Sodium channels have four homologous domains (D1-D4) each with six putative transmembrane segments (S1-$6). The highly charged $4 segments in each domain are postulated voltage sensors for gating. We made 15 charge-neutralizing or -reversing substitutions in the first or third basic residues (arginine or lysine) by replacement with histidine, glutamine, or glutamate in $4 segments of each domain of the human heart Na ÷ channel. Nine of the mutations cause shifts in the conductance-voltage (G-V) midpoints, and all but two significantly decrease the voltage dependence of peak Na + current, consistent with a role of $4 segments in activation. The decreases in voltage dependence of activation were equivalent to a decrease in apparent gating charge of 0.5-2.1 elementary charges (%) per channel for single charge-neutralizing mutations. Three charge-reversing mutations gave decreases of 1.2-1.9 eo per channel in voltage dependence of activation. The steady-state inactivation (h~) curves were fit by single-component Boltzmann functions and show significant decreases in slope for 9 of the 15 mutants and shifts of midpoints in 9 mutants. The voltage dependence of inactivation time constants is markedly decreased by mutations only in $4D4, providing further evidence that this segment plays a unique role in activation-inactivation coupling.
؊ ) to suppress current in Xenopus oocytes. The fragments include portions of the six putative transmembrane segments, S1 through S6, specifically: S1, S1-S2, S1-S2-S3, S2-S3, S2-S3-S4, S3-S4, S3-S4-S5, S2 through COOH, S3 through COOH, S4 through COOH, and S5-S6-COOH. Electrophysiologic experiments show that the fragments S1-S2-S3, S3-S4-S5, S2 through COOH, and S3 through COOH strongly suppress Kv1.3 (T1
TWIK-related acid-sensitive K(+) (K(2P) 9.1, TASK-3) ion channels have the capacity to regulate the activity of neuronal pathways by influencing the resting membrane potential of neurons on which they are expressed. The central nervous system (CNS) expression of these channels suggests potential roles in neurologic disorders, and it is believed that the development of TASK-3 antagonists could lead to the therapeutic treatment of a number of neurological conditions. While a therapeutic potential for TASK-3 channel modulation exists, there are only a few documented examples of potent and selective small-molecule channel blockers. Herein, we describe the discovery and lead optimization efforts for a novel series of TASK-3 channel antagonists based on a 5,6,7,8-tetrahydropyrido[4,3-d]pyrimidine high-throughput screening lead from which a subseries of potent and selective inhibitors were identified. One compound was profiled in detail with respect to its physical properties and demonstrated pharmacological target engagement as indicated by its ability to modulate sleep architecture in rodent electroencephalogram (EEG) telemetry models.
Voltage-gated sodium channels control the upstroke of the action potential in excitable cells of nerve and muscle tissue, making them ideal targets for exogenous toxins that aim to squelch electrical excitability. One such toxin, tetrodotoxin (TTX), blocks sodium channels with nanomolar affinity only when an aromatic Phe or Tyr residue is present at a specific location in the external vestibule of the ion-conducting pore. To test whether TTX is attracted to Tyr 401 of Na V 1.4 through a cation-interaction, this aromatic residue was replaced with fluorinated derivatives of Phe using in vivo nonsense suppression. Consistent with a cation-interaction, increased fluorination of Phe 401 , which reduces the negative electrostatic potential on the aromatic face, caused a monotonic increase in the inhibitory constant for block. Trifluorination of the aromatic ring decreased TTX affinity by ϳ50-fold, a reduction similar to that caused by replacement with the comparably hydrophobic residue Leu. Furthermore, we show that an energetically equivalent cation-interaction underlies both use-dependent and tonic block by TTX. Our results are supported by high level ab initio quantum mechanical calculations applied to a model of TTX binding to benzene. Our analysis suggests that the aromatic side chain faces the permeation pathway where it orients TTX optimally and interacts with permeant ions. These results are the first of their kind to show the incorporation of unnatural amino acids into a voltage-gated sodium channel and demonstrate that a cation-interaction is responsible for the obligate nature of an aromatic at this position in TTX-sensitive sodium channels.Sodium channels control electrical excitability in muscle and nervous tissues by driving membrane depolarization during an action potential. These large (ϳ260 kDa) proteins have four homologous transmembrane domains that are arranged in a clockwise orientation around a central pore (1). Numerous sodium channel isoforms exist and share the common traits of being exceptionally sensitive to voltage and selective for Na ϩ ions (2). They can, however, differ in expression patterns, inactivation time course, sensitivity to toxins, and effects of auxiliary subunits. In terms of extracellular block by the guanidinium toxin tetrodotoxin (TTX), 2 sodium channels have been deemed binary in their sensitivity; they are either TTX-sensitive, blocked by low nanomolar concentrations, or TTX-resistant with blocking constants in the micromolar range (3). Members of the former class include isoforms expressed in brain (Na V 1.1, Na V 1.2, and Na V 1.3), skeletal muscle (Na V 1.4), and peripheral nervous system (Na V 1.6 and Na V 1.7); the latter class includes isoforms of cardiac muscle (Na V 1.5) and sensory neurons (Na V 1.8 and Na V 1.9). A number of point mutations in the external vestibule of the channel can substantially disrupt TTX binding (4 -8), yet many of these residues are conserved between sensitive and resistant isoforms and therefore cannot explain isoform-specific TTX sensi...
Low-voltage-activated (T-type) calcium channels play a role in diverse physiological responses including neuronal burst firing, hormone secretion, and cell growth. To better understand the biological role and therapeutic potential of the target, a number of structurally diverse antagonists have been identified. Multiple drug interaction sites have been identified for L-type calcium channels, suggesting a similar possibility exists for the structurally related T-type channels. Here, we radiolabel a novel amide T-type calcium channel antagonist (TTA-A1) and show that several known antagonists, including mibefradil, flunarizine, and pimozide, displace binding in a concentration-dependent manner. Further, we identify a novel quinazolinone T-type antagonist (TTA-Q4) that enhanced amide radioligand binding, increased affinity in a saturable manner and slowed dissociation. Functional evaluation showed these compounds to be state-dependent antagonists which show a positive allosteric interaction. Consistent with slowing dissociation, the duration of efficacy was prolonged when compounds were co-administered to WAG/Rij rats, a genetic model of absence epilepsy. The development of a T-type calcium channel radioligand has been used to demonstrate structurally distinct TTAs interact at allosteric sites and to confirm the potential for synergistic inhibition of T-type calcium channels with structurally diverse antagonists.
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