The Inhibitory Effects of Ca2+ Channel Blocker Nifedipine on Rat Kv2.1 Potassium Channels

It is well documented that nifedipine, a commonly used dihydropyridine Ca²⁺ channel blocker, has also significant interactions with voltage-gated K⁺ (Kv) channels. But to date, little is known whether nifedipine exerted an action on Kv2.1 channels, a member of the Shab subfamily with slow inactivation. In the present study, we explored the effects of nifedipine on rat Kv2.1 channels expressed in HEK293 cells. Data from whole-cell recording showed that nifedipine substantially reduced Kv2.1 currents with the IC₅₀ value of 37.5 ± 5.7 μM and delayed the time course of activation without effects on the activation curve. Moreover, this drug also significantly shortened the duration of inactivation and deactivation of Kv2.1 currents in a voltage-dependent manner. Interestingly, the half-maximum inactivation potential (V _1/2) of Kv2.1 currents was -11.4 ± 0.9 mV in control and became -38.5 ± 0.4 mV after application of 50 μM nifedipine. The large hyperpolarizing shift (27 mV) of the inactivation curve has not been reported previously and may result in more inactivation for outward delayed rectifier K⁺ currents mediated by Kv2.1 channels at repolarization phases. The Y380R mutant significantly increased the binding affinity of nifedipine to Kv2.1 channels, suggesting an interaction of nifedipine with the outer mouth region of this channel. The data present here will be helpful to understand the diverse effects exerted by nifedipine on various Kv channels.     

Effects of Nitrendipine and Nimodipine on Low-Threshold Ca<Superscript>2+</Superscript> Channels in Thalamic Neurons of the Rat

We compared the effects of two dihydropyridines extensively used in clinical medicine, nimodipine and nitrendipine, on Ca²⁺ channels of two subtypes providing the fast and slow components of low-threshold Ca²⁺ currents in isolated neurons of the nucl. lateralis dorsalis of the thalamus of the rat; a patch-clamp technique in the whole-cell configuration was used. The fast component of the Ca²⁺ current demonstrated a higher sensitivity to nimodipine and nitrendipine (IC₅₀ = 0.6 and 0.1 µM, respectively) than the slow component (IC₅₀ = =1.09 and 0.18 µM, respectively). Maximum decreases in the current amplitude, A _max, caused by nimodipine and nitrendipine were 74 and 94% for the fast component and 55 and 80% for the slow component, respectively. Both tested agents evoked a shift of the inactivation curves of the fast component toward more negative potentials, whereas they did not significantly modify the inactivation characteristics of the slow component. Both dihydropyridines weakly influenced the characteristics of stationary activation of low-threshold Ca²⁺ channels and the blocking intensity demonstrated a voltage dependence.Neirofiziologiya/Neurophysiology, Vol. 37, No. 1, pp. 3–10, January–February, 2005.     

Effects of the Small Molecule HERG Activator NS1643 on Kv11.3 Channels

NS1643 is one of the small molecule HERG (Kv11.1) channel activators and has also been found to increase erg2 (Kv11.2) currents. We now investigated whether NS1643 is also able to act as an activator of Kv11.3 (erg3) channels expressed in CHO cells. Activation of rat Kv11.3 current occurred in a dose-dependent manner and maximal current increasing effects were obtained with 10 µM NS1643. At this concentration, steady-state outward current increased by about 80% and the current increase was associated with a significant shift in the voltage dependence of activation to more negative potentials by about 15 mV. In addition, activation kinetics were accelerated, whereas deactivation was slowed. There was no significant effect on the kinetics of inactivation and recovery from inactivation. The strong current-activating agonistic effect of NS1643 did not result from a shift in the voltage dependence of Kv11.3 channel inactivation and was independent from external Na⁺ or Ca²⁺. At the higher concentration of 20 µM, NS1643 induced clearly less current increase. The left shift in the voltage dependence of activation reversed and the voltage sensitivity of activation dramatically decreased along with a slowing of Kv11.3 channel activation. These data show that, in comparison to other Kv11 family members, NS1643 exerts distinct effects on Kv11.3 channels with especially pronounced partial antagonistic effects at higher concentration.     

The inhibitory effect of curcumin on voltage-dependent K+ channels in rabbit coronary arterial smooth muscle cells

We investigated the effects of curcumin, the principal active compound of turmeric, on voltage-dependent K⁺ (Kv) channels in freshly isolated rabbit coronary arterial smooth muscle cells using the voltage-clamp technique. Curcumin reduced the Kv current in a dose-dependent manner with an apparent K_d value of 1.07 ± 0.03 μM. Although curcumin did not alter the kinetics of Kv current activation, it predominantly accelerated the decay rate of channel inactivation. The association and dissociation rate constants of curcumin were 1.35 ± 0.05 μM^?1 s^?1 and 1.47 ± 0.17 s^?1, respectively. Curcumin did not alter the steady-state activation or inactivation curves. Application of train pulses (1 or 2 Hz) increased curcumin-induced blockade of the Kv current, and the recovery time constant also increased in the presence of curcumin suggesting, that the inhibitory action of Kv currents by curcumin was use-dependent. From these results, we concluded that curcumin inhibited vascular Kv current in a state-, time-, and use-dependent manner.     

K<Superscript>+</Superscript> Channels in Cardiomyocytes of the Pulmonate Snail <Emphasis Type="Italic">Helix</Emphasis>

We used the patch-clamp technique to identify and characterize the electrophysiological, biophysical, and pharmacological properties of K⁺ channels in enzymatically dissociated ventricular cells of the land pulmonate snail Helix. The family of outward K⁺ currents started to activate at –30 mV and the activation was faster at more depolarized potentials (time constants: at 0 mV 17.4 ± 1.2 ms vs. 2.5 ± 0.1 ms at + 60 mV). The current waveforms were similar to those of the A-type family of voltage-dependent K⁺ currents encoded by Kv4.2 in mammals. Inactivation of the current was relatively fast, i.e., 50.2 ± 1.8% of current was inactivated within 250 ms at + 40 mV. The recovery of K⁺ channels from inactivation was relatively slow with a mean time constant of 1.7 ± 0.2 s. Closer examination of steady-state inactivation kinetics revealed that the voltage dependency of inactivation was U-shaped, exhibiting less inactivation at more depolarized membrane potentials. On the basis of this phenomenon, we suggest that a channel encoded by Kv2.1 similar to that in mammals does exist in land pulmonates of the Helix genus. Outward currents were sensitive to 4-aminopyridine and tetraethylammonium chloride. The last compound was most effective, with an IC₅₀ of 336 ± 142 µmol l^–1. Thus, using distinct pharmacological and biophysical tools we identified different types of voltage-gated K⁺ channels. Present address for S.A.K.: Brigham and Womens Hospital, Cardiovascular Division, Harvard Medical School, 75 Francis St., Thorn 1216, Boston, MA 02115.     

The Human Red Cell Voltage-regulated Cation Channel. The Interplay with the Chloride Conductance,the Ca<Superscript>2+</Superscript>-activated K<Superscript>+</Superscript> Channel and the Ca<Superscript>2+</Superscript> Pump

Electrocytes from the electric organ of Electrophorus electricus exhibited sodium action potentials that have been proposed to be repolarized by leak currents and not by outward voltage-gated potassium currents. However, patch-clamp recordings have suggested that electrocytes may contain a very low density of voltage-gated K⁺ channels. We report here the cloning of a K⁺ channel from an eel electric organ cDNA library, which, when expressed in mammalian tissue culture cells, displayed delayed-rectifier K⁺ channel characteristics. The amino-acid sequence of the eel K⁺ channel had the highest identity to Kv1.1 potassium channels. However, different important functional regions of eel Kv1.1 had higher amino-acid identity to other Kv1 members, for example, the eel Kv1.1 S4-S5 region was identical to Kv1.5 and Kv1.6. Northern blot analysis indicated that eel Kv1.1 mRNA was expressed at appreciable levels in the electric organ but it was not detected in eel brain, muscle, or cardiac tissue. Because electrocytes do not express robust outward voltage-gated potassium currents we speculate that eel Kv1.1 channels are chronically inhibited in the electric organ and may be functionally recruited by an unknown mechanism.     

The calmodulin inhibitor and antipsychotic drug trifluoperazine inhibits voltage-dependent K channels in rabbit coronary arterial smooth muscle cells

We investigated the effect of the calmodulin inhibitor and antipsychotic drug trifluoperazine on voltage-dependent K⁺ (Kv) channels. Kv currents were recorded by whole-cell configuration of patch clamp in freshly isolated rabbit coronary arterial smooth muscle cells. The amplitudes of Kv currents were reduced by trifluoperazine in a concentration-dependent manner, with an apparent IC₅₀ value of 1.58 ± 0.48 μM. The rate constants of association and dissociation by trifluoperazine were 3.73 ± 0.33 μM⁻¹ s⁻¹ and 5.84 ± 1.41 s⁻¹, respectively. Application of trifluoperazine caused a positive shift in the activation curve but had no significant effect on the inactivation curve. Furthermore, trifluoperazine provoked use-dependent inhibition of the Kv current under train pulses (1 or 2 Hz). These findings suggest that trifluoperazine interacts with Kv current in a closed state and inhibits Kv current in the open state in a time- and use-dependent manner, regardless of its function as a calmodulin inhibitor and antipsychotic drug.     

The Link between Ion Permeation and Inactivation Gating of Kv4 Potassium Channels

Kv4 potassium channels undergo rapid inactivation but do not seem to exhibit the classical N-type and C-type mechanisms present in other Kv channels. We have previously hypothesized that Kv4 channels preferentially inactivate from the preopen closed state, which involves regions of the channel that contribute to the internal vestibule of the pore. To further test this hypothesis, we have examined the effects of permeant ions on gating of three Kv4 channels (Kv4.1, Kv4.2, and Kv4.3) expressed in Xenopus oocytes. Rb⁺ is an excellent tool for this purpose because its prolonged residency time in the pore delays K⁺ channel closing. The data showed that, only when Rb⁺ carried the current, both channel closing and the development of macroscopic inactivation are slowed (1.5- to 4-fold, relative to the K⁺ current). Furthermore, macroscopic Rb⁺ currents were larger than K⁺ currents (1.2- to 3-fold) as the result of a more stable open state, which increases the maximum open probability. These results demonstrate that pore occupancy can influence inactivation gating in a manner that depends on how channel closing impacts inactivation from the preopen closed state. By examining possible changes in ionic selectivity and the influence of elevating the external K⁺ concentration, additional experiments did not support the presence of C-type inactivation in Kv4 channels.     

T-channels and Na<Superscript>+</Superscript>,Ca<Superscript>2+</Superscript>-exchangers as components of the Ca<Superscript>2+</Superscript>-system of regulation of activity of the heart myocardium of the frog <Emphasis Type="Italic">Rana temporaria</Emphasis>

Earlier we have shown that regulation of rhythm and strength of the frog heart contractions, mediated by transmitters of the autonomic nervous system, is of the Ca²⁺-dependent character. In the present work, we studied chronoand inotropic effect of verapamil—an inhibitor of Ca²⁺-channels of the L-type, of nickel chloride-an inhibitor of Ca²⁺—channels of the T-type and of Na⁺,Ca²⁺exchangers as well as of adrenaline and acetylcholine (ACh) after nickel chloride. It has been found that the intracardially administered NiCh₂ at a dose of 0.01 μg/kg produced a sharp fall of amplitude of action potential (AP) and an almost twofold deceleration of heart rate (HR). The intracardiac administration of NiCh₂ (0.01 μg/kg) on the background of action of verapamil (6 mg/kg, i/m) led as soon as after 3 min to even more prominent HR deceleration and to further fall of the AP amplitude by more than 50% as compared with norm. An intracardiac administration of adrenaline (0.5 mg/kg) partly restored the cardiac activity. However, preservation of the myocardium electrical activity in such animals was brief and its duration did not exceed several minutes. Administration of Ni²⁺ on the background of acetylcholine (3.6 mg/kg) led to almost complete cessation of cardiac activity. As soon as 3 min after injection of this agent the HR decreased to 2 contractions/min. On electrograms (EG), the 10-fold fall of the AP amplitude was recorded. To elucidate role of extraand intracellular Ca²⁺ in regulation of strength of heart contractions, isometric contraction of myocardium preparations was studied in response to action of NiCl₂ (10–200 μM), verapamil (70 μM), adrenaline (5 μM), and acetylcholine (0.2 μM) after NiCl₂. It has been found that Ni²⁺ causes a dose-dependent increase of the muscle contraction amplitude. Minimal change of the contraction amplitude (on average, by 14.9% as compared with control) was recorded at a Ni²⁺ concentration of 100 μM. An increase of Ni²⁺ in the sample to 200 μM increased the cardiac contraction strength, on average, by 41%. The negative inotropic action of verapamil was essentially reduced by 100 μM Ni²⁺. Adrenaline added to the sample after Ni²⁺ produced stimulating effect on the cardiac muscle, with an almost twofold rise of the contraction amplitude. ACh (0.2 μM) decreased the cardiac contraction amplitude, on average, by 56.3%, whereas Ni²⁺ (200 μM) administered after ACh not only restored, but also stimulated partly the myocardial work. Within several parts of percent there was an increase of such isometric contraction parameters as amplitude of the effort developed by muscle, maximal rate, maximal acceleration, time of semirise and semifall. The obtained experimental results indicate that the functional activity of the frog pacemaker and contractile cardiomyocytes is regulated by Ca²⁺-dependent mechanisms. Structure of these mechanisms includes the potential-controlled Land T-channels of the plasma membrane as well as Na⁺,Ca²-exchangers characteristic exclusively of contractile cardiomyocytes. The existence of these differences seems to be due to the cardiomyocyte morphological peculiarities that appeared in evolution at the stage of the functional cell specialization.     

Inhibition of Kv4.3 by genistein via a tyrosine phosphorylation-independent mechanism

The effects of genistein, a protein tyrosine kinase (PTK) inhibitor, on voltage-dependent K(+) (Kv) 4.3 channel were examined using the whole cell patch-clamp techniques. Genistein inhibited Kv4.3 in a reversible, concentration-dependent manner with an IC(50) of 124.78 μM. Other PTK inhibitors (tyrphostin 23, tyrphostin 25, lavendustin A) had no effect on genistein-induced inhibition of Kv4.3. Orthovanadate, an inhibitor of protein phosphatases, did not reverse the inhibition of Kv4.3 by genistein. We also tested the effects of two inactive structural analogs: genistin and daidzein. Whereas Kv4.3 was unaffected by genistin, daidzein inhibited Kv4.3, albeit with a lower potency. Genistein did not affect the activation and inactivation kinetics of Kv4.3. Genistein-induced inhibition of Kv4.3 was voltage dependent with a steep increase over the channel opening voltage range. In the full-activation voltage range positive to +20 mV, no voltage-dependent inhibition was found. Genistein had no significant effect on steady-state activation, but shifted the voltage dependence of the steady-state inactivation of Kv4.3 in the hyperpolarizing direction in a concentration-dependent manner. The K(i) for the interaction between genistein and the inactivated state of Kv4.3, which was estimated from the concentration-dependent shift in the steady-state inactivation curve, was 1.17 μM. Under control conditions, closed-state inactivation was fitted to a single exponential function, and genistein accelerated closed-state inactivation. Genistein induced a weak use-dependent inhibition. These results suggest that genistein directly inhibits Kv4.3 by interacting with the closed-inactivated state of Kv4.3 channels. This effect is not mediated via inhibition of the PTK activity, because other types of PTK inhibitors could not prevent the inhibitory action of genistein.     

The effect of PI3 kinase inhibitor LY294002 on voltage-dependent K+ channels in rabbit coronary arterial smooth muscle cells

AimsWe examined the effect of LY294002, a phosphatidylinositol 3-kinase (PI3K) inhibitor, on voltage-dependent K⁺ (Kv) channels.Main methodsElectrophysiological recordings were performed in freshly isolated rabbit coronary arterial smooth muscle cells.Key findingsThe Kv current amplitude was inhibited by LY294002 in a dose-dependent manner, with a K_d value of 1.48 μM. Without alteration of the kinetics of activation, LY294002 accelerated the decay rate of Kv channel inactivation. The rate constants of association and dissociation for LY294002 were 1.83 ± 0.01 μM^? 1 s^? 1 and 2.59 ± 0.14 s^? 1, respectively. Application of LY294002 had no significant impact on the steady-state activation or inactivation curves. In the presence of LY294002, the recovery time constant from inactivation was increased, and Kv channel inhibition increased under train pulses (1 or 2 Hz). This indicates that LY294002-induced Kv channel inhibition is use-dependent. Furthermore, pretreatment with another PI3K inhibitor, wortmannin (10 μM), did not affect the Kv current, and did not change the inhibitory effect of LY294002.SignificanceBased on these results, we suggest that LY294002 directly blocks Kv current irrespective of PI3K inhibition.     

17β-Estradiol Inhibits Outward Voltage-Gated K+ Currents in Human Osteoblast-Like MG63 Cells

Previous studies have shown that 17β-estradiol has a pivotal function by blocking voltage-gated K⁺ (Kv) channels in several different types of cells such as cardiac myocytes and neurons. Outward Kv currents can also be measured in osteoblasts, although little is known about the effects of 17β-estradiol on these currents. In human osteoblast-like MG63 cells, we found that 17β-estradiol inhibits peak and end Kv currents, with IC₅₀ values of 480 and 325 nM, respectively. To elucidate the mechanism of inhibition, the kinetics of Kv currents were investigated. The half-maximum activation potential (V _1/2) was 1.3 mV and was shifted left to ?4.4 mV after application of 500 nM 17β-estradiol. For steady-state inactivation, the V _1/2 was –55.0 mV and weakly shifted left to –58.2 mV. To identify the molecular basis of outward Kv currents in MG63 cells, we performed RT-PCR analyses. The expression of Kv2.1 channels appeared to dominate over that of other Kv channels in MG63 cells. In COS-7 cells with heterologously expressed Kv2.1 channels, 17β-estradiol also inhibits macroscopic currents of Kv2.1. Our data indicate that 17β-estradiol inhibits Kv currents in human osteoblast-like MG63 cells and that Kv2.1 is a potential molecular correlate of outward Kv currents in these cells.     

Effects of intracellular magnesium on Kv1.5 and Kv2.1 potassium channels

We characterized the effects of intracellular Mg²⁺ (Mg²⁺_i) on potassium currents mediated by the Kv1.5 and Kv2.1 channels expressed in Xenopus oocytes. Increase in Mg²⁺_i caused a voltage-dependent block of the current amplitude, apparent acceleration of the current kinetics (explained by a corresponding shift in the steady-state activation) and leftward shifts in activation and inactivation dependencies for both channels. The voltage-dependent block was more potent for Kv2.1 [dissociation constant at 0 mV, K_d(0), was ~70 mM and the electric distance of the Mg²⁺ binding site, , was 0.2] than for the Kv1.5 channel [K_d(0)~40 mM and =0.1]. Similar shifts in the voltage-dependent parameters for both channels were described by the Gouy-Chapman formalism with the negative charge density of 1 e^–/100 Ų. Additionally, Mg²⁺_i selectively reduced a non-inactivating current and increased the accumulation of inactivation of the Kv1.5, but not the Kv2.1 channel. A potential functional role of the differential effects of Mg²⁺_i on the Kv channels is discussed.     

Comparison of the endogenous IK currents in rat hippocampal neurons and cloned Kv2.1 channels in CHO cells

The Kv2.1 potassium channel is a principal component of the delayed rectifier I(K) current in the pyramidal neurons of cortex and hippocampus. We used whole-cell patch-clamp recording techniques to systemically compare the electrophysiological properties between the native neuronal I(K) current of cultured rat hippocampal neurons and the cloned Kv2.1 channel currents in the CHO cells. The slope factors for the activation curves of both currents obtained at different prepulse holding potentials and holding times were similar, suggesting similar voltage-dependent gating. However, the half-maximal activation voltage for I(K) was approximately 20 mV more negative than the Kv2.1 channel in CHO cells at a given prepulse condition, indicating that the neuronal I(K) current had a lower threshold for activation than that of the Kv2.1 channel. In addition, the neuronal I(K) showed a stronger holding membrane potential and holding time-dependence than Kv2.1. The Kv2.1 channel gave a U-shaped inactivation, while the I(K) current did not. The I(K) current also had much stronger voltage-dependent inactivation than Kv2.1. These results imply that the neuronal factors could make Kv2.1 channels easier to activate. The information obtained from these comparative studies help elucidate the mechanism of molecular regulation of the native neuronal I(K) current in neurons.     

DPP10 is an inactivation modulatory protein of Kv4.3 and Kv1.4

Voltage-gated K⁺ channels exist in vivo as multiprotein complexes made up of pore-forming and ancillary subunits. To further our understanding of the role of a dipeptidyl peptidase-related ancillary subunit, DPP10, we expressed it with Kv4.3 and Kv1.4, two channels responsible for fast-inactivating K⁺ currents. Previously, DPP10 has been shown to effect Kv4 channels. However, Kv1.4, when expressed with DPP10, showed many of the same effects as Kv4.3, such as faster time to peak current and negative shifts in the half-inactivation potential of steady-state activation and inactivation. The exception was recovery from inactivation, which is slowed by DPP10. DPP10 expressed with Kv4.3 caused negative shifts in both steady-state activation and inactivation of Kv4.3, but no significant shifts were detected when DPP10 was expressed with Kv4.3 + KChIP2b (Kv channel interacting protein). DPP10 and KChIP2b had different effects on closed-state inactivation. At –60 mV, KChIP2b nearly abolishes closed-state inactivation in Kv4.3, whereas it developed to a much greater extent in the presence of DPP10. Finally, expression of a DPP10 mutant consisting of its transmembrane and cytoplasmic 58 amino acids resulted in effects on Kv4.3 gating that were nearly identical to those of wild-type DPP10. These data show that DPP10 and KChIP2b both modulate Kv4.3 inactivation but that their primary effects are on different inactivation states. Thus DPP10 may be a general modulator of voltage-gated K⁺ channel inactivation; understanding its mechanism of action may lead to deeper understanding of the inactivation of a broad range of K⁺ channels. potassium channel inactivation; potassium channel ancillary subunits; closed-state inactivation; voltage-gated potassium channels     

The influence of protons and zinc ions on the steady-state inactivation of Kv1.3 potassium channels

Using the whole-cell patch-clamp technique, we investigated the influence of extracellular pH and zinc ions (Zn²⁺) on the steady-state inactivation of Kv1.3 channels expressed in human lymphocytes. The obtained data showed that lowering the extracellular pH from 7.35 to 6.8 shifted the inactivation midpoint (V_i) by 17.4 ± 1.12 mV (n = 6) towards positive membrane potentials. This shift was statistically significant (p < 0.05). Applying 100 μM Zn²⁺ at pH 6.8 further shifted the Vi value by 16.55 ± 1.80 mV (n = 6) towards positive membrane potentials. This shift was also statistically significant (p < 0.05). The total shift of the Vi by protons and Zn²⁺ was 33.95 ± 1.90 mV (n = 6), which was significantly higher (p < 0.05) than the shift caused by Zn²⁺ alone. The Zn²⁺-induced shift of the V_i at pH 6.8 was almost identical to the shift at pH = 7.35. Thus, the proton-and Zn²⁺-induced shifts of the V_i value were additive. The steady-state inactivation curves as a function of membrane voltage were compared with the functions of the steady-state activation. The total shift of the steady-state inactivation was almost identical to the total shift of the steady-state activation (32.01 ± 2.10 mV, n = 10). As a result, the “windows” of membrane potentials in which the channels can be active under physiological conditions were also markedly shifted towards positive membrane potentials. The values of membrane voltage and the normalised chord conductance corresponding to the points of intersection of the curves of steady-state activation and inactivation were also calculated. The possible physiological significance of the observed modulatory effects is discussed herein.     

Forskolin Suppresses Delayed-Rectifier K+ Currents and Enhances Spike Frequency-Dependent Adaptation of Sympathetic Neurons

In signal transduction research natural or synthetic molecules are commonly used to target a great variety of signaling proteins. For instance, forskolin, a diterpene activator of adenylate cyclase, has been widely used in cellular preparations to increase the intracellular cAMP level. However, it has been shown that forskolin directly inhibits some cloned K⁺ channels, which in excitable cells set up the resting membrane potential, the shape of action potential and regulate repetitive firing. Despite the growing evidence indicating that K⁺ channels are blocked by forskolin, there are no studies yet assessing the impact of this mechanism of action on neuron excitability and firing patterns. In sympathetic neurons, we find that forskolin and its derivative 1,9-Dideoxyforskolin, reversibly suppress the delayed rectifier K⁺ current (I_KV). Besides, forskolin reduced the spike afterhyperpolarization and enhanced the spike frequency-dependent adaptation. Given that I_KV is mostly generated by Kv2.1 channels, HEK-293 cells were transfected with cDNA encoding for the Kv2.1 α subunit, to characterize the mechanism of forskolin action. Both drugs reversible suppressed the Kv2.1-mediated K⁺ currents. Forskolin inhibited Kv2.1 currents and I_KV with an IC₅₀ of ~32 μM and ~24 µM, respectively. Besides, the drug induced an apparent current inactivation and slowed-down current deactivation. We suggest that forskolin reduces the excitability of sympathetic neurons by enhancing the spike frequency-dependent adaptation, partially through a direct block of their native Kv2.1 channels.     

Identification and functional characterization of ion channels in CD34<Superscript>+</Superscript> hematopoietic stem cells from human peripheral blood

Hematopoietic stem cells (HSCs) are used therapeutically for hematological diseases and may also serve as a source for nonhematopoietic tissue engineering in the future. In other cell types, ion channels have been investigated as potential targets for the regulation of proliferation and differentiation. However, the ion channels of HSCs remain elusive. Here, we functionally characterized the ion channels of CD34⁺ cells from human peripheral blood. Using fluorescence-activated cell sorting, we confirmed that the CD34⁺ cells also express CD45 and CD133. In the CD34⁺/CD45⁺/CD133^high HSCs, RT-PCR of 58 ion channel mRNAs revealed the coexpression of Kv1.3, Kv7.1, Nav1.7, TASK2, TALK2, TWIK2, TRPC4, TRPC6, TRPM2, TRPM7, and TRPV2. Whole-cell patch clamp recordings identified voltage-gated K⁺ currents (putatively Kv1.3), pH-sensitive TASK2-like back-ground K⁺ currents, ADP-ribose-activated TRPM2 currents, temperature-sensitive TRPV2-like currents, and diacylglycerol-analogue-activated TRPC6-like currents. Our results lend new insight into the physiological role of ion channels in HSCs, the specific implications of which require further investigation.     

Kv1.3 potassium channel-blocking toxin Ctri9577, novel gating modifier of Kv4.3 potassium channel from the scorpion toxin family

Scorpion toxin Ctri9577, as a potent Kv1.3 channel blocker, is a new member of the α-KTx15 subfamily which are a group of blockers for Kv4.x potassium channels. However, the pharmacological function of Ctri9577 for Kv4.x channels remains unknown. Scorpion toxin Ctri9577 was found to effectively inhibit Kv4.3 channel currents with IC₅₀ value of 1.34 ± 0.03 μM. Different from the mechanism of scorpion toxins as the blocker recognizing channel extracellular pore entryways, Ctri9577 was a novel gating modifier affecting voltage dependence of activation, steady-state inactivation, and the recovery process from the inactivation of Kv4.3 channel. However, Ctri9755, as a potent Kv1.3 channel blocker, was found not to affect voltage dependence of activation of Kv1.3 channel. Interestingly, pharmacological experiments indicated that 1 μM Ctri9755 showed less inhibition on Kv4.1 and Kv4.2 channel currents. Similar to the classical gating modifier of spider toxins, Ctri9577 was shown to interact with the linker between the transmembrane S3 and S4 helical domains through the mutagenesis experiments. To the best of our knowledge, Ctri9577 was the first gating modifier of potassium channels among scorpion toxin family, and the first scorpion toxin as both gating modifier and blocker for different potassium channels. These findings further highlighted the structural and functional diversity of scorpion toxins specific for the potassium channels.     

Role of N-terminal domain and accessory subunits in controlling deactivation-inactivation coupling of Kv4.2 channels

We examined the relationship between deactivation and inactivation in Kv4.2 channels. In particular, we were interested in the role of a Kv4.2 N-terminal domain and accessory subunits in controlling macroscopic gating kinetics and asked if the effects of N-terminal deletion and accessory subunit coexpression conform to a kinetic coupling of deactivation and inactivation. We expressed Kv4.2 wild-type channels and N-terminal deletion mutants in the absence and presence of Kv channel interacting proteins (KChIPs) and dipeptidyl aminopeptidase-like proteins (DPPs) in human embryonic kidney 293 cells. Kv4.2-mediated A-type currents at positive and deactivation tail currents at negative membrane potentials were recorded under whole-cell voltage-clamp and analyzed by multi-exponential fitting. The observed changes in Kv4.2 macroscopic inactivation kinetics caused by N-terminal deletion, accessory subunit coexpression, or a combination of the two maneuvers were compared with respective changes in deactivation kinetics. Extensive correlation analyses indicated that modulatory effects on deactivation closely parallel respective effects on inactivation, including both onset and recovery kinetics. Searching for the structural determinants, which control deactivation and inactivation, we found that in a Kv4.2Δ2-10 N-terminal deletion mutant both the initial rapid phase of macroscopic inactivation and tail current deactivation were slowed. On the other hand, the intermediate and slow phase of A-type current decay, recovery from inactivation, and tail current decay kinetics were accelerated in Kv4.2Δ2-10 by KChIP2 and DPPX. Thus, a Kv4.2 N-terminal domain, which may control both inactivation and deactivation, is not necessary for active modulation of current kinetics by accessory subunits. Our results further suggest distinct mechanisms for Kv4.2 gating modulation by KChIPs and DPPs.