Hypertonicity activates nonselective cation channels in mouse cortical collecting duct cells.

We investigated the effect of cell shrinkage on whole-cell currents of M-1 mouse cortical collecting duct cells. Addition of 100 mM sucrose to an isotonic NaCl bath solution induced cell shrinkage and increased whole-cell currents within 5-10 min by approximately 12-fold. The effect was reversible upon return to isotonic solution and could also be elicited by adding 100 mM urea or 50 mM NaCl. Replacement of bath Na+ by K+, Cs+, Li+, or Rb+ did not significantly affect the stimulated inward current, but replacement by N-methyl-D-glucamine reduced it by 88.1 +/- 1.3% (n = 34); this demonstrates that hypertonicity activates a nonselective alkali cation conductance. The activation was independent of extra- and intracellular Ca2+, but 1 or 10 mM ATP in the pipette suppressed it in a concentration-dependent manner, indicating that intracellular ATP levels may modulate the degree of channel activation. Flufenamic acid (0.1 mM) and gadolinium (0.1 mM) inhibited the stimulated current by 68.7 +/- 5.9% (n = 9) and 32.4 +/- 11.7% (n = 6), respectively, whereas 0.1 mM amiloride had no significant effect. During the early phase of hypertonic stimulation single-channel transitions could be detected in whole-cell current recordings, and a gradual activation of 30 and more individual channels with a single-channel conductance of 26.7 +/- 0.4 pS (n = 29) could be resolved. Thus, we identified the nonselective cation channel underlying the shrinkage-induced whole-cell conductance that may play a role in volume regulation.     

Up-regulation of pressure-activated Ca(2+)-permeable cation channel in intact vascular endothelium of hypertensive rats.

In endothelial cells, stretch-activated cation channels have been proposed to act as mechanosensors for changes in hemodynamic forces. We have identified a novel mechanosensitive pressure-activated channel in intact endothelium from rat aorta and mesenteric artery. The 18-pS cation channel responded with a multifold increase in channel activity when positive pressure was applied to the luminal cell surface with the patch pipette and inactivated at negative pipette pressure. Channel permeability ratio for K+, Na+, and Ca2+ ions was 1:0.98:0.23. Ca2+ influx through the channel was sufficient to activate a neighboring Ca2(+)-dependent K+ channel. Hemodynamic forces are chronically disturbed in arterial hypertension. Endothelial cell dysfunction has been implicated in the pathogenesis of arterial hypertension. In two comparative studies, density of the pressure-activated channel was found to be significantly higher in spontaneously hypertensive rats and renovascular hypertensive rats compared with their respective normotensive controls. Channel activity presumably leads to mechanosensitive Ca2+ influx and induces cell hyperpolarization by K+ channel activity. Both Ca2+ influx and hyperpolarization are known to induce a vasodilatory endothelial response by stimulating endothelial nitric oxide (NO) production. Up-regulation of channel density in hypertension could, therefore, represent a counterregulatory mechanism of vascular endothelium.     

Mechanosensitive nonselective cation channel facilitation by endothelin-1 is regulated by protein kinase C in arterial myocytes

OBJECTIVE: The mechanosensitive nonselective cation channel (NSC(MS)) and endothelin-1 (ET-1) play critical roles in the regulation of vascular tone. This study was undertaken to investigate the effect of ET-1 on NSC(MS) and on the myogenic response of arteries. METHODS: Cell-attached patch-clamp techniques were applied to rabbit pulmonary and cerebral arterial smooth muscle cells using a 140 mM CsCl pipette and bath solutions (Ca(2+)-free, 1 mM EGTA). Myogenic responses were determined by video analysis of pressurized arteries. RESULTS: The application of negative pressures through the pipette activated NSC(MS), and this was augmented by bath application of ET-1 (1 pM-30 nM). ET-1 lowered the lowest pressure required for NSC(MS) activation. NSC(MS) facilitation by ET-1 was prevented by BQ-123 (1 microM, an ET(A) antagonist) but not by BQ-788 (1 microM, an ET(B) antagonist). Phorbol 12-myristate 13-acetate (PMA, 100 nM), a protein kinase C activator, also increased the activity of NSC(MS). ET-1- or PMA-induced facilitation of NSC(MS) was abolished by GF109203X (10 microM), a protein kinase C inhibitor. Video analysis of pressurized cerebral artery showed inhibition of the myogenic response by the NSC(MS) channel blockers GsMTx-4 (5 microM) and DIDS (3-100 microM). Treatment with ET-1 (10 pM) augmented the myogenic response and this was inhibited by DIDS (30 microM). CONCLUSION: Stimulation of ET-1 receptor (ET(A)) facilitates NSC(MS) via a protein kinase C-dependent signaling pathway in rabbit arterial myocytes. Our findings suggest that NSC(MS) play a role in the myogenic response and its augmentation by ET-1.     

Behavior of nonselective cation channels and large-conductance Ca2+-activated K+ channels induced by dynamic changes in membrane stretch in cultured smooth muscle cells of human coronary artery

INTRODUCTION: The effects of membrane stretch on ion channels were investigated in cultured smooth muscle cells of human coronary artery. METHODS AND RESULTS: In the cell-attached configuration, membrane stretch with negative pressure induced two types of stretch-activated (SA) ion channels: a nonselective cation channel and a large-conductance Ca2+-activated K+ (BK(Ca)) channel. The single-channel conductances of SA cation and BK(Ca) channels were 26 and 203 pS, respectively. To elucidate the mechanism of activation of these SA channels and to minimize mechanical disruption, a sinusoidal change in pipette pressure was applied to the on-cell membrane patch. During dynamic changes in pipette pressure, increases in SA cation channel activity was found to coincide with increases in BK(Ca) channel activity. In the continued presence of cyclic stretch, the activity of SA cation channels gradually diminished. However, after termination of cyclic stretch, BK(Ca) channel activity was greatly enhanced, but the activity of SA cation channels disappeared. CONCLUSION: This study is the first to demonstrate that the behavior of SA cation and BK(Ca) channels in coronary smooth muscle cells is differentially susceptible to dynamic changes in membrane tension.     

Oxytocin and cAMP stimulate monovalent cation movements through a Ca2+-sensitive, amiloride-insensitive channel in the apical membrane of toad urinary bladder.

The effects of oxytocin and cAMP on ion transport were investigated in toad urinary bladders incubated with Ca2+-free solutions on the apical side. Under these conditions both oxytocin and cAMP markedly stimulated the movements of Na+, K+, Rb+, Cs+, Li+, and NH4+ through a pathway that is insensitive to amiloride. The amiloride-insensitive currents were inhibited by the addition of Ca2+, Sr2+, or Mg2+ to the apical solution. The movement of the monovalent cations was associated with a spontaneous Lorentzian component in the power spectrum of the fluctuation in short-circuit current. The plateau of the Lorentzian component was enhanced by oxytocin and cAMP and was depressed by divalent cations. Methohexital inhibited the stimulation of monovalent cation movements caused by oxytocin. These findings suggest that oxytocin and cAMP activate at least two kinds of ionic channels in the apical membrane of toad urinary bladder: the well-known amiloride-sensitive channel and an amiloride-insensitive channel that allows the movement of several monovalent cations and is blocked by Ca2+ and other divalent cations.     

Pregnancy upregulates large-conductance Ca(2+)-activated K(+) channel activity and attenuates myogenic tone in uterine arteries

Uterine vascular tone significantly decreases whereas uterine blood flow dramatically increases during pregnancy. However, the complete molecular mechanisms remain elusive. We hypothesized that increased Ca(2+)-activated K(+) (BK(Ca)) channel activity contributes to the decreased myogenic tone of uterine arteries in pregnancy. Resistance-sized uterine arteries were isolated from nonpregnant and near-term pregnant sheep. Electrophysiological studies revealed a greater whole-cell K(+) current density in pregnant compared with nonpregnant uterine arteries. Tetraethylammonium and iberiotoxin inhibited K(+) currents to the same extent in uterine arterial myocytes. The BK(Ca) channel current density was significantly increased in pregnant uterine arteries. In accordance, tetraethylammonium significantly increased pressure-induced myogenic tone in pregnant uterine arteries and abolished the difference in myogenic responses between pregnant and nonpregnant uterine arteries. Activation of protein kinase C produced a similar effect to tetraethylammonium by inhibiting BK(Ca) channel activity and increasing myogenic tone in pregnant uterine arteries. Chronic treatment of nonpregnant uterine arteries with physiologically relevant concentrations of 17β-estradiol and progesterone caused a significant increase in the BK(Ca) channel current density. Western blot analyses demonstrated a significant increase of the β1, but not α, subunit of BK(Ca) channels in pregnant uterine arteries. In accordance, steroid treatment of nonpregnant uterine arteries resulted in an upregulation of the β1, but not α, subunit expression. The results indicate that the steroid hormone-mediated upregulation of the β1 subunit and BK(Ca) channel activity may play a key role in attenuating myogenic tone of the uterine artery in pregnancy.     

A common mechanism underlies stretch activation and receptor activation of TRPC6 channels

The TRP family of ion channels transduce an extensive range of chemical and physical signals. TRPC6 is a receptor-activated nonselective cation channel expressed widely in vascular smooth muscle and other cell types. We report here that TRPC6 is also a sensor of mechanically and osmotically induced membrane stretch. Pressure-induced activation of TRPC6 was independent of phospholipase C. The stretch responses were blocked by the tarantula peptide, GsMTx-4, known to specifically inhibit mechanosensitive channels by modifying the external lipid-channel boundary. The GsMTx-4 peptide also blocked the activation of TRPC6 channels by either receptor-induced PLC activation or by direct application of diacylglycerol. The effects of the peptide on both stretch- and diacylglycerol-mediated TRPC6 activation indicate that the mechanical and chemical lipid sensing by the channel has a common molecular mechanism that may involve lateral-lipid tension. The mechanosensing properties of TRPC6 channels highly expressed in smooth muscle cells are likely to play a key role in regulating myogenic tone in vascular tissue.     

Transient receptor potential channels regulate myogenic tone of resistance arteries

Elevation of intravascular pressure causes depolarization and constriction (myogenic tone) of small arteries and arterioles, and this response is a key element in blood flow regulation. However, the nature of pressure-induced depolarization has remained elusive. In the present study, we provide evidence that a transient receptor potential channel (TRPC6) homologue has a major role in this depolarizing response to pressure. Antisense oligodeoxynucleotides to TRPC6 decreased TRPC6 protein expression and greatly attenuated arterial smooth muscle depolarization and constriction caused by elevated pressure in intact cerebral arteries. Suppressing the expression of this channel protein also reduced the current density of a major cation current in resistance artery smooth muscle cells. We propose that TRPC6 channels play an essential role in regulation of myogenic tone.     

Voltage-induced membrane displacement in patch pipettes activates mechanosensitive channels

The patch-clamp technique allows currents to be recorded through single ion channels in patches of cell membrane in the tips of glass pipettes. When recording, voltage is typically applied across the membrane patch to drive ions through open channels and to probe the voltage-sensitivity of channel activity. In this study, we used video microscopy and single-channel recording to show that prolonged depolarization of a membrane patch in borosilicate pipettes results in delayed slow displacement of the membrane into the pipette and that this displacement is associated with the activation of mechanosensitive (MS) channels in the same patch. The membrane displacement, approximately 1 micrometer with each prolonged depolarization, occurs after variable delays ranging from tens of milliseconds to many seconds and is correlated in time with activation of MS channels. Increasing the voltage step shortens both the delay to membrane displacement and the delay to activation. Preventing depolarization-induced membrane displacement by applying positive pressure to the shank of the pipette or by coating the tips of the borosilicate pipettes with soft glass prevents the depolarization-induced activation of MS channels. The correlation between depolarization-induced membrane displacement and activation of MS channels indicates that the membrane displacement is associated with sufficient membrane tension to activate MS channels. Because membrane tension can modulate the activity of various ligand and voltage-activated ion channels as well as some transporters, an apparent voltage dependence of a channel or transporter in a membrane patch in a borosilicate pipette may result from voltage-induced tension rather than from direct modulation by voltage.     

Single K+ channel currents of anomalous rectification in cultured rat myotubes.

The currents through single K+ channels of the anomalous (or inward) rectifier were recorded in tissue cultured rat myotubes by using the "gigohm seal" patch clamp technique developed by Sigworth and Neher. These unitary currents were detected as current fluctuations due to the blocking and unblocking of channels by Ba2+. The single-channel conductance was obtained from the slope of the linear relationship between unitary current amplitude and membrane potential. When the external solution contained 155 mM K+, the single-channel conductance was 10.4 +/- 2.6 pS (+/- SD; n = 6). This value was independent of the the concentration of blocking ions but increased with increasing external K+ concentration. The behavior of the unitary current agreed with that expected from the blocking kinetics of Ba2+ on the macroscopic K+ current of the anomalous rectifier. The density of the channel is likely to be small and may even be less than 1/micrometers 2.     

Critical role for transient receptor potential channel TRPM4 in myogenic constriction of cerebral arteries

Local control of cerebral blood flow is regulated in part through myogenic constriction of resistance arteries. Although this response requires Ca2+ influx via voltage-dependent Ca2+ channels secondary to smooth muscle cell depolarization, the mechanisms responsible for alteration of vascular smooth muscle (VSM) cell membrane potential are not fully understood. A previous study from our laboratory demonstrated a critical role for a member of the transient receptor potential (TRP) superfamily of ion channels, TRPC6, in this response. Several other of the approximately 22 identified TRP proteins are also present in cerebral arteries, but their functions have not been elucidated. Two of these channels, TRPM4 and TRPM5, exhibit biophysical properties that are consistent with a role for control of membrane potential of excitable cells. We hypothesized that TRPM4/TRPM5-dependent currents contribute to myogenic vasoconstriction of cerebral arteries. Cation channels with unitary conductance, ion selectivity and Ca2+-dependence similar to those of cloned TRPM4 and TRPM5 were present in freshly isolated VSM cells. We found that TRPM4 mRNA was detected in both whole cerebral arteries and in isolated VSM cells whereas TRPM5 message was absent from cerebral artery myocytes. We also found that pressure-induced smooth muscle cell depolarization was attenuated in isolated cerebral arteries treated with TRPM4 antisense oligodeoxynucleotides to downregulate channel subunit expression. In agreement with these data, myogenic vasoconstriction of intact cerebral arteries administered TRPM4 antisense was attenuated compared with controls, whereas KCl-induced constriction did not differ between groups. We concluded that activation of TRPM4-dependent currents contributed to myogenic vasoconstriction of cerebral arteries.     

Involvement of a GTP-binding protein in stimulating action of angiotensin II on calcium channels in vascular smooth muscle cells.

The possible involvement of a GTP-binding protein in the regulation of Ca2+ channels by angiotensin II (Ang II) in vascular muscle cells was investigated by the whole-cell voltage-clamp method. Single cells were freshly isolated from guinea pig portal vein. The pipette solution contained high Cs+ to inhibit K+ currents and thereby isolate the Ca2+ channel current. Ba2+ (2 mM) was in the bath solution as a charge carrier for the Ca2+ channel. Application of Ang II (0.1-100 nM) produced an increase in peak amplitude of the Ba2+ current, with a shift of the current-voltage curve in the negative direction. These effects were inhibited by pretreatment with an antagonist of the Ang II receptor, [Sar1,Ile8]-Ang II. Presence of 0.1 mM GTP in the pipette solution stabilized the Ang II action, but 0.3-1.0 mM GDP-beta-S and 1.0 mM GTP-gamma-S inhibited it. GTP-gamma-S alone produced a slowly progressing increase in the basal (unstimulated) current amplitude. Preincubation of muscle tissues with pertussis toxin (1 micrograms/ml, for up to 6 hours at 36 degrees C) or intracellular application of preactivated pertussis toxin (1 micrograms/ml) plus NAD (1 mM) did not inhibit the Ang II action. Cholera toxin (10 micrograms/ml) also had no effect on the Ang II action. These results suggest that the Ang II stimulation of Ca2+ channels in smooth muscle of guinea pig portal vein may be mediated by a G protein that is insensitive to both pertussis toxin and cholera toxin.     

Stretch-activated single K+ channels account for whole-cell currents elicited by swelling

Functionally significant stretch-activated ion channels have been clearly identified in excitable cells. Although single-channel studies suggest their expression in other cell types, their activity in the whole-cell configuration has not been shown. This discrepancy makes their physiological significance doubtful and suggests that their mechanical activation is artifactual. Possible roles for these molecules in nonexcitable cells are acute cell-volume regulation and, in epithelial cells, the complex adjustment of ion fluxes across individual cell membranes when the rate of transepithelial transport changes. We report the results of experiments on isolated epithelial cells expressing in the basolateral membrane stretch-activated K+ channels demonstrable by the cell-attached patch-clamp technique. In these cells, reversible whole-cell currents were elicited by both isosmotic and hyposmotic cell swelling. Cation selectivity and block by inorganic agents were the same for single-channel and whole-cell currents, indicating that the same entity underlies single-channel and whole-cell currents and that the single-channel events are not artifactual. In these cells, when the rate of apical-membrane NaCl entry increases, the cell Na+ content and volume also increase, stimulating the Na+,K+-ATPase at the basolateral membrane, i.e., both Na+ extrusion and K+ uptake increase. We speculate that, under these conditions, the parallel activation of basolateral K+ channels (by the swelling) elevates conductive K+ loss, tending to maintain the cell K+ content constant ("pump-leak parallelism"). This study describes a physiologically relevant stretch-activated channel, at both the single-channel and whole-cell levels, in a nonneural cell type.     

Crosstalk between voltage-independent Ca2+ channels and L-type Ca2+ channels in A7r5 vascular smooth muscle cells at elevated intracellular pH: evidence for functional coupling between L-type Ca2+ channels and a 2-APB-sensitive cation channel

This study was designed to investigate the role of voltage-independent and voltage-dependent Ca2+ channels in the Ca2+ signaling associated with intracellular alkalinization in A7r5 vascular smooth muscle cells. Extracellular administration of ammonium chloride (20 mmol/L) resulted in elevation of intracellular pH and activation of a sustained Ca2+ entry that was inhibited by 2-amino-ethoxydiphenyl borate (2-APB, 200 micromol/L) but not by verapamil (10 micro;mol/L). Alkalosis-induced Ca2+ entry was mediated by a voltage-independent cation conductance that allowed permeation of Ca2+ (PCa/PNa approximately 6), and was associated with inhibition of L-type Ca2+ currents. Alkalosis-induced inhibition of L-type Ca2+ currents was dependent on the presence of extracellular Ca2+ and was prevented by expression of a dominant-negative mutant of calmodulin. In the absence of extracellular Ca2+, with Ba2+ or Na+ as charge carrier, intracellular alkalosis failed to inhibit but potentiated L-type Ca2+ channel currents. Inhibition of Ca2+ currents through voltage-independent cation channels by 2-APB prevented alkalosis-induced inhibition of L-type Ca2+ currents. Similarly, 2-APB prevented vasopressin-induced activation of nonselective cation channels and inhibition of L-type Ca2+ currents. We suggest the existence of a pH-controlled Ca2+ entry pathway that governs the activity of smooth muscle L-type Ca2+ channels due to control of Ca2+/calmodulin-dependent negative feedback regulation. This Ca2+ entry pathway exhibits striking similarity with the pathway activated by stimulation of phospholipase-C-coupled receptors, and may involve a similar type of cation channel. We demonstrate for the first time the tight functional coupling between these voltage-independent Ca2+ channels and classical voltage-gated L-type Ca2+ channels.     

Inward rectification of a potassium channel in cardiac ventricular cells depends on internal magnesium ions.

The mechanism of rectification of the inwardly rectifying potassium channel was examined with single-channel recording techniques in isolated ventricular myocytes from adult guinea pig heart. Inward, or anomalous, rectification describes the property that potassium (K) current can enter the cell at potentials negative to the potassium equilibrium potential, EK, more readily than it can leave the cell at positive potentials. Voltage ramps applied to single inward rectifier channels in cell-attached patches produced single-channel currents that rectified strongly with a marked reduction in current at a potential near EK. At more positive potentials no current could be detected. Rectification was influenced by external and internal K concentrations. Single-channel activity, which usually disappears rapidly in excised patches, could be maintained by removing calcium from the internal solution. Rectification could be eliminated by excision of the patch into an internal solution in which free magnesium (Mg2+) was reduced to less than 1 microM, and it could be restored by the addition of approximately 1 mM Mg2+ to the internal solution. At intermediate concentrations of Mg2+, intermediate degrees of rectification were obtained, and the current at potentials positive to EK was often interrupted by brief closures. These studies suggest that rectification is due to internal block by Mg2+, possibly the result of rapid block of the open channel.     

Stretch-dependent calcium uptake associated with myogenic tone in rabbit facial vein

Many blood vessels possess stretch-dependent myogenic tone. This tone contributes to the active resistance in small arteries, which regulates blood flow in the body. In this report we describe results indicating that stretch-dependent myogenic tone in the rabbit facial vein, a vascular preparation with useful experimental properties, is accompanied by an increased uptake of 45Ca2+. This influx may not occur through voltage-gated Ca2+ channels, since neither the extent of myogenic tone nor 45Ca2+ influx is affected by a concentration of 1,4-dihydropyridine (PN 200-110, 10 nM) that reduces tone and 45Ca2+ influx due to the depolarization by K+. These results indicate that in myogenically active vessels a stretch-activated influx of Ca2+ may occur through distinct Ca2+-entry pathways.     

Enhancement of rabbit cardiac sodium channels by beta-adrenergic stimulation.

Voltage-dependent sodium channels from a variety of tissues are known to be phosphorylated by the cAMP-dependent protein kinase, protein kinase A. However, the functional significance of sodium channel phosphorylation is not clearly understood. Using whole-cell voltage-clamp techniques, we show that sodium currents (INas) in rabbit cardiac myocytes are enhanced by isoproterenol (ISO). This enhancement of INa by ISO 1) is holding potential dependent, 2) can be mimicked by forskolin and dibutyryl cAMP, and 3) is accompanied by an increase in the rate of Na+ channel inactivation. In single-channel, inside-out patch experiments, the catalytic subunit of protein kinase A also enhances INa and increases the rate of inactivation, suggesting that cardiac Na+ channel phosphorylation may be physiologically important. Addition of the protein kinase A inhibitor to the pipette solution in whole-cell experiments blocks the stimulatory effect of forskolin without blocking the effect of ISO, suggesting that ISO also enhances INa through a cAMP-independent pathway. To determine if ISO may stimulate INa through a direct G protein pathway, single channels were recorded in the presence of the Gs-activating GTP analogue, GTP gamma S, and the stimulatory G protein subunit, Gs alpha. Both of these agents enhanced INa without affecting the rate of Na+ channel inactivation. These results suggest that ISO enhances rabbit cardiac INa through a dual (direct and indirect) G protein regulatory pathway.     

Blockage of the sodium current in isolated single cells from rat ventricle with mexiletine and disopyramide

The blocking effects of local anesthetics, mexiletine and disopyramide on the sodium currents (INa) of enzymatically isolated, single cells from rat ventricle were studied under voltage clamp conditions. A suction pipette technique was used for voltage clamp and internal perfusion. Potassium currents were blocked by replacing K+ with Cs+ in the internal and external solutions; calcium currents were blocked by replacing Ca2+ with Co2+ in the external solution to isolate INa. When the cells were stimulated infrequently (less than 1 Hz), both drugs produced dose-dependent depression of INa, which was correlated with one-to-one binding to sodium channel. A half-blocking concentration (KD) of 2.8 X 10(-5) M was observed for both agents. The shape of the current-voltage curve along the voltage axis remained unchanged in the presence of either drug. Both drugs shifted the inactivation curve of INa to more negative potentials. Mexiletine produced a marked use-dependent blockage of INa, whereas disopyramide did not produce significant use-dependent block under similar experimental conditions. Both drugs prolonged the recovery of INa from inactivation. The results suggested that both drugs interact with the inactivation mechanism of the sodium channels of rat myocardial cells.     

Antihypertensive effects of the putative T-type calcium channel antagonist mibefradil are mediated by the L-type calcium channel Cav1.2

The role of T-type Ca2+ channels for cardiovascular physiology, in particular blood pressure regulation, is controversial. Selective blockade of T-type Ca2+ channels in resistance arteries has been proposed to explain the effect of the antihypertensive drug mibefradil. In the present study, we used a third generation, time- and tissue-specific conditional knockout model of the L-type Ca2+ channel Cav1.2 (Cav1.2SMAKO mice) to genetically dissect the effects of mibefradil on T- and L-type Ca2+ channels. Myogenic tone and phenylephrine-induced contraction in hindlimb perfusion experiments were sensitive to mibefradil in control mice, whereas the drug showed no effect in Cav1.2-deficient animals. Mean arterial blood pressure in awake, freely moving control mice was reduced by 38+/-2.5 mm Hg at a dose of 1.25 mg/kg bodyweight mibefradil, but not changed in Cav1.2SMAKO mice. These results demonstrate that the effect of the putative T-type Ca2+ channel-selective blocker mibefradil on blood pressure and small vessel myogenic tone is mediated by the Cav1.2 L-type Ca2+ channel.     

Serotonin increases an anomalously rectifying K+ current in the Aplysia neuron R15.

Previous work has shown that serotonin causes an increase in K+ conductance in the identified Aplysia neuron R15. This response is mediated by cAMP-dependent protein phosphorylation. The results presented here show that the K+ channel modulated by serotonin is an anomalous or inward rectifier (designated IR) that is present in R15 together with the three other distinct K+ channels previously described for this cell. Several lines of evidence indicate that this inward rectifier is partially activated in the resting cell and is further activated by serotonin. Voltage clamp analysis of resting and serotonin-evoked membrane currents at various external K+ concentrations shows that both currents have reversal potentials close to the potassium equilibrium potential, exhibit similar dependences in magnitude on external K+ concentration, and display marked anomalous rectification. The effects of particular monovalent and divalent cations are also similar on the resting and serotonin-evoked currents. Rb+, Cs+, and Ba2+ block both currents while Tl+ can substitute for K+ as a charge carrier and channel activator in both. These properties are characteristics of anomalous rectifiers in other systems. Furthermore, measurement of the voltage dependence of inactivation for the fast transient K+ current shows that this current cannot account for the anomalously rectifying K+ conductance in R15. The inward rectifier is therefore a separate current mediated by its own channels, the activity of which can be modulated by serotonin.