Modulation of the epithelial calcium channel, ECaC, by intracellular Ca2+

We have studied the modulation by intracellular Ca2+ of the epithelial Ca2+ channel, ECaC, heterologously expressed in HEK 293 cells. Whole-cell and inside-out patch clamp current recordings were combined with FuraII-Ca2+ measurements:1. Currents through ECaC were dramatically inhibited if Ca2+ was the charge carrier. This inhibition was dependent on the extracellular Ca2+ concentration and occurred also in cells buffered intracellularly with 10 mM BAPTA.2. Application of 30 mM [Ca(2)]e induced in non-Ca2+] buffered HEK 293 cells at -80 m V an increase in intracellular Ca2+([Ca2]i) with a maximum rate of rise of 241 +/-15nM/s (n= 18 cells) and a peak value of 891 +/- 106 nM. The peak of the concomitant current with a density of 12.3 +/- 2.6 pA/pF was closely correlated with the peak of the first-time derivative of the Ca2+ transient, as expected if the Ca2+ transient is due to influx of Ca2+. Consequently, no Ca2+] signal was observed in cells transfected with the Ca2+ impermeable ECaC mutant, D542A, in which an aspartate in the pore region was neutralized.3. Increasing [Ca2+]i by dialyzing the cell with pipette solutions containing various Ca2+] concentrations, all buffered with 10 mM BAPTA, inhibited currents through ECaC carried by either Na+ or Ca2+] ions. Half maximal inhibition of Ca(2+)currents in the absence of monovalent cations occurred at 67 nM (n between 6 and 8), whereas Na+ currents in the absence of Ca2+] and Mg2+ were inhibited with an IC50 of 89 nM (n between 6 and 10). Currents through ECaC in the presence of 1 mM Ca2+ and Na+, which are mainly carried by Ca2+, are inhibited by [Ca2]i with an IC50of 82 nM (n between 6 and 8). Monovalent cation currents through the Ca2+impermeable D542A ECaC mutant were also inhibited by an elevation of [Ca2]i (IC50 = 123 nM, n between 7 and 18). 4. The sensitivity of ECaC currents in inside-out patches for [Ca2]i was slightly shifted to higher concentrations as compared with whole cell measurements. Half-maximal inhibition occurred at 169 nM if Na+ was the charge carrier (n between 4 and 11) and 228 nM at 1 mM [Ca2]e (n between 4 and 8).5. Recovery from inhibition upon washout of extracellular Ca2+ (whole-cell configuration) or removal of Ca2+ from the inner side of the channel (inside-out patches) was slow in both conditions. Half-maximal recovery was reached after 96 +/- 34 s (n= 15) in whole-cell mode and after 135 +/- 23 s (n = 17) in inside-out patches.6. We conclude that influx of Ca2+ through ECaC and [Ca2]i induce feedback inhibition of ECaC currents, which is controlled by the concentration of Ca2+ in a micro domain near the inner mouth of the channel. Slow recovery seems to depend on dissociation of Ca( 2+ from an internal Ca2+ binding site at ECaC.     

Expression of voltage dependent potassium currents in freshly dissociated rat articular chondrocytes.

The electrophysiological properties of voltage dependent potassium channels from freshly dissociated rat articular chondrocytes were studied. The resting membrane potential (-42.7+/-2.0 mV) was significantly depolarized by increasing concentrations of external potassium. No change was observed when external chloride concentration was varied. Addition of TEA, 4AP, alpha-Dendrotoxin and charybdotoxin depolarized resting membrane potential. Whole cell patch clamp studies revealed the presence of outwardly rectifying currents whose kinetic and pharmacological properties suggest the expression of voltage dependent potassium channels. Two kinds of currents were observed under the same experimental conditions. The first one, most frequently observed (80%), starts activating near -50 mV, with V(1/2)=-18 mV, G(max)=0.30 pS/pF. The second kind was observed in only 10% of cases; It activates near -40 mV, with(1/2)=+28.35 mV, G(max)=0.28 pS/pF pA/pF and does not inactivates. Inactivating currents were significantly inhibited by TEA (IC(50)=1.45 mM), 4AP (IC(50)=0.64 mM), CTX (IC(50) = 10 nM), alpha-Dendrotoxin (IC(50) < 100 nM) and Margatoxin (IC(50)=28.5 nM). These results show that rat chondrocytes express voltage dependent potassium currents and suggest a role of voltage-dependent potassium channels in regulating membrane potential of rat chondrocytes.     

Reverse engineering the L-type Ca2+ channel alpha1c subunit in adult cardiac myocytes using novel adenoviral vectors

The alpha(1c) subunit of the cardiac L-type Ca(2+) channel, which contains the channel pore, voltage- and Ca(2+)-dependent gating structures, and drug binding sites, has been well studied in heterologous expression systems, but many aspects of L-type Ca(2+) channel behavior in intact cardiomyocytes remain poorly characterized. Here, we develop adenoviral constructs with E1, E3 and fiber gene deletions, to allow incorporation of full-length alpha(1c) gene cassettes into the adenovirus backbone. Wild-type (alpha(1c-wt)) and mutant (alpha(1c-D-)) Ca(2+) channel adenoviruses were constructed. The alpha(1c-D-) contained four point substitutions at amino acid residues known to be critical for dihydropyridine binding. Both alpha(1c-wt) and alpha(1c-D-) expressed robustly in A549 cells (peak L-type Ca(2+) current (I(CaL)) at 0 mV: alpha(1c-wt) -9.94+/-1.00pA/pF, n=9; alpha(1c-D-) -10.30pA/pF, n=12). I(CaL) carried by alpha(1c-D-) was markedly less sensitive to nitrendipine (IC(50) 17.1 microM) than alpha(1c-wt) (IC(50) 88 nM); a feature exploited to discriminate between engineered and native currents in transduced guinea-pig myocytes. 10 microM nitrendipine blocked only 51+/-5% (n=9) of I(CaL) in alpha(1c-D-)-expressing myocytes, in comparison to 86+/-8% (n=9) of I(CaL) in control myocytes. Moreover, in 20 microM nitrendipine, calcium transients could still be evoked in alpha(1c-D-)-transduced cells, but were largely blocked in control myocytes, indicating that the engineered channels were coupled to sarcoplasmic reticular Ca(2+) release. These alpha(1c) adenoviruses provide an unprecedented tool for structure-function studies of cardiac excitation-contraction coupling and L-type Ca(2+) channel regulation in the native myocyte background.     

Four kinetically distinct depolarization-activated K+ currents in adult mouse ventricular myocytes

In the experiments here, the time- and voltage-dependent properties of the Ca2+-independent, depolarization-activated K+ currents in adult mouse ventricular myocytes were characterized in detail. In the majority (65 of 72, approximately 90%) of cells dispersed from the ventricles, analysis of the decay phases of the outward currents revealed three distinct K+ current components: a rapidly inactivating, transient outward K+ current, Ito,f (mean +/- SEM taudecay = 85 +/- 2 ms); a slowly (mean +/- SEM taudecay = 1,162 +/- 29 ms) inactivating K+ current, IK,slow; and a non inactivating, steady state current, Iss. In a small subset (7 of 72, approximately 10%) of cells, Ito,f was absent and a slowly inactivating (mean +/- SEM taudecay = 196 +/- 7 ms) transient outward current, referred to as Ito,s, was identified; the densities and properties of IK,slow and Iss in Ito,s-expressing cells are indistinguishable from the corresponding currents in cells with Ito,f. Microdissection techniques were used to remove tissue pieces from the left ventricular apex and from the ventricular septum to allow the hypothesis that there are regional differences in Ito,f and Ito,s expression to be tested directly. Electrophysiological recordings revealed that all cells isolated from the apex express Ito,f (n = 35); Ito,s is not detected in these cells (n = 35). In the septum, by contrast, all of the cells express Ito,s (n = 28) and in the majority (22 of 28, 80%) of cells, Ito,f is also present. The density of Ito,f (mean +/- SEM at +40 mV = 6.8 +/- 0.5 pA/pF, n = 22) in septum cells, however, is significantly (P < 0.001) lower than Ito,f density in cells from the apex (mean +/- SEM at +40 mV = 34.6 +/- 2.6 pA/pF, n = 35). In addition to differences in inactivation kinetics, Ito,f, Ito,s, and IK,slow display distinct rates of recovery (from inactivation), as well as differential sensitivities to 4-aminopyridine (4-AP), tetraethylammonium (TEA), and Heteropoda toxin-3. IK,slow, for example, is blocked selectively by low (10-50 microM) concentrations of 4-AP and by (>/=25 mM) TEA. Although both Ito,f and Ito,s are blocked by high (>100 microM) 4-AP concentrations and are relatively insensitive to TEA, Ito,f is selectively blocked by nanomolar concentrations of Heteropoda toxin-3, and Ito,s (as well as IK,slow and Iss) is unaffected. Iss is partially blocked by high concentrations of 4-AP or TEA. The functional implications of the distinct properties and expression patterns of Ito,f and Ito,s, as well as the likely molecular correlates of these (and the IK,slow and Iss) currents, are discussed.     

Prenatal stress on the kinetic properties of Ca2+ and K+ channels in offspring hippocampal CA3 pyramidal neurons

Prenatal stress is known to cause neuronal loss and oxidative damage in the hippocampus of offspring rats. To further understand the mechanisms, the present study was undertaken to investigate the effects of prenatal stress on the kinetic properties of high-voltage-activated (HVA) Ca(2+) and K(+) channels in freshly isolated hippocampal CA3 pyramidal neurons of offspring rats. Pregnant rats in the prenatal stress group were exposed to restraint stress on days 14-20 of pregnancy three times daily for 45 min. The patch clamp technique was employed to record HVA Ca(2+) and K(+) channel currents. Prenatal stress significantly increased HVA Ca(2+) channel disturbance including the maximal average HVA calcium peak current amplitude (-576.52+/-7.03 pA in control group and -702.05+/-6.82 pA in prenatal stress group, p<0.01), the maximal average HVA Ca(2+) current density (-40.89+/-0.31 pA/pF in control group and -49.44+/-0.37 pA/pF in prenatal stress group, p<0.01), and the maximal average integral current of the HVA Ca(2+) channel (106.81+/-4.20 nA ms in control group and 133.49+/-4.59 nA ms in prenatal stress group, p<0.01). The current-voltage relationship and conductance--voltage relationship of HVA Ca(2+) channels and potassium channels in offspring CA3 neurons were not affected by prenatal stress. These data suggest that exposure of animals to stressful experience during pregnancy can exert effects on calcium ion channels of offspring hippocampal neurons and that the calcium channel disturbance may play a role in prenatal stress-induced neuronal loss and oxidative damage in offspring brain.     

Reduced functional expression of K(+) channels in vascular smooth muscle cells from rats made hypertensive with N{omega}-nitro-L-arginine

Smooth muscle membrane potential is determined, in part, by K(+) channels. In the companion paper to this article, we demonstrated that superior mesenteric arteries from rats made hypertensive with N(omega)-nitro-l-arginine (l-NNA) are depolarized and express less K(+) channel protein compared with those from normotensive rats. In the present study, we used patch-clamp techniques to test the hypothesis that l-NNA-induced hypertension reduces the functional expression of K(+) channels in smooth muscle. In whole cell experiments using a Ca(2+)-free pipette solution, current at 0 mV, largely due to voltage-dependent K(+) (K(V)) channels, was reduced approximately 60% by hypertension (2.7 +/- 0.4 vs. 1.1 +/- 0.2 pA/pF). Current at +100 mV with 300 nM free Ca(2+), largely due to large-conductance Ca(2+)-activated K(+) (BK(Ca)) channels, was reduced approximately 40% by hypertension (181 +/- 24 vs. 101 +/- 28 pA/pF). Current blocked by 3 mM 4-aminopyridine, an inhibitor of many K(V) channel types, was reduced approximately 50% by hypertension (1.0 +/- 0.4 vs. 0.5 +/- 0.2 pA/pF). Current blocked by 1 mM tetraethylammonium, an inhibitor of BK(Ca) channels, was reduced approximately 40% by hypertension (86 +/- 14 vs. 53 +/- 19 pA/pF). Differences in BK(Ca) current magnitude are not attributable to changes in single-channel conductance or Ca(2+)/voltage sensitivity. The data support the hypothesis that l-NNA-induced hypertension reduces K(+) current in vascular smooth muscle. Reduced molecular and functional expression of K(+) channels may partly explain the depolarization and augmented contractile sensitivity of smooth muscle from l-NNA-treated rats.     

The beta subunit increases Ca2+ currents and gating charge movements of human cardiac L-type Ca2+ channels.

The properties of the gating currents (nonlinear charge movements) of human cardiac L-type Ca2- channels and their relationship to the activation of the Ca2+ channel (ionic) currents were studied using a mammalian expression system. Cloned human cardiac alpha1 + rabbit alpha 2 subunits or human cardiac alpha 1 + rabbit alpha 2 + human beta 3 subunits were transiently expressed in HEK293 cells. The maximum Ca2+ current density increased from -3.9 +/- 0.9 pA/pF for the alpha 1 + alpha 2 subunits to -11.6 +/- 2.2 pA/pF for alpha 1 + alpha 2 + beta 3 subunits. Calcium channel gating currents were recorded after the addition of 5 mM Co2+, using a -P/5 protocol. The maximum nonlinear charge movement (Qmax) increased from 2.5 +/- 0.3 nC/muF for alpha 1 + alpha 2 subunit to 12.1 +/- 0.3 nC/muF for alpha 1 + alpha 2 + beta 3 subunit expression. The QON was equal to the QOFF for both subunit combinations. The QON-Vm data were fit by a sum of two Boltzmann expressions and ranged over more negative potentials, as compared with the voltage dependence for activation of the Ca2+ conductance. We conclude that 1) the beta subunit increases the number of functional alpha 1 subunits expressed in the plasma membrane of these cells and 2) the voltage-dependent activation of the human cardiac L-type calcium channel involves the movements of at least two nonidentical and functionally distinct gating structures.     

Deranged Kv channel regulation in fibroblasts from mice lacking the serum and glucocorticoid inducible kinase SGK1

Coexpression of the serum and glucocorticoid inducible kinase 1 (SGK1) up-regulates Kv channel activity in Xenopus oocytes and human embryonic kidney cells. To investigate the physiological impact of SGK1 dependent Kv channel regulation, we recorded whole-cell currents in lung fibroblasts from SGK1 knockout mice (sgk1-/-) and wild-type littermates (sgk1+/+). Serum-grown mouse lung fibroblasts (MLF) from both genotypes exhibited voltage-gated outwardly rectifying K(+)-currents with time-dependent activation (tau(act) approximately 3 msec), slow inactivation (tau(inact) approximately 700 msec), use-dependent inactivation, and (partial) inhibition by K(+) channel blockers TEA, 4-AP, and margatoxin. In serum grown MLF peak Kv current density at +100 mV was significantly lower in sgk1-/- (14 +/- 2 pA/pF, n = 13) than in sgk1+/+ (31 +/- 4 pA/pF, n = 16). PCR amplification of different Kv1 and Kv3 subunits from mouse fibroblasts demonstrated the expression of Kv1.1-1.7, Kv3.1, and Kv3.3 mRNA in both sgk1+/+ and sgk1-/- cells. Upon serum deprivation Kv currents almost disappeared in sgk1+/+ (4 +/- 1 pA/pF, n = 11) but not in sgk1-/- (10 +/- 1 pA/pF, n = 6) MLF. Accordingly, following serum deprivation Kv current density was significantly lower in sgk1+/+ than in sgk1-/-. Stimulation of serum-depleted cells with dexamethasone (dex) (1 microM, 1 day), IGF-1 (6.7 microM, 4-6 h) or both, significantly activated Kv currents in sgk1+/+ but not in sgk1-/- MLF. In the presence of both, dex and IGF-1, the Kv current density was significantly larger in sgk1+/+ (27 +/- 3 pA/pF, n = 12) than in sgk1-/- (13 +/- 3 pA/pF, n = 10) cells. Similar to MLF, Kv currents were significantly higher in sgk1+/+ mouse tail fibroblasts (MTF). In sgk1+/+ but not sgk1-/- MTF the Kv currents were inhibited upon serum deprivation and reincreased after stimulation of serum deprived MTF with dex (1 microM, 1 day) and afterwards with IGF-1 (6.7 microM, 4-6 h). According to Fura-2-fluorescence capacitative Ca(2+) entry was lower in sgk1-/- MTF compared to sgk1+/+ MTF. Upon serum deprivation capacitative Ca(2+) entry decreased significantly in sgk1+/+ but not in sgk1-/- MTF. Stimulation of depleted cells with dex (1 microM, 1 day) and afterwards with IGF-1 (6.7 microM, 4-6 h) reincreased capacitative Ca(2+) entry in sgk1+/+ MTF, whereas in sgk1-/- cells it remained unchanged. In conclusion, lack of SGK1 does not abrogate Kv channel activity but abolishes regulation of those channels by serum, glucocorticoids and IGF-1, an effect influencing capacitative Ca(2+) entry.     

Functional coupling between 'R-type' Ca2+ channels and insulin secretion in the insulinoma cell line INS-1.

Among voltage-gated Ca2+ channels the non-dihydropyridine-sensitive alpha1E subunit is functionally less well characterized than the structurally related alpha1A (omega-agatoxin-IVA sensitive, P- /Q-type) and alpha1B (omega-conotoxin-GVIA sensitive, N-type) subunits. In the rat insulinoma cell line, INS-1, a tissue-specific splice variant of alpha1E (alpha1Ee) has been characterized at the mRNA and protein levels, suggesting that INS-1 cells are a suitable model for investigating the function of alpha1Ee. In alpha1E-transfected human embryonic kidney (HEK-293) cells the alpha1E-selective peptide antagonist SNX-482 (100 nM) reduces alpha1Ed- and alpha1Ee-induced Ba2+ inward currents in the absence and presence of the auxiliary subunits beta3 and alpha2delta-2 by more than 80%. The inhibition is fast and only partially reversible. No effect of SNX-482 was detected on the recombinant T-type Ca2+ channel subunits alpha1G, alpha1H, and alpha1I showing that the toxin from the venom of Hysterocrates gigas is useful as an alpha1E-selective antagonist. After blocking known components of Ca2+ channel inward current in INS-1 cells by 2 microM (+/-)-isradipine plus 0.5 microM omega-conotoxin-MVIIC, the remaining current is reduced by 100 nM SNX-482 from -12.4 +/- 1.2 pA/pF to -7.6 +/- 0.5 pA/pF (n = 9). Furthermore, in INS-1 cells, glucose- and KCl-induced insulin release are reduced by SNX-482 in a dose-dependent manner leading to the conclusion that alpha1E, in addition to L-type and non-L-type (alpha1A-mediated) Ca2+ currents, is involved in Ca2+ dependent insulin secretion of INS-1 cells.     

Vasoactive intestinal peptide-stimulated Cl- secretion: activation of cAMP-dependent K+ channels

Vasoactive intestinal peptide (VIP) stimulates active Cl- secretion by the intestinal epithelium, a process that depends upon the maintenance of a favorable electrical driving force established by a basolateral membrane K+ conductance. To demonstrate the role of this K- conductance, we measured short-circuit current (I(SC)) across monolayers of the human colonic secretory cell line, T84. The serosal application of VIP (50 nM) increased I(SC) from 3 +/- 0.4 microA/cm2 to 75 +/- 11 microA/cm2 (n = 4), which was reduced to a near zero value by serosal applications of Ba2+ (5 mM). The chromanol, 293B (100 microM), reduced I(SC) by 74%, but charybdotoxin (CTX, 50 nM) had no effect. We used the whole-cell voltage-clamp technique to determine whether the K+ conductance is regulated by cAMP-dependent phosphorylation in isolated cells. VIP (300 nM) activated K+ current (131 +/- 26 pA, n = 15) when membrane potential was held at the Cl- equilibrium potential (E(Cl-) = -2 mV), and activated inward current (179 +/- 28 pA, n = 15) when membrane potential was held at the K+ equilibrium potential (E(K+) = -80 mV); however, when the cAMP-dependent kinase (PKA) inhibitor, PKI (100 nM), was added to patch pipettes, VIP failed to stimulate these currents. Barium (Ba2+ , 5 mM), but not 293B, blocked this K+ conductance in single cells. We used the cell-attached membrane patch under conditions that favor K + current flow to demonstrate the channels that underlie this K+ conductance. VIP activated inwardly rectifying channel currents in this configuration. Additionally, we used fura-2AM to show that VIP does not alter the intracellular Ca2+ concentration, [Ca2 +]i. Caffeine (5 mM), a phosphodiesterase inhibitor, also stimulated K+ current (185 +/- 56 pA, n = 8) without altering [Ca2+]i. These results demonstrate that VIP activates a basolateral membrane K+ conductance in T84 cells that is regulated by cAMP-dependent phosphorylation.     

Functional alterations in cerebrovascular K(+) and Ca(2+) channels are comparable between simulated microgravity rat and SHR

Exposure to microgravity leads to a sustained elevation in transmural pressure across the cerebral vasculature due to removal of hydrostatic pressure gradients. We hypothesized that ion channel remodeling in cerebral vascular smooth muscle cells (VSMCs) similar to that associated with hypertension may occur and play a role in upward autoregulation of cerebral vessels during microgravity. Sprague-Dawley rats were subjected to 4-wk tail suspension (Sus) to simulate the cardiovascular effect of microgravity. Large-conductance Ca(2+)-activated K(+) (BK(Ca)), voltage-gated K(+) (K(V)), and L-type voltage-dependent Ca(2+) (Ca(L)) currents of Sus and control (Con) rat cerebral VSMCs were investigated with a whole cell voltage-clamp technique. Under the same experimental conditions, K(V), BK(Ca), and Ca(L) currents of cerebral VSMCs from adult spontaneously hypertensive rats (SHR) and Wistar-Kyoto rats (WKY) were also investigated. K(V) current density decreased in Sus rats vs. Con rats [1.07 +/- 0.14 (n = 22) vs. 1.31 +/- 0.28 (n = 16) pA/pF at +20 mV (P < 0.05)] and BK(Ca) and Ca(L) current densities increased [BK(Ca): 1.70 +/- 0.37 (n = 23) vs. 0.88 +/- 0.22 (n = 19) pA/pF at +20 mV (P < 0.05); Ca(L): -2.17 +/- 0.21 (n = 35) vs. -1.31 +/- 0.10 (n = 26) pA/pF at +10 mV (P < 0.05)]. Similar changes were also observed in SHR vs. WKY cerebral VSMCs: K(V) current density decreased [1.03 +/- 0.33 (n = 9) vs. 1.62 +/- 0.64 (n = 9) pA/pF at +20 mV (P < 0.05)] and BK(Ca) and Ca(L) current densities increased [BK(Ca): 2.54 +/- 0.47 (n = 11) vs. 1.12 +/- 0.33 (n = 12) pA/pF at +20 mV (P < 0.05); Ca(L): -3.99 +/- 0.53 (n = 12) vs. -2.28 +/- 0.20 (n = 10) pA/pF at +20 mV (P < 0.05)]. These findings support our hypothesis, and their impact on space cardiovascular research is discussed.     

Recovery of Ca2+ current, charge movements, and Ca2+ transients in myotubes deficient in dihydropyridine receptor beta 1 subunit transfected with beta 1 cDNA.

The Ca2+ currents, charge movements, and intracellular Ca2+ transients of mouse dihydropyridine receptor (DHPR) beta 1-null myotubes expressing a mouse DHPR beta 1 cDNA have been characterized. In beta 1-null myotubes maintained in culture for 10-15 days, the density of the L-type current was approximately 7-fold lower than in normal cells of the same age (Imax was 0.65 +/- 0.05 pA/pF in mutant versus 4.5 +/- 0.8 pA/pF in normal), activation of the L-type current was significantly faster (tau activation at +40 mV was 28 +/- 7 ms in mutant versus 57 +/- 8 ms in normal), charge movements were approximately 2.5-fold lower (Qmax was 2.5 +/- 0.2 nC/microF in mutant versus 6.3 +/- 0.7 nC/microF in normal), Ca2+ transients were not elicited by depolarization, and spontaneous or evoked contractions were absent. Transfection of beta 1-null cells by lipofection with beta 1 cDNA reestablished spontaneous or evoked contractions in approximately 10% of cells after 6 days and approximately 30% of cells after 13 days. In contracting beta 1-transfected myotubes there was a complete recovery of the L-type current density (Imax was 4 +/- 0.9 pA/pF), the kinetics of activation (tau activation at +40 mV was 64 +/- 5 ms), the magnitude of charge movements (Qmax was 6.7 +/- 0.4 nC/microF), and the amplitude and voltage dependence of Ca2+ transients evoked by depolarizations. Ca2+ transients of transfected cells were unaltered by the removal of external Ca2+ or by the block of the L-type Ca2+ current, demonstrating that a skeletal-type excitation-contraction coupling was restored. The recovery of the normal skeletal muscle phenotype in beta 1-transfected beta-null myotubes shows that the beta 1 subunit is essential for the functional expression of the DHPR complex.     

Reduced Ca2+ current, charge movement, and absence of Ca2+ transients in skeletal muscle deficient in dihydropyridine receptor beta 1 subunit.

The Ca2+ currents, charge movements, and intracellular Ca2+ transients in mouse skeletal muscle cells homozygous for a null mutation in the cchb1 gene encoding the beta 1 subunit of the dihydropyridine receptor have been characterized. I beta null, the L-type Ca2+ current of mutant cells, had a approximately 13-fold lower density than the L-type current of normal cells (0.41 +/- 0.042 pA/pF at + 20 mV, compared with 5.2 +/- 0.38 pA/pF in normal cells). I beta null was sensitive to dihydropyridines and had faster kinetics of activation and slower kinetics of inactivation than the L-type current of normal cells. Charge movement was reduced approximately 2.8-fold, with Qmax = 6.9 +/- 0.61 and Qmax = 2.5 +/- 0.2 nC/microF in normal and mutant cells, respectively. Approximately 40% of Qmax was nifedipine sensitive in both groups. In contrast to normal cells, Ca2+ transients could not be detected in mutant cells at any test potential; however, caffeine induced a robust Ca2+ transient. In homogenates of mutant muscle, the maximum density of [3H]PN200-110 binding sites (Bmax) was reduced approximately 3.9-fold. The results suggest that the excitation-contraction uncoupling of beta 1-null skeletal muscle involves a failure of the transduction mechanism that is due to either a reduced amount of alpha 1S subunits in the membrane or the specific absence of beta 1 from the voltage-sensor complex.     

Role of L-type calcium channels and PKC in active tone development in rabbit coronary artery

The present study investigated active tone development in isolated ring segments of rabbit epicardial coronary artery. Endothelium-denuded (E-) or endothelium-intact (E+) vessels treated with the NO synthase inhibitor N(omega)-nitro-L-arginine (100 microM) developed active tone, which was enhanced by stretch and reversed by the NO donor sodium nitroprusside (SNP; IC(50)=9 nM). Nifedipine abolished active tone and the contractile response to phorbol dibutyrate (PDBu; 10 nM) with the same potency (IC(50)=8 nM), whereas 300 nM PDBu responses were only partially blocked by nifedipine. The classical and novel PKC inhibitors GF-109203X (IC(50)=1-2 microM) and chelerythrine (IC(50)=4-5 microM) and the classical PKC inhibitor G?-6976 (IC(50)=0.3-0.4 microM) blocked both active tone and 10 nM PDBu responses with similar potency. Active tone development was associated with depolarization of membrane potential (E(m)) and a shift to the left of the E(m)-vs.-contraction relationship determined by varying extracellular potassium. The depolarization and leftward shift were reversed by either chelerythrine (10 microM) or SNP (30 nM). PDBu (100-300 nM) increased peak L-type calcium channel (Ca(v)) currents in isolated coronary myocytes, and this effect was reversed by chelerythrine (1 microM) or G?-6976 (200 nM). SNP (500 nM) reduced Ca(v) currents only in the presence of the PKA blocker 8-bromo-2'-O-monobutyryl-cAMPS, Rp isomer (10 microM). In conclusion, active tone development in coronary artery is suppressed by basal NO release and is dependent on both enhanced Ca(v) activity and classical PKC activity. Both E(m)-dependent and -independent processes contribute to contraction. Our results suggest that one E(m)-independent process is direct enhancement of Ca(v) current by PKC.     

大鼠肥厚心肌细胞钙电流及 Na~ /Ca~(2 )交换电流的研究(英文)

本文观察了心肌肥厚对大鼠心肌细胞Na ／Ca2 交换电流的影响。我们采用Goldblatt两肾一夹方法诱发大鼠心肌肥厚,应用全细胞膜片钳技术记录电流。结果表明：肥厚心肌细胞的Ni2 -敏感Na ／Ca2 交换电流密度大于正常细胞。在钳制电压为＋50mV时,正常细胞的外向交换电流密度为1．53±0．31pA／pF,而肥厚细胞则为2．62±0．53pA／pF（P＜0．01）;钳制电压为-100mV时,正常细胞的内向交换电流密度为0．42±0．14pA／pF,肥厚细胞达1．12±0．33pA／pF（P＜0．001）。这些结果提示,肥厚心肌细胞的Na ／Ca2 交换电流发生了改变,其意义有待进一步探讨。     

Developmental differences in L-type calcium current of human atrial myocytes

We investigated differences in L-type Ca2+ current (ICa) between infant (INF, 1-12 mo old), young adult (YAD, 14-18 yr old), and older adult (AD) myocytes from biopsies of right atrial appendages. Basal ICa was smaller in INF myocytes (1.2 +/- 0.1 pA/pF, n = 29, 6 +/- 1 mo old, 11 patients) than in YAD (2.5 +/- 0.2 pA/pF, n = 20, 16 +/- 1 yr old, 5 patients) or AD (2.6 +/- 0.3 pA/pF, n = 19, 66 +/- 3 yr old, 9 patients) myocytes (P < 0.05). Maximal ICa produced by isoproterenol (Iso) was similar in INF, YAD, and AD cells: 8.4 +/- 1.1, 9.6 +/- 1.0, and 9.2 +/- 1.3 pA/pF, respectively. Efficacy (Emax) was larger in INF (607 +/- 50%) than for YAD (371 +/- 29%) or AD (455 +/- 12%) myocytes. Potency (EC50) was 8- to 10-fold higher in AD (0.82 +/- 0.09 nM) or YAD (0.41 +/- 0.14 nM) than in INF (7.6 +/- 3.5 nM) myocytes. Protein levels were similar for Gialpha2 but much greater for Gialpha3 in INF than in AD or YAD atrial tissue. When Gialpha3 activity was inhibited by inclusion of a Gialpha3 COOH-terminal decapeptide in the pipette, basal ICa and the response to 10 nM Iso were increased in INF, but not in YAD, cells. We propose that basal ICa and the response to low-dose beta-adrenergic stimulation are inhibited in INF (but not YAD or AD) cells as a result of constitutive inhibitory effects of Gialpha3.     

Effects of streptozotocin-induced diabetes on Kv channels in rat small coronary smooth muscle cells

Diabetes impairs endothelium dependent vasodilation, but the mechanism of endothelium independent dilation is not well understood. In the present study, we examined the effect of streptozotocin (STZ)-induced diabetes on the vasomotor of small coronary artery and the activity of voltage-dependent K+ channel of vascular smooth muscle cells in STZ rats [corrected] using the videomicroscopy and patch clamp method. STZ-induced diabetes appeared to [corrected] reduce the vasodilation induced by beta-adrenoceptor agonist, isoproterenol (10(-9)-10(-5) mol/l), and adenylyl cyclase activator forskolin (10(-9)-10(-5) mol/l) respectively (isoproterenol: 44.2 +/- 6.7% vs. 82.5 +/- 4.8%, and forskolin: 54.4 +/- 4.5% vs. 94.3 +/- 2.4%). 4-AP, a Kv channel blocker of VSMC, further decreased dilation to isoproterenol (44.2 +/- 6.7% vs. 10.2 +/- 3.5%) and forskolin (54.4 +/- 4.5% vs. 13.8 +/- 11.0%) significantly. Whole cell K+ current recording demonstrated that STZ-induced diabetes decreased isoproterenol and forskolin-induced K+ current (ISO: 55.6 +/- 7.8 pA/pF vs. 28.4 +/- 3.4 pA/pF, forskolin: 61.3 +/- 9.8 pA/pF vs. 32.4 +/- 3.4 pA/pF). 4-AP further reduced the decreased K+ current (ISO: 28.4 +/- 3.4 pA/pF vs. 14.3 +/- 2.1 pA/pF, forskolin: 32.4 +/- 3.4 pA/pF vs. 14.8 +/- 2.9 pA/pF). These results indicated that STZ-induced diabetes impaired cAMP mediated dilation of small coronary artery and suppressed the Kv channel activity of vascular smooth muscle cells. Kv channel of VSMC was shown to play a determinate role reducing dilation of small coronary artery in STZ rats.     

Osmotic shrinkage of human cervical cancer cells induces an extracellular Cl- -dependent nonselective cation channel,which requires p38 MAPK

This study is to integrate a functional role of nonselective cation (NSC) channels into a model of volume regulation on osmotic shrinkage for human cervical cancer cells. Application of a hypertonic solution (400 mosm kg(-1)) induced cell shrinkage, which was accompanied by a 7-fold increase of inward currents at -80 mV from -4.1 +/- 0.4 pA pF(-1) to -29 +/- 1.1 pA pF(-1) (n = 36, p < 0.001). There is a good correlation of channel activity and cell volume changes. Replacement of bath Na(+) by K(+), Cs(+), Li(+), or Rb(+) did not affect the stimulated inward current significantly, but replacement by Ca(2+), Ba(2+), or the impermeable cation N-methyl-d-glucamine abolished the inward current; this demonstrates that the shrinkage-induced currents discriminate poorly between monovalent cations but are not carried by divalent cations. Replacement of extracellular Cl(-) by gluconate abolished the shrinkage-induced currents in a concentration-dependent manner without changing the reversal potential. Gadolinium (Gd(3+)) inhibited the stimulated current, whereas bumetanide and amiloride had no inhibitory effect. Cell shrinkage triggered mitogen-activated protein (MAP) kinase cascades leading to the activation of MAP/extracellular signal-regulated kinase 1/2 (ERK1/2) kinase (MEK1/2), and p38 kinase. Interference with p38 MAPK by either the specific inhibitor (SB202190), or a dominant-negative mutant profoundly suppressed the activation of the shrinkage-induced NSC channels. In contrast, the regulatory mechanism of shrinkage-induced NSC channels was independent of the volume-responsive MEK1/2 signaling pathway. More importantly, the cell volume response to hypertonicity was inhibited significantly in p38 dominant-negative mutant or by SB202190. Therefore, p38 MAPK is critically involved in the activation of a shrinkage-induced NSC channel, which plays an important role in the volume regulation of human cervical cancer cells.     

Effects of carvedilol on transient outward and ultra-rapid delayed rectifier potassium currents in human atrial myocytes

Carvedilol is a beta- and alpha(1)-adrenoceptor antagonist. It is widely used in the treatment of cardiovascular diseases including atrial arrhythmias. However, it is unclear whether carvedilol may affect the repolarization currents, transient outward K(+) current (I(to)) and ultra-rapid delayed rectifier K(+) current (I(Kur)) in the human atrium. The present study evaluated effects of carvedilol on I(to) and I(Kur) in isolated human atrial myocytes by whole-cell patch-clamp recording technique. We found that carvedilol reversibly inhibited I(to) and I(Kur) in a concentration-dependent manner. Carvedilol (0.3 microM) suppressed I(to) from 9.2+/-0.5 pA/pF to 4.8+/-0.5 pA/pF (P<0.01) and I(Kur) from 3.6+/-0.5 pA/pF to 1.9+/-0.3 pA/pF (P<0.01) at +50 mV. I(to) was inhibited in a voltage-dependent manner, being significantly attenuated at test potentials from +10 to +50 mV, whereas the inhibition of I(Kur) was independent. The concentration giving a 50% inhibition was 0.50 microM for I(to) and 0.39 microM for I(Kur). Voltage-dependence of activation, inactivation and time-dependent recovery from inactivation of I(to) were not altered by carvedilol. However, time to peak and time-dependent inactivation of I(to) were significantly accelerated, indicating an open channel blocking action. The findings indicate that carvedilol significantly inhibits the major repolarization K(+) currents I(to) and I(Kur) in human atrial myocytes.     

Gamma-dendrotoxin blocks large conductance Ca2+-activated K+ channels in neuroblastoma cells

In N1E 115 neuroblastoma cells, gamma-dendrotoxin (DTX, 200 nM) blocked the outward K(+) current by 31.1 +/- 3.5% (n = 4) with approximately 500 nM Ca(2+) in the pipet solution, but had no effect on the outward K(+) current when internal Ca(2+) was reduced. Using a ramp protocol, iberiotoxin (IbTX, 100 nM) inhibited a component of the whole cell current, but in the presence of 200 nM gamma-DTX, no further inhibition by IbTX was observed. Two types of single channels were seen using outside-out patches when the pipette free Ca(2+) concentration was approximately 500 nM; a 63 pS and a 187 pS channel. The 63 pS channel was TEA-, IbTX- and gamma-DTX-insensitive, while the 187 pS channel was blocked by 1 mM TEA, 100 nM IbTX or 200 nM gamma-DTX. Both channels were activated by external application of ionomycin, when the pipet calcium concentration was reduced. gamma-DTX (200 nM) reduced the probability of openings of the 187 pS channel, with an IC(50) of 8.5 nM. In GH(3) cells gamma-DTX (200 nM) also blocked an IbTX-sensitive component of whole-cell K(+) currents. These results suggest that gamma-DTX blocks a large conductance Ca(2+) activated K(+) current in N1E 115 cells. This is the first indication that any of the dendrotoxins, which have classically been known to block voltage-gated (Kv) channels, can also block Ca(2+) activated K(+) channels.