Search Results (9)

KB-R7943, an isothiourea derivative, has been recognized as an inhibitor in the reverse mode of the Na⁺-Ca²⁺ exchanging process. This compound was demonstrated to prevent intracellular Na⁺-dependent Ca²⁺ uptake in intact cells; however, it is much less effective at preventing extracellular Na⁺-dependent Ca²⁺ efflux. Therefore, whether or how this compound may produce any perturbations on other types of ionic currents, particularly on voltage-gated Na⁺ current (I_Na), needs to be further studied. In this study, the whole-cell current recordings demonstrated that upon abrupt depolarization in pituitary GH₃ cells, the exposure to KB-R7943 concentration-dependently depressed the transient (I_Na(T)) or late component (I_Na(L)) of I_Na with an IC₅₀ value of 11 or 0.9 μM, respectively. Likewise, the dissociation constant for the KB-R7943-mediated block of I_Na on the basis of a minimum reaction scheme was estimated to be 0.97 μM. The presence of benzamil or amiloride could suppress the I_Na(L) magnitude. The instantaneous window Na⁺ current (I_Na(W)) activated by abrupt ascending ramp voltage (V_ramp) was suppressed by adding KB-R7943; however, subsequent addition of deltamethrin or tefluthrin (Tef) effectively reversed KB-R7943-inhibted I_Na(W). With prolonged duration of depolarizing pulses, the I_Na(L) amplitude became exponentially decreased; moreover, KB-R7943 diminished I_Na(L) magnitude. The resurgent Na⁺ current (I_Na(R)) evoked by a repolarizing V_ramp was also suppressed by adding this compound; moreover, subsequent addition of ranolazine or Tef further diminished or reversed, respectively, its reduction in I_Na(R) magnitude. The persistent Na⁺ current (I_Na(P)) activated by sinusoidal voltage waveform became enhanced by Tef; however, subsequent application of KB-R7943 counteracted Tef-stimulated I_Na(P). The docking prediction reflected that there seem to be molecular interactions of this molecule with the hNa_V1.2 or hNa_V1.7 channels. Collectively, this study highlights evidence showing that KB-R7943 has the propensity to perturb the magnitude and gating kinetics of I_Na (e.g., I_Na(T), I_Na(L), I_Na(W), I_Na(R), and I_Na(P)) and that the Na_V channels appear to be important targets for the in vivo actions of KB-R7943 or other relevant compounds. Full article

Picaridin (icaridin), a member of the piperidine chemical family, is a broad-spectrum arthropod repellent. Its actions have been largely thought to be due to its interaction with odorant receptor proteins. However, to our knowledge, to what extent the presence of picaridin can modify the magnitude, gating, and/or the strength of voltage-dependent hysteresis (Hys_(V)) of plasmalemmal ionic currents, such as, voltage-gated Na⁺ current [I_Na], has not been entirely explored. In GH₃ pituitary tumor cells, we demonstrated that with exposure to picaridin the transient (I_Na(T)) and late (I_Na(L)) components of voltage-gated Na⁺ current (I_Na) were differentially stimulated with effective EC₅₀’s of 32.7 and 2.8 μM, respectively. Upon cell exposure to it, the steady-state current versus voltage relationship I_Na(T) was shifted to more hyperpolarized potentials. Moreover, its presence caused a rightward shift in the midpoint for the steady-state inactivate curve of the current. The cumulative inhibition of I_Na(T) induced during repetitive stimuli became retarded during its exposure. The recovery time course from the I_Na block elicited, following the conditioning pulse stimulation, was satisfactorily fitted by two exponential processes. Moreover, the fast and slow time constants of recovery from the I_Na block by the same conditioning protocol were noticeably increased in the presence of picaridin. However, the fraction in fast or slow component of recovery time course was, respectively, increased or decreased with an increase in picaridin concentrations. The Hys_(V)’s strength of persistent I_Na (I_Na(P)), responding to triangular ramp voltage, was also enhanced during cell exposure to picaridin. The magnitude of resurgent I_Na (I_Na(R)) was raised in its presence. Picaritin-induced increases of I_Na(P) or I_Na(R) intrinsically in GH₃ cells could be attenuated by further addition of ranolazine. The predictions of molecular docking also disclosed that there are possible interactions of the picaridin molecule with the hNa_V1.7 channel. Taken literally, the stimulation of I_Na exerted by the exposure to picaridin is expected to exert impacts on the functional activities residing in electrically excitable cells. Full article

Paramyotonia congenita (PMC) is a rare skeletal muscle disorder characterized by muscle stiffness upon repetitive exercise and cold exposure. PMC was reported to be caused by dominant mutations in the SCN4A gene encoding the α subunit of the Na_v1.4 channel. Recently, we identified two missense mutations of the SCN4A gene, p.V781I and p.A1737T, in two PMC families. To evaluate the changes in electrophysiological properties caused by the mutations, both mutant and wild-type (WT) SCN4A genes were expressed in CHO-K1 and HEK-293T cells. Then, whole-cell patch-clamp recording was employed to study the altered gating of mutant channels. The activation curve of transient current showed a hyperpolarizing shift in both mutant Na_v1.4 channels as compared to the WT channel, whereas there was a depolarizing shift in the fast inactivation curve. These changes confer to an increase in window current in the mutant channels. Further investigations demonstrated that the mutated channel proteins generate significantly larger resurgent currents as compared to the WT channel and take longer to attain the peak of resurgent current than the WT channel. In conclusion, the current study demonstrates that p.V781I and p.A1737T mutations in the Na_v1.4 channel increase both the sustained and the resurgent Na⁺ current, leading to membrane hyperexcitability with a lower firing threshold, which may influence the clinical phenotype. Full article

Mirogabalin (MGB, Tarlige^®), an inhibitor of the α₂δ-1 subunit of voltage-gated Ca²⁺ (Ca_V) channels, is used as a way to alleviate peripheral neuropathic pain and diabetic neuropathy. However, to what extent MGB modifies the magnitude, gating, and/or hysteresis of various types of plasmalemmal ionic currents remains largely unexplored. In pituitary tumor (GH₃) cells, we found that MGB was effective at suppressing the peak (transient, I_Na(T)) and sustained (late, I_Na(L)) components of the voltage-gated Na⁺ current (I_Na) in a concentration-dependent manner, with an effective IC₅₀ of 19.5 and 7.3 μM, respectively, while the K_D value calculated on the basis of minimum reaction scheme was 8.2 μM. The recovery of I_Na(T) inactivation slowed in the presence of MGB, although the overall current–voltage relation of I_Na(T) was unaltered; however, there was a leftward shift in the inactivation curve of the current. The magnitude of the window (I_Na(W)) or resurgent I_Na (I_Na(R)) evoked by the respective ascending or descending ramp pulse (V_ramp) was reduced during cell exposure to MGB. MGB-induced attenuation in I_Na(W) or I_Na(R) was reversed by the further addition of tefluthrin, a pyrethroid insecticide known to stimulate I_Na. MGB also effectively lessened the strength of voltage-dependent hysteresis of persistent I_Na in response to the isosceles triangular V_ramp. The cumulative inhibition of I_Na(T), evoked by pulse train stimulation, was enhanced in its presence. Taken together, in addition to the inhibition of Ca_V channels, the Na_V channel attenuation produced by MGB might have an impact in its analgesic effects occurring in vivo. Full article

GV-58 ((2R)-2-[(6-{[(5-methylthiophen-2-yl)methyl]amino}-9-propyl-9H-purin-2-yl)amino]butan-1-ol) is recognized to be an activator of N- and P/Q-type Ca²⁺ currents. However, its modulatory actions on other types of ionic currents in electrically excitable cells remain largely unanswered. This study was undertaken to explore the possible modifications caused by GV-58 in ionic currents (e.g., voltage-gated Na⁺ current [I_Na], A-type K⁺ current [I_K(A)], and erg-mediated K⁺ current [I_K(erg)]) identified from pituitary GH₃ lactotrophs. GH₃ cell exposure to GV-58 enhanced the transient and late components of I_Na with varying potencies; consequently, the EC₅₀ values of GV-58 required for its differential increase in peak and late I_Na in GH₃ cells were estimated to be 8.9 and 2.6 μM, respectively. The I_Na in response to brief depolarizing pulse was respectively stimulated or suppressed by GV-58 or tetrodotoxin, but it failed to be altered by ω-conotoxin MVIID. Cell exposure to this compound increased the recovery of I_Na inactivation evoked by two-pulse protocol based on a geometrics progression; however, in its presence, there was a slowing in the inactivation rate of current decay evoked by a train of depolarizing pulses. The existence of GV-58 also resulted in an increase in the amplitude of ramp-induced resurgent and window I_Na. The presence of this compound inhibited I_K(A) magnitude, accompanied by a shortening in inactivation time course of the current; however, it mildly decreased I_K(erg). Under current-clamp conditions, GV-58 increased the frequency of spontaneous action potentials in GH₃ cells. Moreover, in NSC-34 motor neuron-like cells, the presence of GV-58 not only raised I_Na amplitude but also reduced current inactivation. Taken together, the overall work provides a noticeable yet unidentified finding which implies that, in addition to its agonistic effect on Ca²⁺ currents, GV-58 may concertedly modify the amplitude and gating kinetics of I_Na in electrically excitable cells, hence modifiying functional activities in these cells. Full article

Sparsentan is viewed as a dual antagonist of endothelin type A (ET_A) receptor and angiotensin II (AngII) receptor and it could be beneficial in patients with focal segmental glomerulosclerosis. Moreover, it could improve glomerular filtration rate and augment protective tissue remodeling in mouse models of focal segmental glomerulosclerosis. The ionic mechanisms through which it interacts with the magnitude and/or gating kinetics of ionic currents in excitable cells were not thoroughly investigated. Herein, we aimed to examine the effects of varying sparsentan concentrations on ionic currents residing in pituitary GH₃ somatolactotrophs. From whole-cell current recordings made in GH₃ cells, sparsentan (0.3–100 μM) differentially inhibited the peak and late components of voltage-gated Na⁺ current (I_Na). The IC₅₀ value of sparsentan required to exert a reduction in peak and late I_Na in GH₃ cells was 15.04 and 1.21 μM, respectively; meanwhile, the K_D value estimated from its shortening in the slow component of I_Na inactivation time constant was 2.09 μM. The sparsentan (10 μM) presence did not change the overall current–voltage relationship of I_Na; however, the steady-state inactivation curve of the current was shifted to more negative potential in its presence (10 μM), with no change in the gating charge of the curve. The window I_Na activated by a brief upsloping ramp was decreased during exposure to sparsentan (10 μM); moreover, recovery of peak I_Na became slowed in its presence. The Tefluthrin (Tef)-stimulated resurgent I_Na activated in response to abrupt depolarization followed by the descending ramp pulse was additionally attenuated by subsequent application of sparsentan. In continued presence of Tef (3 μM) or β-pompilidotoxin (3 μM), further application of sparsentan (3 μM) reversed their stimulation of I_Na. However, sparsentan-induced inhibition of I_Na failed to be overcome by subsequent application of either endothelin 1 (1 μM) or angiotensin II (1 μM); moreover, in continued presence of endothelin (1 μM) or angiotensin II (1 μM), further addition of sparsentan (3 μM) effectively decreased peak I_Na. Additionally, the application of sparsentan (3 μM) inhibited the peak and late components of erg-mediated K⁺ current in GH₃ cells, although it mildly decreased the amplitude of delayed-rectifier K⁺ current. Altogether, this study provides a distinct yet unidentified finding that sparsentan may perturb the amplitude or gating of varying ionic currents in excitable cells. Full article

Paramyotonia congenita (PMC) is a rare hereditary skeletal muscle disorder. The major symptom, muscle stiffness, is frequently induced by cold exposure and repetitive exercise. Mutations in human SCN4A gene, which encodes the α-subunit of Na_v1.4 channel, are responsible for PMC. Mutation screening of SCN4A gene from two PMC families identified two missense mutations, p.T1313M and p.R1448H. To elucidate the electrophysiological abnormalities caused by the mutations, the p.T1313M, p.R1448H, and wild-type (WT) SCN4A genes were transient expressed on Chinese hamster ovary (CHO-K1) cells. The detailed study on the gating defects of the mutant channels using the whole-cell patch clamping technique was performed. The mutant Na_v1.4 channels impaired the basic gating properties with increasing sustained and window currents during membrane depolarization and facilitated the genesis of resurgent currents during repolarization. The mutations caused a hyperpolarization shift in the fast inactivation and slightly enhanced the slow inactivation with an increase in half-maximal inactivation voltage. No differences were found in the decay kinetics of the tail current between mutant and WT channels. In addition to generating the larger resurgent sodium current, the time to peak in the mutant channels was longer than that in the WT channels. In conclusion, our results demonstrated that the mutations p.T1313M and p.R1448H in Na_v1.4 channels can enhance fast inactivation, slow inactivation, and resurgent current, revealing that subtle changes in gating processes can influence the clinical phenotype. Full article

Sesamin (SSM) and sesamolin (SesA) are the two major furofuran lignans of sesame oil and they have been previously noticed to exert various biological actions. However, their modulatory actions on different types of ionic currents in electrically excitable cells remain largely unresolved. The present experiments were undertaken to explore the possible perturbations of SSM and SesA on different types of ionic currents, e.g., voltage-gated Na⁺ currents (I_Na), erg-mediated K⁺ currents (I_K(erg)), M-type K⁺ currents (I_K(M)), delayed-rectifier K⁺ currents (I_K(DR)) and hyperpolarization-activated cation currents (I_h) identified from pituitary tumor (GH₃) cells. The exposure to SSM or SesA depressed the transient and late components of I_Na with different potencies. The IC₅₀ value of SSM needed to lessen the peak or sustained I_Na was calculated to be 7.2 or 0.6 μM, while that of SesA was 9.8 or 2.5 μM, respectively. The dissociation constant of SSM-perturbed inhibition on I_Na, based on the first-order reaction scheme, was measured to be 0.93 μM, a value very similar to the IC₅₀ for its depressant action on sustained I_Na. The addition of SSM was also effective at suppressing the amplitude of resurgent I_Na. The addition of SSM could concentration-dependently inhibit the I_K(M) amplitude with an IC₅₀ value of 4.8 μM. SSM at a concentration of 30 μM could suppress the amplitude of I_K(erg), while at 10 μM, it mildly decreased the I_K(DR) amplitude. However, the addition of neither SSM (10 μM) nor SesA (10 μM) altered the amplitude or kinetics of I_h in response to long-lasting hyperpolarization. Additionally, in this study, a modified Markovian model designed for SCN8A-encoded (or Na_V1.6) channels was implemented to evaluate the plausible modifications of SSM on the gating kinetics of Na_V channels. The model demonstrated herein was well suited to predict that the SSM-mediated decrease in peak I_Na, followed by increased current inactivation, which could largely account for its favorable decrease in the probability of the open-blocked over open state of Na_V channels. Collectively, our study provides evidence that highlights the notion that SSM or SesA could block multiple ion currents, such as I_Na and I_K(M), and suggests that these actions are potentially important and may participate in the functional activities of various electrically excitable cells in vivo. Full article

Myotonia congenita (MC) is a rare disorder characterized by stiffness and weakness of the limb and trunk muscles. Mutations in the SCN4A gene encoding the alpha-subunit of the voltage-gated sodium channel Na_v1.4 have been reported to be responsible for sodium channel myotonia (SCM). The Na_v1.4 channel is expressed in skeletal muscles, and its related channelopathies affect skeletal muscle excitability, which can manifest as SCM, paramyotonia and periodic paralysis. In this study, the missense mutation p.V445M was identified in two individual families with MC. To determine the functional consequences of having a mutated Na_v1.4 channel, whole-cell patch-clamp recording of transfected Chinese hamster ovary cells was performed. Evaluation of the transient Na⁺ current found that a hyperpolarizing shift occurs at both the activation and inactivation curves with an increase of the window currents in the mutant channels. The Na_v1.4 channel’s co-expression with the Na_vβ4 peptide can generate resurgent Na⁺ currents at repolarization following a depolarization. The magnitude of the resurgent currents is higher in the mutant than in the wild-type (WT) channel. Although the decay kinetics are comparable between the mutant and WT channels, the time to the peak of resurgent Na⁺ currents in the mutant channel is significantly protracted compared with that in the WT channel. These findings suggest that the p.V445M mutation in the Na_v1.4 channel results in an increase of both sustained and resurgent Na⁺ currents, which may contribute to hyperexcitability with repetitive firing and is likely to facilitate recurrent myotonia in SCM patients. Full article