Time Constant Of Inactivation Research Articles

Role of the transient outward potassium current in the genesis of early afterdepolarizations in cardiac cells

The transient outward potassium current (I(to)) plays important roles in action potential (AP) morphology and dynamics; however, its role in the genesis of early afterdepolarizations (EADs) is not well understood. We aimed to study the effects and mechanisms of I(to) on EAD genesis in cardiac cells using combined experimental and computational approaches. We first carried out patch-clamp experiments in isolated rabbit ventricular myocytes exposed to H(2)O(2) (0.2 or 1 mM), in which EADs were induced at a slow pacing rate. EADs were eliminated by either increasing the pacing rate or blocking I(to) with 2 mM 4-aminopyridine. In addition to enhancing the L-type calcium current (I(Ca,L)) and the late sodium current, H(2)O(2) also increased the conductance, slowed inactivation, and accelerated recovery from the inactivation of I(to). Computer simulations showed that I(to) promoted EADs under the condition of reduced repolarization reserve, consistent with the experimental observations. However, EADs were only promoted in the intermediate ranges of the I(to) conductance and the inactivation time constant. The underlying mechanism is that I(to) lowers the AP plateau voltage into the range at which the time-dependent potassium current (namely I(Ks)) activation is further slowed and I(Ca,L) is available for reactivation, leading to voltage oscillations to manifest EADs. Further experimental studies in cardiac cells of other species validated the theoretical predictions. In cardiac cells, I(to), with a proper conductance and inactivation speed, potentiates EADs by setting the AP plateau into the voltage range where I(Ca,L) reactivation is facilitated and I(Ks) activation is slowed.

Reduced Na + and higher K + channel expression and function contribute to right ventricular origin of arrhythmias in Scn5a+/− mice

Brugada syndrome (BrS) is associated with ventricular tachycardia originating particularly in the right ventricle (RV). We explore electrophysiological features predisposing to such arrhythmic tendency and their possible RV localization in a heterozygotic Scn5a+/− murine model. Nav1.5 mRNA and protein expression were lower in Scn5a+/− than wild-type (WT), with a further reduction in the RV compared with the left ventricle (LV). RVs showed higher expression levels of Kv4.2, Kv4.3 and KChIP2 in both Scn5a+/− and WT. Action potential upstroke velocity and maximum Na+ current (INa) density were correspondingly decreased in Scn5a+/−, with a further reduction in the RV. The voltage dependence of inactivation was shifted to more negative values in Scn5a+/−. These findings are predictive of a localized depolarization abnormality leading to slowed conduction. Persistent Na+ current (IpNa) density was decreased in a similar pattern to INa. RV transient outward current (Ito) density was greater than LV in both WT and Scn5a+/−, and had larger time constants of inactivation. These findings were also consistent with the observation that AP durations were smallest in the RV of Scn5a+/−, fulfilling predictions of an increased heterogeneity of repolarization as an additional possible electrophysiological mechanism for arrhythmogenesis in BrS.

E Right ventricular origin of arrhythmias inScn5a+/− mice is due to reduced Na+ and higher K+ channel expression and function

<h3>Introduction</h3> Brugada syndrome is associated with ventricular tachycardia originating in the right ventricle (RV); this has been attributed to either depolarisation abnormalities or increased repolarisation heterogeneities. We have used a heterozygotic <i>Scn5a+/−</i> murine model to investigate the underlying mechanisms for the predisposition of the RV to arrhythmias. <h3>Methods and Results</h3> Na_v1.5 mRNA and protein expression were lower in <i>Scn5a+/−</i> than wild-type (WT) hearts, with a further reduction in the RV compared to left ventricle (LV) (Abstract E figure 1A,B, n=4, significant differences: * = WT vs <i>Scn5a+/−</i>; # = LV vs RV). There were higher expression levels of K_v4.2, K_v4.3 and KChIP₂ in RV than LV in both groups. Action potential (AP) upstroke velocity was decreased in <i>Scn5a+/−</i> (RV: 59.43±2.70 V/s to 30.26±4.03 V/s, p&lt;0.0001, n=20), and furthermore was smaller in RV than LV. AP durations were smallest in the RV of <i>Scn5a+/−</i> myocytes. RV transient outward current density (<i>I_to</i>) was greater than LV in both WT and <i>Scn5a+/−</i> (Abstract E figure 1C, n=17), with similar voltage dependence of activation. Time constants of inactivation were larger in RV than LV, and voltage dependence of inactivation was shifted to more negative values in RV compared to LV, but to more positive values in <i>Scn5a+/−</i> compared to WT. Maximum Na⁺ current density (<i>I_Na</i>) was decreased in <i>Scn5a+/−</i>, with a further reduction in the RV (Abstract E figure 2A–C, n=17). Voltage dependence of activation was unchanged, but inactivation was shifted to more negative values in <i>Scn5a+/−</i>. Maximum persistent Na⁺ current density (<i>I_pNa</i>) was decreased in a similar pattern to <i>I_Na</i> (RV: −0.30±0.03 pA/pF, n=15 vs −0.17±0.02 pA/pF, n=22, p=0.0009). <h3>Conclusion</h3> Our findings show preferential upregulation of the single Scn5a gene in the LV of the <i>Scn5a+/−</i> mice compared to RV. The reduced expression of Na⁺ channels in RV leads to smaller <i>I_Na</i>, resulting in slowed conduction, and smaller <i>I_pNa</i>, which in combination with increased <i>I_to</i>, results in shorter AP durations and greater heterogeneity of repolarisation, thus suggesting arrhythmogenesis may be initiated by both abnormal depolarisation and repolarisation in the RV of <i>Scn5a+/−</i> hearts. Insight into the molecular mechanisms of arrhythmias could prove crucial in planning possible new pharmacological therapies for a disease where the mainstay of treatment is cardioverter-defibrillator implantation.

Properties of IA in a neuron of the dorsal raphe nucleus

Voltage clamp data were analyzed in order to characterize the properties of the fast potassium transient current IA for a presumed serotonergic neuron of the rat dorsal raphe nucleus (DRN). We obtain maximal conductance, time constants of activation and inactivation, and the steady state activation and inactivation functions m∞ and h∞, as Boltzmann curves, defined by half-activation potentials and slope factors.IA is estimated as g¯(V−Vrev)m4h, with g¯=20.5nS. For activation, the half-activation potential is Va=−52.5mV with slope factor ka=16.5mV, whereas for inactivation the corresponding quantities are −91.5mV and −9.3mV. We discuss the results in terms of the corresponding properties of IA in other cell types and their possible relevance to pacemaking activity in cells of the DRN. Methods of identification of serotonergic DRN neurons and the nature of the Kv channels underlying the A-type current are also discussed.

The Effect of Substrate Stiffness on Cardiomyocyte Calcium Currents

The stiffness of myocardial tissue changes significantly during development and at birth, concurrent with significant changes in contractile and electrical maturation of cardiomyocytes. Previous studies by our group have shown that cardiomyocytes generate maximum contractile force when cultured on a substrate with a stiffness approximating native cardiac tissue. The force is correlated with the size of calcium transients, and on very stiff substrates this is due to lower amounts of stored calcium. However, the same relationship with stored calcium does not exist on softer substrates, and we therefore hypothesized that substrate stiffness also modifies calcium currents during cardiomyocyte action potentials. Neonatal rat ventricular cardiomyocytes (NRVMs) were isolated and cultured on polyacrylamide hydrogels of varying elastic modulus (1, 5, 10, 25, and 50 kPa). Using whole-cell patch clamping, action potential voltages were recorded in the presence and absence of BDM, a myosin inhibitor, calcium and potassium currents were recorded at voltages between −80 and 40 mV, and calcium current inactivation dynamics were determined. No significant difference was observed in potassium currents. However, the voltage at which a calcium current was at its maximum was shown to decrease with increasing stiffness, and the inactivation time constant was shown to be shorter on stiffer substrates. This can lead to less calcium influx in cells on softer substrates. Interestingly, the calcium current dynamics in NRVM on stiff substrates resemble those in adult rat cardiomyocytes. These results may explain functional differences in cardiomyocytes resulting from changes in the elastic modulus of the extracellular matrix, as observed during embryonic development, in ischemic regions of the heart after myocardial infarction, and during dilated cardiomyopathy.

Abstract 8633: Right Ventricular Origin of Arrhythmias in Scn5a+/- Mice Modeling Brugada Syndrome is Due to Reduced Na + and Higher K + Channel Expression and Function in the Right Ventricle

Brugada syndrome is associated with ventricular tachycardia originating in the right ventricle (RV); this has been attributed to either depolarization abnormalities or increased repolarization heterogeneities. We use a heterozygotic Scn5a+/- murine model to test these hypotheses. Na v 1.5 mRNA and protein expression were lower in Scn5a+/- than wild-type (WT) hearts, with a further reduction in the RV (panel A, n = 4, significant differences: * = WT vs Scn5a+/- ; # = LV vs RV). There were higher expression levels of K v 4.2, K v 4.3 and KChIP2 in RV than LV in both groups. Action potential (AP) upstroke velocity was decreased in Scn5a+/- (RV: 59.43 +/- 2.70 V/s to 30.26 +/- 4.03 V/s, p &lt; 0.0001, n = 20), and furthermore was smaller in RV than LV. AP durations were smallest in the RV of Scn5a+/- myocytes. Transient outward current density ( I to ) was greater in the RV of both WT and Scn5a+/- (panel B, n = 17), with similar voltage dependence of activation. Time constants of inactivation were larger in RV than LV, and voltage dependence of inactivation was shifted to more negative values in RV compared to LV, but to more positive values in Scn5a+/- compared to WT. Maximum Na + current density ( I Na ) was decreased in Scn5a+/- , with a further reduction in the RV (panels C, D, n = 17). Voltage dependence of activation was unchanged, but inactivation was shifted to more negative values in Scn5a+/- . Maximum persistent Na + current density ( I pNa ) was decreased in a similar pattern to I Na (RV: -0.30 +/- 0.03 pA/pF, n = 15 vs -0.17+/- 0.02 pA/pF, n = 22, p =0.0009). Our findings show preferential upregulation of the single Scn5a gene in the LV of the Scn5a+/- mice compared to RV. The reduced expression of Na + channels in RV leads to smaller I Na , resulting in slowed conduction, and smaller I pNa , which in combination with increased I to , results in shorter AP durations and greater heterogeneity of repolarization, thus suggesting arrhythmogenesis may be initiated by both mechanisms in the RV of Scn5a+/- hearts.

Extracellular chloride regulation of Kv2.1, contributor to the major outward Kv current in mammalian outer hair cells

Outer hair cells (OHC) function as both receptors and effectors in providing a boost to auditory reception. Amplification is driven by the motor protein prestin, which is under anionic control. Interestingly, we now find that the major, 4-AP-sensitive, outward K(+) current of the OHC (I(K)) is also sensitive to Cl(-), although, in contrast to prestin, extracellularly. I(K) is inhibited by reducing extracellular Cl(-) levels, with a linear dependence of 0.4%/mM. Other voltage-dependent K(+) (Kv) channel conductances in supporting cells, such as Hensen and Deiters' cells, are not affected by reduced extracellular Cl(-). To elucidate the molecular basis of this Cl(-)-sensitive I(K), we looked at potential molecular candidates based on Cl(-) sensitivity and/or similarities in kinetics. For I(K), we identified three different Ca(2+)-independent components of I(K) based on the time constant of inactivation: a fast, transient outward current, a rapidly activating, slowly inactivating current (Ik(1)), and a slowly inactivating current (Ik(2)). Extracellular Cl(-) differentially affects these components. Because the inactivation time constants of Ik(1) and Ik(2) are similar to those of Kv1.5 and Kv2.1, we transiently transfected these constructs into CHO cells and found that low extracellular Cl(-) inhibited both channels with linear current reductions of 0.38%/mM and 0.49%/mM, respectively. We also tested heterologously expressed Slick and Slack conductances, two intracellularly Cl(-)-sensitive K(+) channels, but found no extracellular Cl(-) sensitivity. The Cl(-) sensitivity of Kv2.1 and its robust expression within OHCs verified by single-cell RT-PCR indicate that these channels underlie the OHC's extracellular Cl(-) sensitivity.

Multiple T-type Ca2+ current subtypes in electrophysiologically characterized hamster dorsal horn neurons: possible role in spinal sensory integration

Whole cell patch-clamp recordings were used to investigate the contribution of transient, low-threshold calcium currents (I(T)) to firing properties of hamster spinal dorsal horn neurons. I(T) was widely, though not uniformly, expressed by cells in Rexed's laminae I-IV and correlated with the pattern of action potential discharge evoked under current-clamp conditions: I(T) in neurons responding to constant membrane depolarization with one or two action potentials was nearly threefold larger than I(T) in cells responding to the same activation with continuous firing. I(T) was evoked by depolarizing voltage ramps exceeding 46 mV/s and increased with ramp slope (240-2,400 mV/s). Bath application of 200 μM Ni(2+) depressed ramp-activated I(T). Phasic firing recorded in current clamp could only be activated by membrane depolarizations exceeding ∼43-46 mV/s and was blocked by Ni(2+) and mibefradil, suggesting I(T) as an underlying mechanism. Two components of I(T), "fast" and "slow," were isolated based on a difference in time constant of inactivation (12 ms and 177 ms, respectively). The amplitude of the fast subtype depended on the slope of membrane depolarization and was twice as great in burst-firing cells than in cells having a tonic discharge. Post hoc single-cell RT-PCR analyses suggested that the fast component is associated with the Ca(V)3.1 channel subtype. I(T) may enhance responses of phasic-firing dorsal horn neurons to rapid membrane depolarizations and contribute to an ability to discriminate between afferent sensory inputs that encode high- and low-frequency stimulus information.

Effects of Resibufogenin and Cinobufagin on voltage-gated potassium channels in primary cultures of rat hippocampal neurons

Outward delayed rectifier potassium channel and outward transient potassium channel have multiple important roles in maintaining the excitability of hippocampal neurons. The present study investigated the effects of two bufadienolides, Resibufogenin (RBG) and Cinobufagin (CBG), on the outward delayed rectifier potassium current (IK) and outward transient potassium current (IA) in rat hippocampal neurons. RBG and CBG have similar structures and both were isolated from the venom gland of toad skin. RBG inhibited both IK and IA, whereas CBG inhibited IK without noticeable effect on IA. Moreover, at 1μM concentration both RBG and CBG could alter some channel kinetics and gating properties of IK, such as steady-state activation and inactivation curves, open probability and time constants. These findings suggested that IK is probably a target of bufadienolides, which may explain the mechanisms of bufadienolides’ pathological effects on central nervous system.

Na+ Channel Scn1b Gene Regulates Dorsal Root Ganglion Nociceptor Excitability in Vivo

Nociceptive dorsal root ganglion (DRG) neurons express tetrodotoxin-sensitive (TTX-S) and -resistant (TTX-R) Na(+) current (I(Na)) mediated by voltage-gated Na(+) channels (VGSCs). In nociceptive DRG neurons, VGSC β2 subunits, encoded by Scn2b, selectively regulate TTX-S α subunit mRNA and protein expression, ultimately resulting in changes in pain sensitivity. We hypothesized that VGSCs in nociceptive DRG neurons may also be regulated by β1 subunits, encoded by Scn1b. Scn1b null mice are models of Dravet Syndrome, a severe pediatric encephalopathy. Many physiological effects of Scn1b deletion on CNS neurons have been described. In contrast, little is known about the role of Scn1b in peripheral neurons in vivo. Here we demonstrate that Scn1b null DRG neurons exhibit a depolarizing shift in the voltage dependence of TTX-S I(Na) inactivation, reduced persistent TTX-R I(Na), a prolonged rate of recovery of TTX-R I(Na) from inactivation, and reduced cell surface expression of Na(v)1.9 compared with their WT littermates. Investigation of action potential firing shows that Scn1b null DRG neurons are hyperexcitable compared with WT. Consistent with this, transient outward K(+) current (I(to)) is significantly reduced in null DRG neurons. We conclude that Scn1b regulates the electrical excitability of nociceptive DRG neurons in vivo by modulating both I(Na) and I(K).

The mechanisms underlying ICa heterogeneity across murine left ventricle

L-type calcium current (I(Ca)) plays a critical role in excitation-contraction coupling (ECC). Unlike transient outward K(+) current (I(to)), it is controversial whether I(Ca) transmural gradient exists in left ventricle. Although previous studies have shown some evidences for I(Ca) heterogeneity, the mechanism is still unknown. In this study, the authors recorded I(Ca) from epicardial (EPI) and endocardial (ENDO) myocytes isolated from murine left ventricle using patch-clamp technique. It was found that I(Ca) density was obviously larger in EPI than in ENDO (7.3 ± 0.3 pA/pF vs. 6.2 ± 0.2 pA/pF, at test potential of +10 mV, P < 0.05). The characteristics of I(Ca) showed no difference between these two regions except for the fast inactivation time constants (9.9 ± 0.9 ms in EPI vs. 13.5 ± 0.9 ms in ENDO, at test potential of +10 mV, P < 0.05). In addition, it was explored the molecular mechanism underlying I(Ca) transmural gradient by Western blot. The authors demonstrated that a higher activity of CaMKII in ENDO cells induced more nuclear translocation of p65, a component of nuclear factor-kappa B (NF-kB). Consequently, p65 in ENDO inhibited more transcription of Cav1.2, the main encoding gene for L-type calcium channels (LTCCs). These results reveal a difference in CaMKII/p65 signal pathway between EPI and ENDO that underlies this mechanism of I(Ca) heterogeneity in murine left ventricle.

Resurgent currents turn painfully exciting

Resurgent currents turn painfully exciting

Effects of Raloxifene on Voltage- Dependent T-Type Ca2+ Channels in Mouse Spermatogenic Cells

Voltage-dependent T-type Ca²⁺ channels have eminent roles in sperm function. In the present study, we investigated the effects of raloxifene, a selective estrogen receptor modulator, on T-type Ca²⁺ channels in mouse spermatogenic cells by using both electrophysiological and molecular techniques. We found that T-type calcium currents (I_T-Ca) were inhibited by raloxifene in a concentration-dependent manner with an IC₅₀ of 2.97 µM, as assessed with the patch clamp technique. Application of raloxifene at 2 µM inhibited I_T-Ca by 54.9 ± 2.1% at –20 mV (n = 10, p < 0.05). Furthermore, raloxifene-induced inhibition of I_T-Ca was associated with a negative shift of both the activation and the steady-state inactivation properties. The time constants of activation and inactivation were decreased, the time constant of deactivation was increased, but the time constant of recovery was not affected. In addition, the inhibitory effects of raloxifene and 17β-estradiol on I_T-Ca were unaffected by the estrogen receptor antagonist ICI 182,780. We also found that raloxifene treatment decreased the mRNA expression of Ca_V3.2 and Ca_V3.3, but not Ca_V3.1 in GC-2spd (ts) cells (mouse spermatocyte cell line), as assessed by real-time RT-PCR. Taken together, these data indicate that in mouse spermatogenic cells, raloxifene decreases I_T-Ca independent of classical estrogen, and the mRNA expression of T-type calcium channels and therefore may affect male reproductive function.

Hirsutenone Directly Blocks Human ether-a-go-go Related Gene K+ Channels

The aim of the present study was to investigate whether hirsutenone affects the human ether-a-go-go related gene (hERG) K(+) channels. Many drugs promote formation of the acquired form of long QT syndrome (LQTS) by blocking the hERG K(+) channels. Hirsutenone, a new candidate for the treatment inflammatory skin lesions, induced a concentration-dependent decrease in hERG K(+) current amplitudes. Hirsutenone significantly decreased the time constants at the onset of inactivation. However, the reductions in the time constants of steady-state inactivation and the recovery from inactivation after hirsutenone treatment were not significant. In addition, the drug had no effect on the voltage-dependent activation curve or the steady-state inactivation curve. In summary, hirsutenone potentially acts as a blocker of hERG K(+) channels functioning by modifying the channel inactivation kinetics.

Mechanism of the negative inotropic effect of naringin in mouse heart

Context: Naringin (NRG) is the major flavonoid (flavanone glycoside) in grapefruit juice. Its biological activity has been only partially characterized and little is known about the mechanism of the negative inotropic action of this flavonoid. Aims: To evaluate the effects of NRG on the surface electrogram (ECG) and the force of contraction (FC) of mice hearts as well as on the sodium (INa), calcium (ICaL) and Na+ – Ca2+ exchange (INaCaX) currents of enzymatically isolated mouse ventricular cardiomyocytes. Methods: ECG and FC were recorded on mouse hearts perfused in a Langendorff column. Ventricular cardiomyocytes were enzimatically dissociated and ionic currents recorded with the patch-clamp technique. Results: NRG increased RR interval and shortened corrected QT only at high concentrations (30-100 µM). However, at a fixed heart rate, it decreased FC with an IC50 of 0.4 µM. NRG reduced INa with an IC50 of 0.07 µM but with a maximal inhibition of 60 %. NRG also depressed ICaL with an IC50 of 0.013 µM and increased its fast inactivation time constant. The effects on ICaL were not voltage-dependent. INaCaX was not affected by NRG. Conclusions: Our results indicate that NRG exerts a negative inotropic effect in mice hearts that could be explained by a decrease in INa and ICaL. These actions should be taken into account when considering this molecule either as a dietetic supplement or as a template to develop therapeutic agents for human diseases.

The N Terminus of a Schistosome β Subunit Regulates Inactivation and Current Density of a Cav2 Channel

The β subunit of high voltage-activated Ca(2+) (Ca(v)) channels targets the pore-forming α(1) subunit to the plasma membrane and tunes the biophysical phenotype of the Ca(v) channel complex. We used a combination of molecular biology and whole-cell patch clamp to investigate the functional role of a long N-terminal polyacidic motif (NPAM) in a Ca(v)β subunit of the human parasite Schistosoma mansoni (β(Sm)), a motif that does not occur in other known Ca(v)β subunits. When expressed in human embryonic kidney cells stably expressing Ca(v)2.3, β(Sm) accelerates Ca(2+)/calmodulin-independent inactivation of Ca(v)2.3. Deleting the first 44 amino acids of β(Sm), a region that includes NPAM, significantly slows the predominant time constant of inactivation (τ(fast)) under conditions that prevent Ca(2+)/CaM-dependent inactivation (β(Sm): τ(fast) = 66 ms; β(SmΔ2-44): τ(fast) = 111 ms, p < 0.01). Interestingly, deleting the amino acids that are N-terminal to NPAM (2-24 or 2-17) results in faster inactivation than with an intact N terminus (τ(fast) = 42 ms with β(SmΔ2-17); τ(fast) = 40 ms with β(SmΔ2-24), p < 0.01). This suggests that NPAM is the structural determinant for accelerating Ca(2+)/calmodulin-independent inactivation. We also created three chimeric subunits that contain the first 44 amino acids of β(Sm) attached to mammalian β(1b), β(2a), and β(3) subunits. For any given mammalian β subunit, inactivation was faster if it contained the N terminus of β(Sm) than if it did not. Co-expression of the mammalian α(2)δ-1 subunit resulted in doubling of the inactivation rate, but the effects of NPAM persisted. Thus, it appears that the schistosome Ca(v) channel complex has acquired a new function that likely contributes to reducing the amount of Ca(2+) that enters the cells in vivo. This feature is of potential interest as a target for new antihelminthics.

A helix-breaking mutation in the epithelial Ca2+ channel TRPV5 leads to reduced Ca2+-dependent inactivation

TRPV5, a member of transient receptor potential (TRP) superfamily of ion channels, plays a crucial role in epithelial calcium transport in the kidney. This channel has a high selectivity for Ca2+ and is tightly regulated by intracellular Ca2+ concentrations. Recently it was shown that the molecular basis of deafness in varitint-waddler mouse is the result of hair cell death caused by the constitutive activity of transient receptor potential mucolipin 3 (TRPML3) channel carrying a helix breaking mutation, A419P, at the intracellular proximity of the fifth transmembrane domain (TM5). This mutation significantly elevates intracellular Ca2+ concentration and causes rapid cell death. Here we show that substituting the equivalent location in TRPV5, the M490, to proline significantly modulates Ca2+-dependent inactivation of TRPV5. The single channel conductance, time constant of inactivation (τ) and half maximal inhibition constant (IC50) of TRPV5(M490P) were increased compared to TRPV5(WT). Moreover TRPV5(M490P) showed lower Ca2+ permeability. Out of different point mutations created to characterize the importance of M490 in Ca2+-dependent inactivation, only TRPV5(M490P)-expressing cells showed apoptosis and extremely altered Ca2+-dependent inactivation. In conclusion, the TRPV5 channel is susceptible for helix breaking mutations and the proximal intracellular region of TM5 of this channel plays an important role in Ca2+-dependent inactivation.

Open channel block of the fast transient outward K + current by primaquine and chloroquine in rat left ventricular cardiomyocytes

Anti-malarial drugs may have severe adverse cardiac effects as a result of their ion channel blocking properties. Here we investigate the effect of the aminoquinolines primaquine and chloroquine on the fast transient outward K + current (I to) of single epicardial myocytes isolated from the left ventricular free wall of female Wistar rats. The ruptured-patch whole-cell configuration of the patch-clamp technique was used to investigate I to. At + 60 mV, primaquine blocked I to amplitude (defined as the current inactivating during a test pulse of 600 ms duration) with an IC 50 of 118 ± 8 μM. I to charge was blocked with an IC 50 of 33 ± 2 μM (n = 42), indicating open channel block. Chloroquine blocked I to amplitude with an IC 50 of 4.6 ± 0.9 mM, while the IC 50 for I to charge was 439 ± 63 μM (n = 23). The kinetic analysis of the onset of block revealed K d values of 52 ± 8 μM (n = 18) and 520 ± 60 μM (n = 11) for primaquine and chloroquine, respectively. Both drugs significantly accelerated the apparent inactivation time constant of I to. Steady-state inactivation of I to was not altered by 30 μM primaquine. In contrast, I to recovery from inactivation was prolonged with the appearance of an additional long time constant without a change of the short time constant. Exposure to 1 mM chloroquine resulted in a right shift of steady-state inactivation, whereas recovery from inactivation was only mildly affected. Both substances exhibited considerable use dependence. In X. laevis oocytes heterologously expressing hKv4.2 + hKChIP2b channels the block by the aminoquinolines was voltage dependent. We conclude that primaquine and chloroquine are open-channel blockers of I to.

Estradiol attenuates multiple tetrodotoxin-sensitive sodium currents in isolated gonadotropin-releasing hormone neurons

Secretion from gonadotropin-releasing hormone (GnRH) neurons is necessary for the production of gametes and hormones from the gonads. Subsequently, GnRH release is regulated by steroid feedback. However, the mechanisms by which steroids, specifically estradiol, modulate GnRH secretion are poorly understood. We have previously shown that estradiol administered to the female mouse decreases inward currents in fluorescently labeled GnRH neurons. The purpose of this study was to examine the contribution of sodium currents in the negative feedback action of estradiol. Electrophysiology was performed on GnRH neurons dissociated from young, middle-aged, or old female mice. All mice were ovariectomized; half were estradiol replaced. The amplitude of the sodium current underlying the action potential was significantly decreased in GnRH neurons from young estradiol-treated animals. In addition, in vivo estradiol significantly decreased the transient sodium current amplitude, but prolonged the sodium current inactivation time constant. Estradiol decreased the persistent sodium current amplitude, and induced a significant negative shift in peak current potential. In contrast to results obtained from cells from young reproductive animals, estradiol did not significantly attenuate the sodium current underlying the action potential in cells isolated from middle-aged or old mice. Sodium channels can modulate cell threshold, latency of firing, and action potential characteristics. The reduction of sodium current amplitude by estradiol suggests a negative feedback on GnRH neurons, which could lead to a downregulation of cell excitability and hormone release. The attenuation of estradiol regulation in peripostreproductive and postreproductive animals could lead to dysregulated hormone release with advancing age.

Voltage-gated K+ currents in mouse articular chondrocytes regulate membrane potential

Membrane currents and resting potential of isolated primary mouse articular chondrocytes maintained in monolayer cell culture for 1-9 days were recorded using patch clamp methods. Quantitative RT-PCR showed that the most abundantly expressed transcript of voltage-gated K+ channels was for Kv1.6, and immunological methods confirmed the expression of Kv1.6 a-subunit proteins. These chondrocytes expressed a large time- and potential-dependent, Ca2+-independent 'delayed rectifier' K+ current. Steady-state activation was well-fit by a Boltzmann function with a threshold near -50 mV, and a half-activation potential of -35.0 mV. The current was 50% blocked by 1.48 mM tetraethylammonium, 0.66 mM 4-aminopyridine, and 20.6 nM a-dendrotoxin. The current inactivated very slowly at membrane potentials in the range of the resting potential of the chondrocytes. Resting membrane potential of the chondrocytes at room temperature (19-21 oC) and in 5 mM external K+ was -46.4 ± 1.3 mV (mean ± s.e.m; n=23), near the 'foot' of the activation curve of this K+ current. Resting potential was depolarized by an average of 4.2 ± 0.8 mV by 25 mM TEA, which blocked about 95% of the K+ current. At a membrane potential of -50 mV, the apparent time constant of inactivation (tauin) was 37.9 s, and the 'steady-state' current level was 19% of that at a holding potential of -90 mV; at -40 mV, tauin was 20.3 s, and 'steady-state' current was 5% of that at -90 mV. These results demonstrate that in these primary cultured, mouse articular chondrocytes steady-state activation of a voltage-gated K+ current contributes to resting membrane potential. However, this current is also likely to have a significant physiological role in repolarizing the chondrocyte following depolarizing stimuli that might occur in conditions of membrane stretch. For example, activation of TRP ('transient receptor potential') non-specific cation channels in these cells during cyclic loading and unloading of the joint cartilage, or in response to hypertonic challenge is expected to result in depolarization and Ca2+ entry. Potassium currents are required to maintain the resting membrane potential.