Ya-Chin Yang

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.

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.

Lidocaine, Carbamazepine, and Imipramine Have Partially Overlapping Binding Sites and Additive Inhibitory Effect on Neuronal Na+Channels

Background:Despite the structural differences, local anesthetics, anticonvulsants, and tricyclic antidepressants exert similar use-dependent actions against voltage-gated Na+ channels, which may be contributory to pain control. The authors explore whether these drugs could doubly occupy the channel and exert synergic clinical effect. Methods:The authors performed electrophysiologic recordings and quantitative analyses in mutant and native neuronal Na+ channels to investigate molecular interactions between different drugs. Results:The authors demonstrate significant interactions between F1764 and W1716, two residues reported for local anesthetic binding, indicating uncertainties to conclude a common drug-binding site by mutation data. Therefore, the authors performed detailed functional studies in native neurons. Quantitative analyses of the inactivation curve shift argue against effective double occupancy of different drugs. For example, the shift of 20.9 ± 1.3 mV in the simultaneous presence of 10 &mgr;m imipramine, 100 &mgr;m lidocaine, and 100 &mgr;m phenytoin is consistent with the one-site (21.5 mV) rather than the two-site (30.5-33.8 mV) or three-site (42.7 mV) predictions. However, there is a deviation from the recovery courses predicted by one site if lidocaine or imipramine coexists with anticonvulsants. Moreover, gating state dependence of macroscopic-binding rates markedly differs between imipramine and carbamazepine. Conclusions:Carbamazepine, lidocaine, and imipramine bind to a common site with the common aromatic motif. External to the aromatic site, there is another weaker and less gating-dependent site for the tertiary amine chain in the latter two drugs. Concomitant clinical use of these drugs, thus, should have at most a simple additive but not a synergistic inhibitory action on Na+ currents.

The external pore loop interacts with S6 and S3-S4 linker in domain 4 to assume an essential role in gating control and anticonvulsant action in the Na+ channel

Carbamazepine, phenytoin, and lamotrigine are widely prescribed anticonvulsants in neurological clinics. These drugs bind to the same receptor site, probably with the diphenyl motif in their structure, to inhibit the Na+ channel. However, the location of the drug receptor remains controversial. In this study, we demonstrate close proximity and potential interaction between an external aromatic residue (W1716 in the external pore loop) and an internal aromatic residue (F1764 in the pore-lining part of the sixth transmembrane segment, S6) of domain 4 (D4), both being closely related to anticonvulsant and/or local anesthetic binding to the Na+ channel. Double-mutant cycle analysis reveals significant cooperativity between the two phenyl residues for anticonvulsant binding. Concomitant F1764C mutation evidently decreases the susceptibility of W1716C to external Cd2+ and membrane-impermeable methanethiosulfonate reagents. Also, the W1716E/F1764R and G1715E/F1764R double mutations significantly alter the selectivity for Na+ over K+ and markedly shift the activation curve, respectively. W1716 and F1764 therefore very likely form a link connecting the outer and inner compartments of the Na+ channel pore (in addition to the selectivity filter). Anticonvulsants and local anesthetics may well traverse this “S6 recess” without trespassing on the selectivity filter. Furthermore, we found that Y1618K, a point mutation in the S3-4 linker (the extracellular extension of D4S4), significantly alters the consequences of carbamazepine binding to the Na+ channel. The effect of Y1618K mutation, however, is abolished by concomitant point mutations in the vicinity of Y1618, but not by those in the internally located inactivation machinery, supporting a direct local rather than a long-range allosteric action. Moreover, Y1618 could interact with D4 pore residues W1716 and L1719 to have a profound effect on both channel gating and anticonvulsant action. We conclude that there are direct interactions among the external S3-4 linker, the external pore loop, and the internal S6 segment in D4, making the external pore loop a pivotal point critically coordinating ion permeation, gating, and anticonvulsant binding in the Na+ channel.

Ionic flow enhances low-affinity binding: a revised mechanistic view into Mg2+ block of NMDA receptors

The N‐methyl‐d‐aspartate receptor (NMDAR) channel is one of the major excitatory amino acid receptors in the mammalian brain. Since external Mg2+ blocks the channel in an apparently voltage‐dependent fashion, this ligand‐gated channel displays intriguing voltage‐dependent control of Na+ and Ca2+ permeability and thus plays an important role in synaptic physiology. We found that the essential features of Mg2+ block could not be solely envisaged by binding of a charged blocker in the membrane electric field. Instead, the blocking effect of Mg2+ is critically regulated by, and quantitatively correlated with, the relative tendency of outward and inward ionic fluxes. The ‘intrinsic’ affinity of Mg2+ to the binding sites, however, is low (in the millimolar range) in the absence of net ionic flow at 0 mV. Besides, extracellular and intracellular Mg2+ blocks the channel at distinct sites of electrical distances ∼0.7 and ∼0.95 from the outside, respectively. The two sites are separated by a high energy barrier for the movement of Mg2+ (but not Na+ or the other ions), and functionally speaking, each could accommodate ∼1.1 and ∼0.8 coexisting permeating ions, respectively. Mg2+ block of the ionic flow thus is greatly facilitated by the flux‐coupling effect or the ionic flow (the preponderant direction of permeant ion movement) per se, as if the poorly permeable Mg2+ is ‘pushed’ against a high energy barrier by the otherwise permeating ions. Extracellular and intracellular Mg2+ block then is in essence ‘use dependent’, more strongly inhibiting both Na+ and Ca2+ fluxes with stronger tendencies of influx and efflux, respectively. In conclusion, although permeant ions themselves could compete with Mg2+, the flow or the tendency of movement of the permeant ions may actually enhance rather than interfere with Mg2+ block, making the unique current–voltage relationship of NMDAR and the molecular basis of many important neurobiological phenomena.

The T-type calcium channel as a new therapeutic target for Parkinson’s disease

Parkinson’s disease (PD) is one of the most prevalent movement disorder caused by degeneration of the dopaminergic neurons in substantia nigra pars compacta. Deep brain stimulation (DBS) at the subthalamic nucleus (STN) has been a new and effective treatment of PD. It is interesting how a neurological disorder caused by the deficiency of a specific chemical substance (i.e., dopamine) from one site could be so successfully treated by a pure physical maneuver (i.e., DBS) at another site. STN neurons could discharge in the single-spike or the burst modes. A significant increase in STN burst discharges has been unequivocally observed in dopamine-deprived conditions such as PD, and was recently shown to have a direct causal relation with parkinsonian symptoms. The occurrence of burst discharges in STN requires enough available T-type Ca2+ currents, which could bring the relatively negative membrane potential to the threshold of firing Na+ spikes. DBS, by injection of negative currents into the extracellular space, most likely would depolarize the STN neuron and then inactivate the T-type Ca2+ channel. Burst discharges are thus decreased and parkinsonian locomotor deficits ameliorated. Conversely, injection of positive currents into STN itself could induce parkinsonian locomotor deficits in animals without dopaminergic lesions. Local application of T-type Ca2+ channel blockers into STN would also dramatically decrease the burst discharges and improve parkinsonian locomotor symptoms. Notably, zonisamide, which could inhibit T-type Ca2+ currents in STN, has been shown to benefit PD patients in a clinical trial. From the pathophysiological perspectives, PD can be viewed as a prototypical disorder of “brain arrhythmias”. Modulation of relevant ion channels by physical or chemical maneuvers may be important therapeutic considerations for PD and other diseases related to deranged neural rhythms.

An Inactivation Stabilizer of the Na+ Channel Acts as an Opportunistic Pore Blocker Modulated by External Na+

The Na+ channel is the primary target of anticonvulsants carbamazepine, phenytoin, and lamotrigine. These drugs modify Na+ channel gating as they have much higher binding affinity to the inactivated state than to the resting state of the channel. It has been proposed that these drugs bind to the Na+ channel pore with a common diphenyl structural motif. Diclofenac is a widely prescribed anti-inflammatory agent that has a similar diphenyl motif in its structure. In this study, we found that diclofenac modifies Na+ channel gating in a way similar to the foregoing anticonvulsants. The dissociation constants of diclofenac binding to the resting, activated, and inactivated Na+ channels are ∼880 μM, ∼88 μM, and ∼7 μM, respectively. The changing affinity well depicts the gradual shaping of a use-dependent receptor along the gating process. Most interestingly, diclofenac does not show the pore-blocking effect of carbamazepine on the Na+ channel when the external solution contains 150 mM Na+, but is turned into an effective Na+ channel pore blocker if the extracellular solution contains no Na+. In contrast, internal Na+ has only negligible effect on the functional consequences of diclofenac binding. Diclofenac thus acts as an “opportunistic” pore blocker modulated by external but not internal Na+, indicating that the diclofenac binding site is located at the junction of a widened part and an acutely narrowed part of the ion conduction pathway, and faces the extracellular rather than the intracellular solution. The diclofenac binding site thus is most likely located at the external pore mouth, and undergoes delicate conformational changes modulated by external Na+ along the gating process of the Na+ channel.

Subthalamic discharges as a causal determinant of parkinsonian motor deficits.

We have reported that intrinsic membrane properties, especially T‐type Ca2+ channels, play a key role in the genesis of burst discharges in the subthalamic nucleus (STN) and parkinsonian locomotor symptoms. Whether deep brain stimulation (DBS) exerts its clinical benefits on Parkinson disease (PD) with changes in T currents or other conductances, however, remains elusive.

Thalamic synaptic transmission of sensory information modulated by synergistic interaction of adenosine and serotonin

The thalamic synapses relay peripheral sensory information to the cortex, and constitute an important part of the thalamocortical network that generates oscillatory activities responsible for different vigilance (sleep and wakefulness) states. However, the modulation of thalamic synaptic transmission by potential sleep regulators, especially by combination of regulators in physiological scenarios, is not fully characterized. We found that somnogen adenosine itself acts similar to wake‐promoting serotonin, both decreasing synaptic strength as well as short‐term depression, at the retinothalamic synapse. We then combined the two modulators considering the coexistence of them in the hypnagogic (sleep‐onset) state. Adenosine plus serotonin results in robust synergistic inhibition of synaptic strength and dramatic transformation of short‐term synaptic depression to facilitation. These synaptic effects are not achievable with a single modulator, and are consistent with a high signal‐to‐noise ratio but a low level of signal transmission through the thalamus appropriate for slow‐wave sleep. This study for the first time demonstrates that the sleep‐regulatory modulators may work differently when present in combination than present singly in terms of shaping information flow in the thalamocortical network. The major synaptic characters such as the strength and short‐term plasticity can be profoundly altered by combination of modulators based on physiological considerations.

Functional Extension of Amino Acid Triads from the Fourth Transmembrane Segment (S4) into Its External Linker in Shaker K+ Channels

Background: The S4 voltage sensor in voltage-gated channels comprises triads of amino acids. Results: The microenvironment of the S3–4 linker is also arranged in similar triads. Conclusion: S4 initially moves in a canal following the S3–4 linker but may subsequently take a more liberal move like a paddle. Significance: This provides a novel synthesis reconciling the currently controversial models of S4 movement. The highly conserved fourth transmembrane segment (S4) is the primary voltage sensor of the voltage-dependent channel and would move outward upon membrane depolarization. S4 comprises repetitive amino acid triads, each containing one basic (presumably charged and voltage-sensing) followed by two hydrophobic residues. We showed that the triad organization is functionally extended into the S3–4 linker right external to S4 in Shaker K+ channels. The arginine (and lysine) substitutes for the third and the sixth residues (Ala-359 and Met-356, respectively) external to the outmost basic residue (Arg-362) in S4 dramatically and additively stabilize S4 in the resting conformation. Also, Leu-361 and Leu-358 play a very similar role in stabilization of S4 in the resting position, presumably by their hydrophobic side chains. Moreover, the double mutation A359R/E283A leads to a partially extruded position of S4 and consequently prominent closed-state inactivation, suggesting that Glu-283 in S2 may coordinate with the arginines in the extruded S4 upon depolarization. We conclude that the triad organization extends into the S3–4 linker for about six amino acids in terms of their microenvironment. These approximately six residues should retain the same helical structure as S4, and their microenvironment serves as part of the “gating canal” accommodating the extruding S4. Upon depolarization, S4 most likely moves initially as a sliding helix and follows the path that is set by the approximately six residues in the S3–4 linker in the resting state, whereas further S4 translocation could be more like, for example, a paddle, without orderly coordination from the contiguous surroundings.