological blockade of T-type Ca(2+) channels reduces motor deficits in a rat model of PD. Ni(2+), mibefradil, NNC 55-0396, and efonidipine, which inhibited T-type Ca(2+) currents in acutely dissociated STN neurons, but not Cd(2+) and nifedipine, which preferentially inhibited L-type or the other non–T-type Ca(2+) currents, effectively diminished burst activity in STN slices. Topical administration of inhibitors of T-type Ca(2+) channels decreased in vivo STN burst activity and dramatically reduced the locomotor deficits in a rat model of PD. Cd(2+) and nifedipine showed no such electrophysiological and behavioral effects. While low-frequency deep brain stimulation (DBS) has been considered ineffective in PD, we found that lengthening the duration of the low-frequency depolarizing pulse effectively improved behavioral measures of locomotion in the rat model of PD, presumably by decreasing the availability of T-type Ca(2+) channels. We therefore conclude that modulation of subthalamic T-type Ca(2+) currents and consequent burst discharges may provide new strategies for the treatment of PD.

Postnatal development of cortical acetylcholinesterase-rich neurons in the rat brain: permanent and transient patterns.

The development of acetylcholinesterase (AChE) activity within cortical neurons of the rat brain was investigated using a histochemical method. The fate of these neurons in later stages of development was studied in animals in which AChE within cortical axons (mostly cholinergic) had been depleted by lesions of the cholinergic neurons of the basal forebrain or by injections of diisopropyl fluorophosphate. We designated neurons with medium to high intensity of reaction product as AChEH and neurons with a low intensity of reaction product as AChEL. Four groups of AChEH cortical neurons were detected: (1) AChEH Cajal-Retzius cells were present in layer I at birth (P0) and decreased steadily in number until none could be detected at P17 or thereafter. (2) AChEH neurons within layer VI and underlying white matter were present at P0, peaked in number and staining intensity at P8-P9, showed a moderate decrease in number at P11-P13 and a further decrease into adulthood. (3) AChEH polymorphic intracortical neurons appeared at P3-P4 in deep cortical layers and by P9 were present in layers II-VI. They continued to increase in number through P11-P14 at which time they displayed the adult pattern and were found in all cortical areas. (4) A large population of AChEH pyramidal neurons appeared at P1-P4, peaked at P8-P10 and was no longer visible at P21. In the adult cerebral cortex, few pyramidal neurons displayed AChE activity and these were almost always of the AChEL type. These results indicate that the AChE within cortical neurons is developmentally regulated and that the content of this enzyme helps to differentiate cortical neurons into distinct populations. The transient expression of AChE activity within cortical neurons suggests a role for this enzyme in the development of the cerebral cortex.

Block of tetrodotoxin-resistant Na+ channel pore by multivalent cations: gating modification and Na+ flow dependence.

Tetrodotoxin-resistant (TTX-R) Na+ channels are much less susceptible to external TTX but more susceptible to external Cd2+ block than tetrodotoxin-sensitive (TTX-S) Na+ channels. Both TTX and Cd2+ seem to block the channel near the “DEKA” ring, which is probably part of a multi-ion single-file region adjacent to the external pore mouth and is involved in the selectivity filter of the channel. In this study we demonstrate that other multivalent transitional metal ions such as La3+, Zn2+, Ni2+, Co2+, and Mn2+ also block the TTX-R channels in dorsal root ganglion neurons. Just like Cd2+, the blocking effect has little intrinsic voltage dependence, but is profoundly influenced by Na+ flow. The apparent dissociation constants of the blocking ions are always significantly smaller in inward Na+ currents than those in outward Na+ current, signaling exit of the blocker along with the Na+ flow and a high internal energy barrier for “permeation” of these multivalent blocking ions through the pore. Most interestingly, the activation and especially the inactivation kinetics are slowed by the blocking ions. Moreover, the gating changes induced by the same concentration of a blocking ion are evidently different in different directions of Na+ current flow, but can always be correlated with the extent of pore block. Further quantitative analyses indicate that the apparent slowing of channel activation is chiefly ascribable to Na+ flow–dependent unblocking of the bound La3+ from the open Na+ channel, whereas channel inactivation cannot happen with any discernible speed in the La3+-blocked channel. Thus, the selectivity filter of Na+ channel is probably contiguous to a single-file multi-ion region at the external pore mouth, a region itself being nonselective in terms of significant binding of different multivalent cations. This region is “open” to the external solution even if the channel is “closed” (“deactivated”), but undergoes imperative conformational changes during the gating (especially the inactivation) process of the channel.

Recovery from Inactivation of T-Type Ca2+ Channels in Rat Thalamic Neurons

We studied the gating kinetics, especially the kinetics of recovery from inactivation, of T-type Ca2+ channels (T-channels) in thalamic neurons. The recovery course is associated with no discernible Ca2+ current and is characterized by an initial delay, as well as a subsequent exponential phase. These findings are qualitatively similar to previous observations on neuronal Na+ channels and suggest that T-channels also must deactivate to recover from inactivation. In contrast to Na+ channels in which both the delay and the time constant of the exponential phase are shortened with increasing hyperpolarization, in T-channels the time constant of the exponential recovery phase remains unchanged between −100 and −200 mV, although the initial delay is still shortened e-fold per 43 mV hyperpolarization over the same voltage range. The deactivating kinetics of tail T-currents also show a similar voltage dependence between −90 and −170 mV. According to the hinged-lid model of fast inactivation, these findings suggest that the affinity difference between inactivating peptide binding to the activated channel and binding to the fully deactivated channel is much smaller in T-channels than in Na+ channels. Moreover, the inactivating peptide in T-channels seems to have much slower binding and unbinding kinetics, and the unbinding rates probably remain unchanged once the inactivated T-channel has gone through the initial steps of deactivation and “closes” the pore (with the activation gate). T-channels thus might have a more rigid hinge and a more abrupt conformational change in the inactivation machinery associated with opening and closing of the pore.

A functional view of the entrances of L-type Ca2+ channels: estimates of the size and surface potential at the pore mouths

At extreme membrane potentials, the unitary inward and outward currents through L-type Ca2+ channels become diffusion controlled and saturate. The magnitudes of these currents indicate that the pore entrances are asymmetric, with the external mouth being much larger than the internal one. On the other hand, negative surface potentials at the two ends of the pore are rather similar. Both would be significant only when the ambient ionic strength is 110 mM or less. We conclude that the surface charges will not help much in concentrating the channels favorite divalent cations in the physiological condition. However, the pore does possess an external mouth large enough to make the important inward Ca2+ flow not limited by diffusion, even with only 1 mM external Ca2+.