Akt activation is necessary for growth factor-induced trafficking of functional K(Ca) channels in developing parasympathetic neurons

The protein kinase Akt is a crucial regulator of neuronal survival and apoptosis. Here we show that Akt activation is necessary for mobilization of large-conductance K(Ca) channels in ciliary ganglion (CG) neurons evoked by beta-neuregulin-1 (NRG1) and transforming growth factor-beta1 (TGFbeta1). Application of NRG1 to embryonic day 9 (E9) CG neurons increased Akt phosphorylation, as observed previously for TGF(beta)1. NRG1- and TGF(beta)1-evoked stimulation of K(Ca) is blocked by inhibitors of PI3K by overexpression of a dominant-negative form of Akt, by overexpression of CTMP, an endogenous negative regulator of Akt, and by application of the Akt inhibitor 1L-6-hydroxymethyl-chiro-inositol 2-(R)-2-O-methyl-3-O-octadecylcarbonate (HIMO). Conversely, overexpression of a constitutively-active form of Akt was sufficient by itself to increase mobilization of functional K(Ca) channels. NRG1 and TGF(beta)1 evoked an Akt-dependent increase in cell-surface SLO alpha-subunits. These procedures have no effect on voltage-activated Ca2+ currents. Thus Akt plays an essential role in the developmental regulation of excitability in CG neurons.     

ATP-sensitive K+ and Ca2+-activated K+ channels in lamprey ( Petromyzon marinus) red blood cell membrane

The patch-clamp technique was used to demonstrate the presence of ATP-sensitive K(+) channels and Ca(2+)-activated K(+) channels in lamprey ( Petromyzon marinus) red blood cell membrane. Whole-cell experiments indicated that the membrane current under isosmotic (285 mosmol l(-1)) conditions is carried by K(+). In the inside-out configuration an ATP-sensitive K(+) channel (70-80 pS inward, 35-40 pS outward) was present in 35% of patches. Application of ATP to the intracellular side reduced unitary current with half-maximal inhibition in the range 10-100 microM. A block was obtained with 100 microM lidocaine and inhibition was obtained with 0.5 mM barium acetate. A Ca(2+)-activated K(+) channel (25-30 pS inward, 10-15 pS outward) was present in 57% of patches. Inhibition was produced by 10 mM TEA and 500 nM apamin and sensitivity to Ba(2+) was lower than for ATP-sensitive channels. No spontaneous channel activity was recorded in the cell-attached configuration under isotonic conditions. With hypotonic saline 68% of patches showed spontaneous single-channel activity, and, of 75 active patches, 66 cell-attached patches showed channel activity corresponding to Ca(2+)-activated K(+) channels.     

Acetylcholine and ATP hyperpolarize endothelium via activation of different types of Ca(2+)-activated K+ channels

Experiments on rat thoracic aorta showed that ATP and acetylcholine hyperpolarize endothelial cell via selective activation of low (SK) and intermediate (IR) conductance Ca²⁺-activated K⁺ channels, respectively. It was hypothesized that ATP- and acetylcholine-activated Ca²⁺-signals are spatially separated and generated in plasma membrane regions with predominant localization of SK- and IR-channels, respectively.     

Whole-cell and unitary Ca channel currents in mammalian intestinal smooth muscle cells: evidence for the existence of two types of Ca channels

Whole-cell and single Ca channel currents were recorded in smooth muscle cells isolated from guinea-pig taenia coli to examine whether multiple types of Ca channels exist. Two different types of voltage-dependent Ca channels with different conductances and inactivation kinetics were identified from cell-attached patch clamp recordings using 50 mM Ba in the patch pipettes. One type of channel, with a large conductance of 25 pS, had a threshold of activation near –40 mV and the mean current reconstructed by averaging individual current responses inactivated slowly. A second type of channel, with a small conductance of 12 pS, had a similar threshold value to that of the 25 pS channel, but the averaged current inactivated rapidly. The steady state inactivation of the 12 pS channel was complete at a holding potential of –40 mV. We concluded that both channel types represent fast and slow inactivating voltage-dependent Ca channels which have been found in many excitable cells.     

Number of K(Ca) channels underlying spontaneous miniature outward currents (SMOCs) in mudpuppy cardiac neurons

Spontaneous miniature outward currents (SMOCs) in parasympathetic neurons from mudpuppy cardiac ganglia are caused by activation of TEA- and iberiotoxin-sensitive, Ca(2+)-dependent K(+) (BK) channels. Previously we reported that SMOCs are activated by Ca(2+)-induced Ca(2+) release (CICR) from caffeine- and ryanodine-sensitive intracellular Ca(2+) stores. In the present study, we analyzed the single channel currents that contribute to SMOC generation in mudpuppy cardiac neurons. The slope conductance of BK channels, determined from the I-V relationship of single-channel currents recorded with cell-attached patches in physiological K(+) concentrations, was 84 pS. The evidence supporting the identity of this channel as the channel involved in SMOC generation was its sensitivity to internal Ca(2+), external TEA, and caffeine. In cell-attached patch recordings, 166 microM TEA applied in the pipette reduced single-channel current amplitude by 32%, and bath-applied caffeine increased BK channel activity. The ratio between the averaged SMOC amplitude and the single-channel current amplitude was used to estimate the average number of channels involved in SMOC generation. The estimated number of channels involved in generation of an averaged SMOC ranged from 18 to 23 channels. We also determined that the Po of the BK channels at the peak of a SMOC remains constant at voltages more positive than -20 mV, suggesting that the transient rise in intracellular Ca(2+) from ryanodine-sensitive intracellular stores in the vicinity of the BK channel reached concentrations most likely exceeding 40 microM.     

Modulation of Ca(2+) signaling by K(+) channels in a hypothalamic neuronal cell line (GT1-1)

The pulsatile release of gonadotropin releasing hormone (GnRH) is driven by the intrinsic activity of GnRH neurons, which is characterized by bursts of action potentials correlated with oscillatory increases in intracellular Ca(2+). The role of K(+) channels in this spontaneous activity was studied by examining the effects of commonly used K(+) channel blockers on K(+) currents, spontaneous action currents, and spontaneous Ca(2+) signaling. Whole-cell recordings of voltage-gated outward K(+) currents in GT1-1 neurons revealed at least two different components of the current. These included a rapidly activating transient component and a more slowly activating, sustained component. The transient component could be eliminated by a depolarizing prepulse or by bath application of 1.5 mM 4-aminopyridine (4-AP). The sustained component was partially blocked by 2 mM tetraethylammonium (TEA). GT1-1 cells also express inwardly rectifying K(+) currents (I(K(IR))) that were activated by hyperpolarization in the presence of elevated extracellular K(+). These currents were blocked by 100 microM Ba(2+) and unaffected by 2 mM TEA or 1.5 mM 4-AP. TEA and Ba(2+) had distinct effects on the pattern of action current bursts and the resulting Ca(2+) oscillations. TEA increased action current burst duration and increased the amplitude of Ca(2+) oscillations. Ba(2+) caused an increase in the frequency of action current bursts and Ca(2+) oscillations. These results indicate that specific subtypes of K(+) channels in GT1-1 cells can have distinct roles in the amplitude modulation or frequency modulation of Ca(2+) signaling. K(+) current modulation of electrical activity and Ca(2+) signaling may be important in the generation of the patterns of cellular activity responsible for the pulsatile release of GnRH.     

Serotonin modulated Ca++ dependent K+ channels in alloimmune effector cell lytic function

Potassium channel activity has been implicated in the lytic function of cloned murine effector T lymphocytes (5) and human NK cells (12) as well as in the initiation of the injury process in tumor cells (23). In the present studies, the effects of various K+ channel blockers on the cytolytic function of in vivo derived alloimmune lymphocytes towards P815 tumor cells were evaluated. The classical K+ channel blocker 4-aminopyridine (4-AP), the naturally occurring monoamine serotonin (5-hydroxytryptamine, 5-HT) and its agonist, quipazine, as well as the Ca++ dependent K+ channel blocker quinidine were chosen for investigation based on their known ion channel gating properties. These agents, when present in the assay medium, inhibited in a dose dependent manner the lysis of P815 tumor cells as measured by specific 51Cr release. Preincubation of effector lymphocytes with the various K+ channel blockers resulted in greater inhibition of lysis than did the preincubation of target cells. The 5-HT agonist quipazine was of particular interest in that it inhibited the lytic process with equal effectiveness when continuously present in the assay medium or when the effector cells alone were preincubated. Quinidine was used to investigate whether Ca++ dependent K+ channels were the predominant ion channel involved in the lytic process. When present during the lytic assay, quinidine was similar to quipazine in terms of their dose range at which they inhibited the lytic process. These results indicate that 5-HT sensitive Ca++ dependent K+ channels are likely to be involved in the delivery of lytic signal(s) by immune effector lymphocytes and suggests that neuroendocrine products may modulate the functional activity of in vivo derived lymphocytes.     

Sarcolemmal K(ATP) channels in ageing

This review highlights some recent research addressing sarcolemmal K(ATP) channels in ageing. These channels are abundant in cardiac myocytes where they are essential in coupling the cellular metabolic state with membrane excitability. The opening of sarcolemmal ATP-sensitive K+ (K(ATP)) channels occurs during ischaemia and protect the heart against injury. Age-dependent changes in the myocardial susceptibility to ischemia have been observed in different species, including humans. Recent research has demonstrated that ageing is associated with decrease in numbers of sarcolemmal K(ATP) in hearts from females, but not males. This phenomenon seems to be associated with age-dependent decrease in concentration of circulating estrogens. In the heart, SUR2A, a regulatory subunit of K(ATP) channels, is present in excess over Kir6.2, a pore-forming K(ATP) channel subunit. The consequence of this is that SUR2A is a subunit that controls the number of sarcolemmal K(ATP) channels. Estrogens specifically up-regulate SUR2A and, thereby, control the number of sarcolemmal K(ATP) channels. Age-dependent loss of sarcolemmal K(ATP) channels creates a cardiac phenotype more sensitive to ischaemia, which may explain, at least in part, an ageing-associated decrease of myocardial tolerance to stress that occurs in elderly women.     

Small (SKCa) Ca2+-activated K+ channels in cultured rat hippocampal pyramidal neurones

 Small (SK_Ca) Ca²⁺-activated K⁺ channels were identified in membrane patches excised from cultured CA1-CA3 pyramidal neurones of the neonatal rat hippocampus. When recorded in low-K⁺ extracellular solution ([K⁺]_o=2.5 mM), SK_Ca channels had a low conductance (@3 pS at 0 mV), were activated by ≥175 nM Ca²⁺ (P _o=0.54 at 500 nM Ca²⁺) and there were two open-time components (2.1 and @70 ms) to their activity. These properties of single SK_Ca channels are similar to those of slow after-hyperpolarization channels (sAHP) previously inferred from fluctuation analysis of the sAHP current. It is concluded that the SK_Ca channel reported here may be the channel that generates the sAHP in hippocampal pyramidal neurones. Received: 9 July 1998 / Received after revision: 5 October 1998 / Accepted: 7 October 1998     

High sensitivity to internal tetraethylammonium in K(Ca) channels of cerebrovascular smooth muscle cells.

Patch clamp methods were used to study the properties of calcium-dependent K(Ca) channels in enzyme dissociated smooth muscle cells from the cerebral arteries of adult rats. Dissociated muscle cells were maintained at 4 degrees C for up to 48 h prior to use. Inside-out membrane patches excised from these cells contained a K(Ca) channel with a conductance of 92 +/- 2.6 pS in symmetrical 140 mM potassium solutions. This channel was activated by membrane depolarization and by cytoplasmic calcium, and showed negligible permeability to sodium or cesium ions. Single channel currents were reduced by internal application of tetraethylammonium ions, with a Kd = 0.31 mM.     

Facilitation of Ca2+-activated K+ channels (IKCa1) by mibefradil in B lymphocytes

K⁺ channels play critical roles in the proliferation and activation of lymphocytes. Mouse B cells express large-conductance background K⁺ channel (LK_bg) in addition to the voltage-gated K⁺ channel (Kv) and Ca²⁺-activated K⁺ channel current (IKCa1). Mibefradil, a blocker of T-type Ca²⁺ channels, has been reported to affect the proliferation of immune cells. In this study, we investigated the effects of mibefradil on the membrane potential and ion channels in murine B cell lines, WEHI-231 and Bal-17. In the whole-cell patch clamp experiments, mibefradil blocked Kv and LK_bg current with half inhibitory concentration (IC₅₀), 1.9 and 2.3 μM, respectively. Interestingly, IKCa1 current was increased by mibefradil. In the inside-out patch clamp study with cloned murine IKCa1 (mIKCa1) in HEK-293, mibefradil increased both Ca²⁺ sensitivity and maximum activity of mIKCa1. At high concentrations (>10 μM), mibefradil inhibited mIKCa1 in a voltage-dependent manner. Application of anti-IgM antibody to stimulate B cell receptors (BCR-ligation) induced transient hyperpolarization of Bal-17 and WEHI-231 cells, which became persistent with 1 μM mibefradil. The hyperpolarizing response was abolished by charybdotoxin, a selective blocker for SK4/IKCa1. In summary, our study firstly reports the ion channel-activating effects of mibefradil. The selective potent activation of IKCa1 suggests that mibefradil-derived drugs might be useful in the control of cell responses related with IKCa1. HY Yoo, H Zheng, and JH Nam contributed equally to this study.     

Biophysical,pharmacological and developmental properties of ATP-sensitive K+ channels in cultured myotomal muscle cells from Xenopus embryos

Unlike mammalian muscle cells in culture, cultured myotomal muscle cells of Xenopus embryos express ATP-sensitive K⁺ (K_ATP) channels. The K_ATP channels are blocked by internal ATP (half-maximal inhibition K _0.5 = 16 M) and to a lesser extent by internal ADP, are voltage independent, have an inward rectification at positive potentials and are inhibited by glibenclamide (K _0.5 = 2 M). Surprisingly, these K_ATP channels are not sensitive to K⁺ channel openers such as cromakalim. Opening of these K_ATP channels does not occur under normal physiological conditions. It is elicited by metabolic exhaustion of the muscle cell and it precedes the development of an irreversible rigor state. Neither intracellular acidosis nor an increase of intracellular Ca²⁺ are involved in K_ATP channel opening. Different types of K⁺ channels are successively expressed after plating of myotomal muscle cells: (1) sustained delayed-rectifier K⁺ channels; (2) K_ATP channels; (3) inward-rectifier K⁺ channels; (4) transient delayed-rectifier K⁺ channels. The current density associated with K_ATP channels far exceeds that of voltage-dependent K⁺ channels. Innervation controls the expression of these K_ATP channels. Co-culture of muscle cells with neurons from the neural tube decreases the number of active K_ATP channels per patch. Similarly, in situ innervated submaxillaris muscle of tadpoles at stage 50–55 has a very low density of K_ATP channels.     

Ca2+ channels,ryanodine receptors and Ca2+-activated K+ channels: a functional unit for regulating arterial tone

Local calcium transients (‘Ca²⁺ sparks’) are thought to be elementary Ca²⁺ signals in heart, skeletal and smooth muscle cells. Ca²⁺ sparks result from the opening of a single, or the coordinated opening of many, tightly clustered ryanodine receptor (RyR) channels in the sarcoplasmic reticulum (SR). In arterial smooth muscle, Ca²⁺ sparks appear to be involved in opposing the tonic contraction of the blood vessel. Intravascular pressure causes a graded membrane potential depolarization to approximately ?40 mV, an elevation of arterial wall [Ca²⁺]_i and contraction (‘myogenic tone’) of arteries. Ca²⁺ sparks activate calcium-sensitive K⁺ (K_Ca) channels in the sarcolemmal membrane to cause membrane hyperpolarization, which opposes the pressure induced depolarization. Thus, inhibition of Ca²⁺ sparks by ryanodine, or of K_Ca channels by iberiotoxin, leads to membrane depolarization, activation of L -type voltage-gated Ca²⁺ channels, and vasoconstriction. Conversely, activation of Ca²⁺ sparks can lead to vasodilation through activation of K_Ca channels. Our recent work is aimed at studying the properties and roles of Ca²⁺ sparks in the regulation of arterial smooth muscle function. The modulation of Ca²⁺ spark frequency and amplitude by membrane potential, cyclic nucleotides and protein kinase C will be explored. The role of local Ca²⁺ entry through voltage-dependent Ca²⁺ channels in the regulation of Ca²⁺ spark properties will also be examined. Finally, using functional evidence from cardiac myocytes, and histological evidence from smooth muscle, we shall explore whether Ca²⁺ channels, RyR channels, and K_Ca channels function as a coupled unit, through Ca²⁺ and voltage, to regulate arterial smooth muscle membrane potential and vascular tone.     

Acetylcholine activates two types of ion channels in sarcolemma from adult muscular dystrophic (mdx) mice.

Dissociated interosseus muscle fibres of wildtype and mdx mice were investigated to characterize acetylcholine (ACh) receptors with the single channel recording patch-clamp technique. On the muscle fibres of mdx mutants, two types of nicotinic acetylcholine receptor (nAChR) channels were found. One channel (29 pS, mean open time 2.1 ms) resembles channels on denervated and embryonic muscle and was not found on wildtype muscle where exclusively a 48 pS channel (mean open time 1.3 ms) was seen.     

Ca(2+)-induced Ca(2+) release activates spontaneous miniature outward currents (SMOCs) in parasympathetic cardiac neurons.

Mudpuppy parasympathetic cardiac neurons exhibit spontaneous miniature outward currents (SMOCs) that are thought to be due to the activation of clusters of large conductance Ca(2+)-activated K(+) channels (BK channels) by localized release of Ca(2+) from internal stores close to the plasma membrane. Perforated-patch whole cell recordings were used to determine whether Ca(2+)-induced Ca(2+) release (CICR) is involved in SMOC generation. We confirmed that BK channels are involved by showing that SMOCs are inhibited by 100 nM iberiotoxin or 500 microM tetraethylammonium (TEA), but not by 100 nM apamin. SMOC frequency is decreased in solutions that contain 0 Ca(2+)/3.6 mM Mg(2+), and also in the presence of 1 microM nifedipine and 3 microM omega-conotoxin GVIA, suggesting that SMOC activation is dependent on calcium influx. However, Ca(2+) influx alone is not sufficient; SMOC activation is also dependent on Ca(2+) release from the caffeine- and ryanodine-sensitive Ca(2+) store, because exposure to 2 mM caffeine consistently caused an increase in SMOC frequency, and 10-100 microM ryanodine altered the configuration of SMOCs and eventually inhibited SMOC activity. Depletion of intracellular Ca(2+) stores by the Ca-ATPase inhibitor cyclopiazonic acid (10 microM) inhibited SMOC activity, even when Ca(2+) influx was not compromised. We also tested the effects of the membrane-permeable Ca(2+) chelators, bis-(o-aminophenoxy)-N,N,N', N'-tetraacetic acid-AM (BAPTA-AM) and EGTA-AM. EGTA-AM (10 microM) caused no inhibition of SMOC activation, whereas 10 microM BAPTA-AM consistently inhibited SMOCs. After SMOCs were completely inhibited by BAPTA, 3 mM caffeine caused SMOC activity to resume. This effect was reversible on removal of caffeine and suggests that the source of Ca(2+) that triggers the internal Ca(2+) release channel is different from the source of Ca(2+) that activates clusters of BK channels. We propose that influx of Ca(2+) through voltage-dependent Ca(2+) channels is required for SMOC generation, but that the influx of Ca(2+) triggers CICR from intracellular stores, which then activates the BK channels responsible for SMOC generation.     

Pharmacological evidence for two types of Ca2+-dependent K+-conductance in bullfrog sympathetic ganglion cells

The effect of apamin, a bee venom toxin, on the action potential and the spike after hyperpolarization was studied in bullfrog sympathetic ganglion cells. Apamin reduced the duration of the afterhyperpolarization but did not affect the maximum rates of rise and fall of Na+- and Ca2+-dependent action potentials. In the presence of apamin and Co2+, the maximum rate of fall of the action potential was decreased, and the spike duration was prolonged. These results suggest that at least two types of Ca2+-dependent K+-conductance co-exist in bullfrog sympathetic ganglion cells.     

The Pars tuberalis of the monkey (Macaca fascicularis) hypophysis: cell types and hormone expression

The pars tuberalis (Pt) of most mammalian species contains specific cells which are structurally and functionally different from the pars distalis (Pd) cells. Pt-specific cells possess melatonin receptors and reveal morphological changes dependent on the duration of the photoperiod. Furthermore, in hamsters the transmission of photoperiodic stimuli to the endocrine system is influenced by melatonin, an effect which is likely to be mediated by Pt-specific cells. In monkeys, however, only little is known about this cell type. Therefore, we studied the ultrastructural differentiation of Pt-specific cells and describe the expression of different hormones and their mRNA by immunohistochemistry and in situ hybridization. Apparently the Pt consists of (1) cells similar to gonadotropic cells of the Pd, (2) folliculostellate cells and (3) a cell population which is morphologically and functionally clearly distinct from all other cell types found in the Pd. Morphologically they resemble the Pt-specific cells found in other species. Regarding the expression of secretory products there is evidence that they transcribe and translate the beta-TSH subunit. Although there is a strong signal for the mRNA of the common alpha-chain, protein staining is much weaker. POMC mRNA is expressed in the Pt while there is no evidence for PRL mRNA. The present results lead to the conclusion that the Pt of the monkey contains Pt-specific cells which express different hormonal subunits as was already shown for other species. In context with previous findings of melatonin receptors in the monkey Pt further investigations are necessary to establish the possible role of Pt-specific cells in the photoperiod-dependent generation of endocrine rhythms.