Functional evidence for Na+-activated K+ channels in circular smooth muscle of the opossum lower esophageal sphincter

Na(+) reduction induces contraction of opossum lower esophageal sphincter (LES) circular smooth muscle strips in vitro; however, the mechanism(s) by which this occurs is unknown. The purpose of the present study was to investigate the electrophysiological effects of low Na(+) on opossum LES circular smooth muscle. In the presence of atropine, quanethidine, nifedipine, and substance P, conventional intracellular electrodes recorded a resting membrane potential (RMP) of -37.5 +/- 0.9 mV (n = 4). Decreasing [Na(+)] from 144.1 to 26.1 mM by substitution of equimolar NaCl with choline Cl depolarized the RMP by 7.1 +/- 1.1 mV. Whole cell patch-clamp recordings revealed outward K(+) currents that began to activate at -60 mV using 400-ms stepped test pulses (-120 to +100 mV) with increments of 20 mV from holding potential of -80 mV. Reduction of [Na(+)] in the bath solution inhibited K(+) currents in a concentration-dependent manner. Single channels with conductance of 49-60 pS were recorded using cell-attached patch-clamp configurations. The channel open probability was significantly decreased by substitution of bath Na(+) with equimolar choline. A 10-fold increase of [K(+)] in the pipette shifted the reversal potential of the single channels to the positive by -50 mV. These data suggest that Na(+)-activated K(+) channels exist in the circular smooth muscle of the opossum LES.     

Opposing roles of K(+) and Cl(-) channels in maintenance of opossum lower esophageal sphincter tone

The ionic basis underlying the maintenance of myogenic tone of lower esophageal sphincter circular muscle (LES) was investigated in opossum with the use of standard isometric tension and conventional intracellular microelectrode recordings in vitro. In tension recording studies, nifedipine (1 microM) reduced basal tone to 27.7 +/- 3.8% of control. The K(+) channel blockers tetraethylammonium (TEA, 2 mM), charybdotoxin (100 nM), and 4-aminopyridine (4-AP, 2 mM) enhanced resting tone, whereas apamin and glibenclamide were without affect. Cl(-) channel blockers DIDS (500 microM) and 5-nitro-2-(3-phenylpropylamino)-benzoic acid (500 microM), as well as niflumic acid (0.1-300 microM), decreased basal tone, but tamoxifen was without effect. Intracellular microelectrode recordings revealed ongoing, spontaneous, spike-like action potentials (APs). Nifedipine abolished APs and depolarized resting membrane potential (RMP). Both TEA and 4-AP significantly depolarized RMP and augmented APs, whereas niflumic acid dose-dependently hyperpolarized RMP and abolished APs. These data suggest that, in the opossum, basal tone is associated with continuous APs and that K(+) and Ca(2+)-activated Cl(-) channels have important opposing roles in the genesis of LES tone.     

Actions of putative chloride channel blocking agents on canine lower esophageal sphincter (LES)

Niflumic acid (NA), a putative Cl(-)-channel blocker, has provided pharmacological evidence that Cl(-)-channel closures mediate hyperpolarization caused by NO in gastrointestinal smooth muscle. However, NA caused concentration-dependent relaxation of canine lower esophageal sphincter (LES) and failed to inhibit NO-mediated relaxations. DIDS also did not inhibit NO-mediated relaxations, but did abolish them when present with 20 mM TEA (tetraethyl ammonium ion), which was also ineffective alone. TEA reversed NA-induced relaxations, but with NA it did not inhibit NO-mediated relaxations. We investigated the modes of action of these agents further. Neither nerve-function block nor block of NOS activity affected the inhibition of LES tone by NA. In patch-clamp studies, NA increased outward currents from -30 to + 90 mV when [Ca2+]pipette was 50 nM. This was prevented by 20 mM TEA, but not by prior inhibition of NOS. At 200 nM [Ca2+]pipette, TEA markedly reduced outward currents, but did not prevent the increase from subsequent NA. In contrast, under similar conditions, application of DIDS after 20 mM TEA further reduced outward currents. When the patch pipette contained CsCl and TEA to block K+ currents, NA had no significant effect on currents between -50 and +90 mV. Thus, NA acted by opening K+ channels: some TEA-sensitive and some not. It had no detectable effect on currents when K+ channels were blocked. We conclude that NA is an unreliable pharmacological tool to evaluate Cl(-)-channel contributions to smooth muscle function. DIDS did not open K+ channels. Decreases in outward currents from DIDS may result from inhibition of K+ currents or currents carried by Cl- at depolarized membrane potentials.     

A Fast Transient Outward Monovalent Current in Rat Saphenous Myocytes Passing Through Ca2+ Channels

Transient outward currents in rat saphenous arterial myocytes were studied using the perforated configuration of the patch-clamp method. When myocytes were bathed in a Na-gluconate solution containing TEA to block large-conductance Ca2+-activated K+ (BK) currents, depolarizing pulses positive to +20 mV from a holding potential of -100 mV induced fast transient outward currents. The activation and inactivation time constants of the current were voltage dependent, and at +40 mV were 3.6 +/- 0.8 ms and 23.9 +/- 6.4 ms (n = 4), respectively. The steady-state inactivation of the transient outward current was steeply voltage dependent (z = 1.7), with 50% of the current inactivated at -55 mV. The current was insensitive to the A-type K+ channel blocker 4-AP (1-5 mM), and was modulated by external Ca, decreasing to approximately 0.85 of control values upon raising Ca2+ from 1 to 10 mM, and increasing approximately 3-fold upon lowering it to 0.1 mM. Transient outward currents were also recorded following replacement of internal K+ with either Na+ or Cs+, raising the possibility that the current was carried by monovalent ions passing through voltage-gated Ca2+ channels. This hypothesis was supported by the finding that the transient outward current had the same inactivation rate as the inward Ba2+ current, and that both currents were effectively blocked by the L-type Ca2+ channel blocker, nifedipine and enhanced by the agonist BAYK8644.     

Potassium channel diversity within the muscular components of the feline lower esophageal sphincter

We hypothesized that regional differences in electrophysiological properties exist within the musculature of the feline lower esophageal sphincter (LES) and that they may potentially contribute to functional asymmetry within the LES. Freshly isolated esophageal smooth muscle cells (SMCs) from the circular muscle and sling regions within the LES were studied under a patch clamp. The resting membrane potential (RMP) of the circular SMCs was significantly more depolarized than was the RMP of the sling SMCs, resulting from a higher Na+ and Cl- permeability in circular muscle than in sling muscle. Large conductance Ca2+-activated K+ (BKCa) set the RMP at both levels, since specific BKCa inhibitors caused depolarization; however, BKCa density was greatest in the circular region. A significant portion of the outward current was due to non-BKCa, especially in sling muscle, and likely delayed rectifier K+ channels (KDR). There was a large reduction in outward current with 4-aminopyridine (4-AP) in sling muscle, while BKCa blockers had a limited effect on the voltage-activated outward current in sling muscle. Differences in BKCa:KDR channel ratios were also manifest by a leftward shift in the voltage-dependent activation curve in circular cells compared to sling cells. The electrophysiological differences seen between the circular and sling muscles provide a basis for their different contributions to LES activities such as resting tone and neurotransmitter responsiveness, and in turn could impart asymmetric drug responses and provide specific therapeutic targets.     

Whole cell current and membrane potential regulation by a human smooth muscle mechanosensitive calcium channel

Mechanotransduction is required for a wide variety of biological functions. The aim of this study was to determine the effect of activation of a mechanosensitive Ca(2+) channel, present in human jejunal circular smooth muscle cells, on whole cell currents and on membrane potential. Currents were recorded using patch-clamp techniques, and perfusion of the bath (10 ml/min, 30 s) was used to mechanoactivate the L-type Ca(2+) channel. Perfusion resulted in activation of L-type Ca(2+) channels and an increase in outward current from 664 +/- 57 to 773 +/- 72 pA at +60 mV. Membrane potential hyperpolarized from -42 +/- 4 to -50 +/- 5 mV. In the presence of nifedipine (10 microM), there was no increase in outward current or change in membrane potential with perfusion. In the presence of charybdotoxin or iberiotoxin, perfusion of the bath did not increase outward current or change membrane potential. A model is proposed in which mechanoactivation of an L-type Ca(2+) channel current in human jejunal circular smooth muscle cells results in increased Ca(2+) entry and cell contraction. Ca(2+) entry activates large-conductance Ca(2+)-activated K(+) channels, resulting in membrane hyperpolarization and relaxation.     

Functional and molecular analysis of L-type calcium channels in human esophagus and lower esophageal sphincter smooth muscle

Excitation of human esophageal smooth muscle involves the release of Ca(2+) from intracellular stores and influx. The lower esophageal sphincter (LES) shows the distinctive property of tonic contraction; however, the mechanisms by which this is maintained are incompletely understood. We examined Ca(2+) channels in human esophageal muscle and investigated their contribution to LES tone. Functional effects were examined with tension recordings, currents were recorded with patch-clamp electrophysiology, channel expression was explored by RT-PCR, and intracellular Ca(2+) concentration was monitored by fura-2 fluorescence. LES muscle strips developed tone that was abolished by the removal of extracellular Ca(2+) and reduced by the application of the L-type Ca(2+) channel blocker nifedipine (to 13 +/- 6% of control) but was unaffected by the inhibition of sarco(endo)plasmic reticulum Ca(2+)-ATPase by cyclopiazonic acid (CPA). Carbachol increased tension above basal tone, and this effect was attenuated by treatment with CPA and nifedipine. Voltage-dependent inward currents were studied using patch-clamp techniques and dissociated cells. Similar inward currents were observed in esophageal body (EB) and LES smooth muscle cells. The inward currents in both tissues were blocked by nifedipine, enhanced by Bay K8644, and transiently suppressed by acetylcholine. The molecular form of the Ca(2+) channel was explored using RT-PCR, and similar splice variant combinations of the pore-forming alpha(1C)-subunit were identified in EB and LES. This is the first characterization of Ca(2+) channels in human esophageal smooth muscle, and we establish that L-type Ca(2+) channels play a critical role in maintaining LES tone.     

Delayed rectifying and calcium-activated K+ channels and their significance for action potential repolarization in mouse pancreatic beta-cells

The contribution of Ca2(+)-activated and delayed rectifying K+ channels to the voltage-dependent outward current involved in spike repolarization in mouse pancreatic beta-cells (Rorsman, P., and G. Trube. 1986. J. Physiol. 374:531-550) was assessed using patch-clamp techniques. A Ca2(+)-dependent component could be identified by its rapid inactivation and sensitivity to the Ca2+ channel blocker Cd2+. This current showed the same voltage dependence as the voltage-activated (Cd2(+)-sensitive) Ca2+ current and contributed 10-20% to the total beta-cell delayed outward current. The single-channel events underlying the Ca2(+)-activated component were investigated in cell-attached patches. Increase of [Ca2+]i invariably induced a dramatic increase in the open state probability of a Ca2(+)-activated K+ channel. This channel had a single-channel conductance of 70 pS [( K+]o = 5.6 mM). The Ca2(+)-independent outward current (constituting greater than 80% of the total) reflected the activation of an 8 pS [( K+]o = 5.6 mM; [K+]i = 155 mM) K+ channel. This channel was the only type observed to be associated with action potentials in cell-attached patches. It is suggested that in mouse beta-cells spike repolarization results mainly from the opening of the 8-pS delayed rectifying K+ channel.     

Developmental differences in Ca2+-activated K+ channel activity in ovine basilar artery

A primary determinant of vascular smooth muscle (VSM) tone and contractility is the resting membrane potential, which, in turn, is influenced heavily by K+ channel activity. Previous studies from our laboratory and others have demonstrated differences in the contractility of cerebral arteries from near-term fetal and adult animals. To test the hypothesis that these contractility differences result from maturational changes in voltage-gated K+ channel function, we compared this function in VSM myocytes from adult and fetal sheep cerebral arteries. The primary current-carrying, voltage-gated K+ channels in VSM myocytes are the large conductance Ca2+-activated K+ channels (BKCa) and voltage-activated K+ (KV) channels. We observed that at voltage-clamped membrane potentials of +60 mV in perforated whole cell studies, the normalized outward current densities in fetal myocytes were >30% higher than in those of the adult (P < 0.05) and that these were predominantly due to iberiotoxin-sensitive currents from BKCa channels. Excised, insideout membrane patches revealed nearly identical unitary conductances and Hill coefficients for BKCa channels. The plot of log intracellular [Ca2+] ([Ca2+]i) versus voltage for half-maximal activation (V(1/2)) yielded linear and parallel relationships, and the change in V(1/2) for a 10-fold change in [Ca2+] was also similar. Channel activity increased e-fold for a 19 +/- 2-mV depolarization for adult myocytes and for an 18 +/- 1-mV depolarization for fetal myocytes (P > 0.05). However, the relationship between BKCa open probability and membrane potential had a relative leftward shift for the fetal compared with adult myocytes at different [Ca2+]i. The [Ca2+] for half-maximal activation (i.e., the calcium set points) at 0 mV were 8.8 and 4.7 microM for adult and fetal myocytes, respectively. Thus the increased BKCa current density in fetal myocytes appears to result from a lower calcium set point.     

Mechanisms of coronary artery depolarization by uridine triphosphate

We sought to define the basic mechanisms by which pyrimidine nucleotides constrict rat coronary resistance arteries. Uridine triphosphate (UTP) caused a dose-dependent constriction in coronary arteries stripped of endothelium. UTP also depolarized and increased cytosolic Ca2+ in coronary smooth muscle cells. Nisoldipine, an antagonist of voltage-operated Ca2+ channels, blocked the rise in cytosolic Ca2+ and reduced UTP-induced vasoconstriction by approximately 75% which suggests a prominent role for depolarization in this constrictor response. The ionic basis of UTP-induced depolarization was subsequently explored in coronary smooth muscle cells using whole-cell patch-clamp electrophysiology. In the absence of K+ and with CsCl in the pipette, UTP (40 microM) activated a sustained inwardly rectifying current (-0.66 +/- 0.10 pA/pF at -60 mV). A 100 mM reduction in bath Na+ shifted the reversal potential of this current (from -2 +/- 1 to -28 +/- 4 mV) and reduced the magnitude (from -2.26 +/- 0.61 to -0.51 +/- 0.11 pA/pF). In addition to activating a depolarizing cation current, UTP inhibited hyperpolarizing outward currents. Specifically, UTP inhibited ATP-sensitive and voltage-dependent K+ currents yet had no effect on inwardly rectifying and Ca2+-activated K+ channels. This study indicates that electromechanical coupling is integral to pyrimidine-induced constriction in coronary resistance arteries.     

Properties of calcium and potassium currents of clonal adrenocortical cells

The ionic currents of clonal Y-1 adrenocortical cells were studied using the whole-cell variant of the patch-clamp technique. These cells had two major current components: a large outward current carried by K ions, and a small inward Ca current. The Ca current depended on the activity of two populations of Ca channels, slow (SD) and fast (FD) deactivating, that could be separated by their different closing time constants (at -80 mV, SD, 3.8 ms, and FD, 0.13 ms). These two kinds of channels also differed in (a) activation threshold (SD, approximately -50 mV; FD, approximately -20 mV), (b) half-maximal activation (SD, between -15 and -10 mV; FD between +10 and +15 mV), and (c) inactivation time course (SD, fast; FD, slow). The total amplitude of the Ca current and the proportion of SD and FD channels varied from cell to cell. The amplitude of the K current was strongly dependent on the internal [Ca2+] and was almost abolished when internal [Ca2+] was less than 0.001 microM. The K current appeared to be independent, or only slightly dependent, of Ca influx. With an internal [Ca2+] of 0.1 microM, the activation threshold was -20 mV, and at +40 mV the half-time of activation was 9 ms. With 73 mM external K the closing time constant at -70 mV was approximately 3 ms. The outward current was also modulated by internal pH and Mg. At a constant pCa gamma a decrease of pH reduced the current amplitude, whereas the activation kinetics were not much altered. Removal of internal Mg produced a drastic decrease in the amplitude of the Ca-activated K current. It was also found that with internal [Ca2+] over 0.1 microM the K current underwent a time-dependent transformation characterized by a large increase in amplitude and in activation kinetics.     

Voltage- and calcium-activated currents in cultured olfactory receptor neurons of male Mamestra brassicae (Lepidoptera)

Insect olfactory receptor neurons (ORNs) grown in primary cultures were studied using the patch-clamp technique in both conventional and amphotericin B perforated whole-cell configurations under voltage-clamp conditions. After 10-24 days in vitro, ORNs had a mean resting potential of -62 mV and an average input resistance of 3.2 GOmega. Five different voltage-dependent ionic currents were isolated: one Na(+), one Ca(2+) and three K(+) currents. The Na(+) current (35-300 pA) activated between -50 and -30 mV and was sensitive to 1 microM tetrodotoxin (TTX). The sustained Ca(2+) current activated between -30 and -20 mV, reached a maximum amplitude at 0 mV (-4.5 +/- 6.0 pA) that increased when Ba(2+) was added to the bath and was blocked by 1 mM Co(2+). Total outward currents were composed of three K(+) currents: a Ca(2+)-activated K(+) current activated between -40 and -30 mV and reached a maximum amplitude at +40 mV (605 +/- 351 pA); a delayed-rectifier K(+) current activated between -30 and -10 mV, had a mean amplitude of 111 +/- 67 pA at +60 mV and was inhibited by 20 mM tetraethylammonium (TEA); and, finally, more than half of ORNs exhibited an A-like current strongly dependent on the holding potential and inhibited by 5 mM 4-aminopyridine (4-AP). Pheromone stimulation evoked inward current as measured by single channel recordings.     

Membrane currents in canine bronchial artery and their regulation by excitatory agonists

The bronchial vasculature plays an important role in airway physiology and pathophysiology. We investigated the ion currents in canine bronchial smooth muscle cells using patch-clamp techniques. Sustained outward K(+) current evoked by step depolarizations was significantly inhibited by tetraethylamonium (1 and 10 mM) or by charybdotoxin (10(-6) M) but was not significantly affected by 4-aminopyridine (1 or 5 mM), suggesting that it was primarily a Ca(2+)-activated K(+) current. Consistent with this, the K(+) current was markedly increased by raising external Ca(2+) to 4 mM but was decreased by nifedipine (10(-6) M) or by removing external Ca(2+). When K(+) currents were blocked (by Cs(+) in the pipette), step depolarizations evoked transient inward currents with characteristics of L-type Ca(2+) current as follows: 1) activation that was voltage dependent (threshold and maximal at -50 and -10 mV, respectively); 2) inactivation that was time dependent and voltage dependent (voltage causing 50% maximal inactivation of -26 +/- 22 mV); and 3) blockade by nifedipine (10(-6) M). The thromboxane mimetic U-46619 (10(-6) M) caused a marked augmentation of outward K(+) current (as did 10 mM caffeine) lasting only 10-20 s; this was followed by significant suppression of the K(+) current lasting several minutes. Phenylephrine (10(-4) M) also suppressed the K(+) current to a similar degree but did not cause the initial transient augmentation. None of these three agonists elicited inward current of any kind. We conclude that bronchial arterial smooth muscle expresses Ca(2+)-dependent K(+) channels and voltage-dependent Ca(2+) channels and that its excitation does not involve activation of Cl(-) channels.     

Ca(2+)-activated K+ channels in human leukemic T cells

Using the patch-clamp technique, we have identified two types of Ca(2+)-activated K+ (K(Ca)) channels in the human leukemic T cell line. Jurkat. Substances that elevate the intracellular Ca2+ concentration ([Ca2+]i), such as ionomycin or the mitogenic lectin phytohemagglutinin (PHA), as well as whole-cell dialysis with pipette solutions containing elevated [Ca2+]i, activate a voltage-independent K+ conductance. Unlike the voltage-gated (type n) K+ channels in these cells, the majority of K(Ca) channels are insensitive to block by charybdotoxin (CTX) or 4-aminopyridine (4-AP), but are highly sensitive to block by apamin (Kd less than 1 nM). Channel activity is strongly dependent on [Ca2+]i, suggesting that multiple Ca2+ binding sites may be involved in channel opening. The Ca2+ concentration at which half of the channels are activated is 400 nM. These channels show little voltage dependence over a potential range of -100 to 0 mV and have a unitary conductance of 4-7 pS in symmetrical 170 mM K+. In the presence of 10 nM apamin, a less prevalent type of K(Ca) channel with a unitary conductance of 40-60 pS can be observed. These larger-conductance channels are sensitive to block by CTX. Pharmacological blockade of K(Ca) channels and voltage-gated type n channels inhibits oscillatory Ca2+ signaling triggered by PHA. These results suggest that K(Ca) channels play a supporting role during T cell activation by sustaining dynamic patterns of Ca2+ signaling.     

A whole-cell and single-channel study of the voltage-dependent outward potassium current in avian hepatocytes

Voltage-dependent membrane currents were studied in dissociated hepatocytes from chick, using the patch-clamp technique. All cells had voltage-dependent outward K+ currents; in 10% of the cells, a fast, transient, tetrodotoxin-sensitive Na+ current was identified. None of the cells had voltage-dependent inward Ca2+ currents. The K+ current activated at a membrane potential of about -10 mV, had a sigmoidal time course, and did not inactivate in 500 ms. The maximum outward conductance was 6.6 +/- 2.4 nS in 18 cells. The reversal potential, estimated from tail current measurements, shifted by 50 mV per 10-fold increase in the external K+ concentration. The current traces were fitted by n2 kinetics with voltage-dependent time constants. Omitting Ca2+ from the external bath or buffering the internal Ca2+ with EGTA did not alter the outward current, which shows that Ca2+-activated K+ currents were not present. 1-5 mM 4-aminopyridine, 0.5-2 mM BaCl2, and 0.1-1 mM CdCl2 reversibly inhibited the current. The block caused by Ba was voltage dependent. Single-channel currents were recorded in cell-attached and outside-out patches. The mean unitary conductance was 7 pS, and the channels displayed bursting kinetics. Thus, avian hepatocytes have a single type of K+ channel belonging to the delayed rectifier class of K+ channels.     

Differential activation of potassium channels in cerebral and hindquarter arteries of rats during simulated microgravity

The purpose of this study was to test the hypothesis that differential autoregulation of cerebral and hindquarter arteries during simulated microgravity is mediated or modulated by differential activation of K(+) channels in vascular smooth muscle cells (VSMCs) of arteries in different anatomic regions. Sprague-Dawley rats were subjected to 1- and 4-wk tail suspension to simulate the cardiovascular deconditioning effect due to short- and medium-term microgravity. K(+) channel function of VSMCs was studied by pharmacological methods and patch-clamp techniques. Large-conductance Ca(2+)-activated K(+) (BK(Ca)) and voltage-gated K(+) (K(v)) currents were determined by subtracting the current recorded after applications of 1 mM tetraethylammonium (TEA) and 1 mM TEA + 3 mM 4-aminopyridine (4-AP), respectively, from that of before. For cerebral vessels, the normalized contractility of basilar arterial rings to TEA, a BK(Ca) blocker, and 4-AP, a K(v) blocker, was significantly decreased after 1- and 4-wk simulated microgravity, respectively. VSMCs isolated from the middle cerebral artery branches of suspended rats had a more depolarized membrane potential (E(m)) and a smaller K(+) current density compared with those of control rats. Furthermore, the reduced total current density was due to smaller BK(Ca) and smaller K(v) current density in cerebral VSMCs after 1- and 4-wk tail suspension, respectively. For hindquarter vessels, VSMCs isolated from second- to sixth-order small mesenteric arteries of both 1- and 4-wk suspended rats had a more negative E(m) and larger K(+) current densities for total, BK(Ca), and K(v) currents. These results indicate that differential activation of K(+) channels occur in cerebral and hindquarter VSMCs during short- and medium-term simulated microgravity. It is further suggested that different profiles of channel remodeling might occur in VSMCs as one of the important underlying cellular mechanisms to mediate and modulate differential vascular adaptation during microgravity.     

Multiple channels mediate calcium leakage in the A7r5 smooth muscle-derived cell line.

Ca2+ entry under resting conditions may be important for contraction of vascular smooth muscle, but little is known about the mechanisms involved. Ca2+ leakage was studied in the A7r5 smooth muscle-derived cell line by patch-clamp techniques. Two channels that could mediate calcium influx at resting membrane potentials were characterized. In 110 mM Ba2+, one channel had a slope conductance of 6.0 +/- 0.6 pS and an extrapolated reversal potential of +41 +/- 13 mV (mean +/- SD, n = 8). The current rectified strongly, with no detectable outward current, even at +90 mV. Channel gating was voltage independent. A second type of channel had a linear current-voltage relationship, a slope conductance of 17.0 +/- 3.2 pS, and a reversal potential of +7 +/- 4 mV (n = 9). The open probability increased e-fold per 44 +/- 10 mV depolarization (n = 5). Both channels were also observed in 110 mM Ca2+. Noise analysis of whole-cell currents indicates that approximately 100 6-pS channels and 30 17-pS channels are open per cell. These 6-pS and 17-pS channels may contribute to resting calcium entry in vascular smooth muscle cells.     

Effects of 4-aminopyridine on potassium currents in a molluscan neuron

The effects of 4-aminopyridine (4-AP) on the delayed K+ current and on the Ca2+-activated K+ current of the Aplysia pacemaker neurons R-15 and L-6 were studied. The delayed outward K+ current was measured in Ca2+- free artificial seawater (ASW) containing tetrodotoxin (TTX), using brief depolarizing clamp pulses. External (and internal) 4-AP blocks the delayed K+ current in a dose-dependent manner but does not block the leakage current. Our results show that one 4-AP molecule combines with a single receptor site and that the block is voltage dependent with an apparent dissociation constant (K4-AP) of approximately 0.8 mM at 0 mV. K4-AP increases e-fold for a 32-mV change in potential, which is consistent with the block occurring approximately 0.8 of the distance through the membrane electrical field. The 4-AP block appears to depend upon stimulus frequency as well as upon voltage. The greater speed of onset of the block produced by internal 4-AP relative to when it is used externally suggests that 4-AP acts from inside the cell. The Ca2+-activated K+ current was measured in Ca2+-free ASW containing TTX, using internal Ca2+-ion injection to directly activate the K+ conductance. Low external 4-AP concentrations (less than 2 mM) have no effect on the Ca2+-activated K+ current, but concentrations of 5 mM or greater increase the K+ current. Internal 4-AP has the same effect. The opposing effects of 4-AP on the two components of the K+ current can be seen in measurements of the total outward K+ current at different membrane potentials in normal ASW and during the repolarizing phase of the action potential.     

Kinetic analysis of a transient potassium current in rat cerebellar granule cells in vitro]

Thin slices were prepared from cerebella of 10-24 day old rats and examined with whole-cell patch-clamp methods. Depolarizing steps from holding potentials negative to -60 mV elicited an early transient outward current, identified as IA, and a late outward K+ current. Depolarizations from -50 mV failed to evoke any A current and gave only a slowly rising component similar to the delayed K+ current, which inactivated thereafter with a time constant of 2.5 s at -30 mV. The IA peaked in 1-2 ms, decayed following a double exponential with time constants of 8.1 and 53.2 ms at +20 mV and was half-inactivated at -82.5 mV. 4-AP 4 mM depressed both K+ currents showing little specificity between them, while TEA 20 mM selectively abolished only the delayed K+ current.     

Electrophysiological effects of basolateral [Na+] in Necturus gallbladder epithelium

In Necturus gallbladder epithelium, lowering serosal [Na+] ([Na+]s) reversibly hyperpolarized the basolateral cell membrane voltage (Vcs) and reduced the fractional resistance of the apical membrane (fRa). Previous results have suggested that there is no sizable basolateral Na+ conductance and that there are apical Ca(2+)-activated K+ channels. Here, we studied the mechanisms of the electrophysiological effects of lowering [Na+]s, in particular the possibility that an elevation in intracellular free [Ca2+] hyperpolarizes Vcs by increasing gK+. When [Na+]s was reduced from 100.5 to 10.5 mM (tetramethylammonium substitution), Vcs hyperpolarized from -68 +/- 2 to a peak value of -82 +/- 2 mV (P less than 0.001), and fRa decreased from 0.84 +/- 0.02 to 0.62 +/- 0.02 (P less than 0.001). Addition of 5 mM tetraethylammonium (TEA+) to the mucosal solution reduced both the hyperpolarization of Vcs and the change in fRa, whereas serosal addition of TEA+ had no effect. Ouabain (10(-4) M, serosal side) produced a small depolarization of Vcs and reduced the hyperpolarization upon lowering [Na+]s, without affecting the decrease in fRa. The effects of mucosal TEA+ and serosal ouabain were additive. Neither amiloride (10(-5) or 10(-3) M) nor tetrodotoxin (10(-6) M) had any effects on Vcs or fRa or on their responses to lowering [Na+]s, suggesting that basolateral Na+ channels do not contribute to the control membrane voltage or to the hyperpolarization upon lowering [Na+]s. The basolateral membrane depolarization upon elevating [K+]s was increased transiently during the hyperpolarization of Vcs upon lowering [Na+]s. Since cable analysis experiments show that basolateral membrane resistance increased, a decrease in basolateral Cl- conductance (gCl-) is the main cause of the increased K+ selectivity. Lowering [Na+]s increases intracellular free [Ca2+], which may be responsible for the increase in the apical membrane TEA(+)-sensitive gK+. We conclude that the decrease in fRa by lowering [Na+]s is mainly caused by an increase in intracellular free [Ca2+], which activates TEA(+)-sensitive maxi K+ channels at the apical membrane and decreases apical membrane resistance. The hyperpolarization of Vcs is due to increase in: (a) apical membrane gK+, (b) the contribution of the Na+ pump to Vcs, (c) basolateral membrane K+ selectivity (decreased gCl-), and (d) intraepithelial current flow brought about by a paracellular diffusion potential.