Muscarinic modulation of a transient K+ conductance in rat neostriatal neurons

Neurons of the neostriatum are richly innervated by cholinergic neurons of intrinsic origin. Both pre- and post-synaptic muscarinic receptors mediate the effects of acetylcholine (ACh). Activation of these receptors is functionally significant, particularly in Parkinson's disease. Current-clamp studies indicate that muscarinic receptors serve to decrease the responsiveness of neostriatal neurons to excitatory inputs. Here we present evidence that this effect is caused, in part, by the muscarinic modulation of the A-current, a transient outward potassium current. The voltage dependence of this current suggests that normally it enhances spike repolarization and slows discharge rate, but does not affect 'synaptic integration'. We find that under the influence of muscarinic agonists, the voltage dependence of A-current activation and inactivation is shifted towards more negative membrane potentials and the peak conductance is increased. Therefore, at relatively hyperpolarized resting potentials, ACh transiently alters the functional role of the A-current, allowing it to suppress excitatory inputs and further slow the discharge rate. But at relatively depolarized resting potentials, ACh increases excitability by removing the A-current through inactivation.     

Single apamin-blocked Ca-activated K+ channels of small conductance in cultured rat skeletal muscle

Action potentials in many excitable cells are followed by a prolonged afterhyperpolarization that modulates repetitive firing. Although it is established that the afterhyperpolarization is produced by Ca-activated K+ currents, the basis of these currents is not known. The large conductance (250 pS) Ca-activated K+ channel (BK channel) is not a major contributor to the afterhyperpolarization in non-innervated skeletal muscle and some nerve cells, because apamin, a neurotoxic component of bee venom, abolishes the afterhyperpolarization but does not block BK channels, and 5 mM extracellular tetraethylammonium ion (TEA) blocks BK channels but does not reduce the afterhyperpolarization. We now report single-channel currents from small conductance (10-14 pS) Ca-activated K+ channels (SK channels) with the necessary properties to account for the afterhyperpolarization. SK channels are blocked by apamin but not by 5 mM external TEA (TEAo). They are also highly Ca-sensitive at the negative membrane potentials associated with the afterhyperpolarization.     

ATP-sensitive K+ channel in the mitochondrial inner membrane.

Mitochondria take up and extrude various inorganic and organic ions, as well as larger substances such as proteins. The technique of patch clamping should provide real-time information on such transport and on energy transduction in oxidative phosphorylation. It has been applied to detect microscopic currents from mitochondrial membranes and conductances of ion channels in the 5-1,000 pS range in the outer and inner membranes. These pores are not, however, selective for particular ions. Here we use fused giant mitoplasts prepared from rat liver mitochondria to identify a small conductance channel highly selective for K+ in the inner mitochondrial membrane. This channel can be reversibly inactivated by ATP applied to the matrix side under inside-out patch configuration; it is also inhibited by 4-aminopyridine and by glybenclamide. The slope conductance of the unitary currents measured at negative membrane potentials was 9.7 +/- 1.0 pS (mean +/- s.d., n = 6) when the pipette solution contained 100 mM K+ and the bathing solution 33.3 mM K+. Our results indicate that mitochondria depolarize by generating a K+ conductance when ATP in the matrix is deficient.     

Reduced Na+ and K+ permeability of K+ channel in plasma membrane isolated from roots of salt-tolerant mutant of wheat

The Na⁺ and K⁺ permeability of K⁺ channel in plasma membrane, isolated from roots of the salt-tolerant mutant of wheat, was lower than that of wild type in 100 mmol/L KCl and NaCl solution. The opening frequency of K⁺ channel of the mutant reduced more significantly than that of wild type in two kinds of solution mentioned above. It is assumed that the reduction of opening frequency mainly contributes to the Na⁺ and K⁺ permeability of K⁺ channel of the mutant. The electric conductance of single-channel of the mutant was similar to that of wild type and the main difference between them was exhibited as the opening frequency. Their K⁺/Ka⁺ selectivity of K⁺ channel had no significant difference. The K⁺/Na⁺ selectivity of the mutant and wild type was 3.35 and 3.18 respectively.     

Ohmic conductance through the inwardly rectifying K channel and blocking by internal Mg2+

The inwardly rectifying K channel provides the resting K conductance in a variety of cells. This channel acts as a valve or diode, permitting entry of K+ under hyperpolarization, but not its exit under depolarization. This behaviour, termed inward rectification, permits long depolarizing responses which are of physiological significance for the pumping function of the heart and for fertilization of egg cells. Little is known about the outward currents through the inwardly rectifying K channel, despite their great physiological importance, and the mechanism of inward rectification itself is unknown. We have used improved patch clamp techniques to control the intracellular media, and have recorded the outward whole-cell and single-channel currents. We report here that the channel conductance is ohmic and that the well-known inward rectification of the resting K conductance is caused by rapid closure of the channel accompanied by a voltage-dependent block by intracellular Mg2+ ions at physiological concentrations.     

Yeast plasma membrane ATPase is essential for growth and has homology with (Na+ + K+), K+- and Ca2+-ATPases

The plasma membrane ATPase of plants and fungi is a hydrogen ion pump. The proton gradient generated by the enzyme drives the active transport of nutrients by H+-symport. In addition, the external acidification in plants and the internal alkalinization in fungi, both resulting from activation of the H+ pump, have been proposed to mediate growth responses. This ATPase has a relative molecular mass (Mr) similar to those of the Na+-, K+- and Ca2+-ATPases of animal cells and, like these proteins, forms an aspartylphosphate intermediate. We have cloned, mapped and sequenced the gene encoding the yeast plasma membrane ATPase (PMA1) and report here that it maps to chromosome VII adjacent to LEU1. The strong homology between the amino-acid sequence encoded by PMA1 and those of (Na+ + K+), Na+-, K+- and Ca2+- ATPases is consistent with the notion that the family of cation pumps which form a phosphorylated intermediate evolved from a common ancestral ATPase. The function of the PMA1 gene is essential because a null mutation is lethal in haploid cells.     

Inositol trisphosphate-dependent periodic activation of a Ca(2+)-activated K+ conductance in glucose-stimulated pancreatic beta-cells.

Glucose-stimulated insulin secretion is associated with the appearance of electrical activity in the pancreatic beta-cell. At intermediate glucose concentrations, beta-cell electrical activity follows a characteristic pattern of slow oscillations in membrane potential on which bursts of action potentials are superimposed. The electrophysiological background of the bursting pattern remains unestablished. Activation of Ca(2+)-activated large-conductance K+ channels (KCa channel) has been implicated in this process but seems unlikely in view of recent evidence demonstrating that the beta-cell electrical activity is unaffected by the specific KCa channel blocker charybdotoxin. Another hypothesis postulates that the bursting arises as a consequence of two components of Ca(2+)-current inactivation. Here we show that activation of a novel Ca(2+)-dependent K+ current in glucose-stimulated beta-cells produces a transient membrane repolarization. This interrupts action potential firing so that action potentials appear in bursts. Spontaneous activity of this current was seen only rarely but could be induced by addition of compounds functionally related to hormones and neurotransmitters present in the intact pancreatic islet. K+ currents of the same type could be evoked by intracellular application of GTP, the effect of which was mediated by mobilization of Ca2+ from inositol 1,4,5-trisphosphate (InsP3)-sensitive intracellular Ca2+ stores. These observations suggest that oscillatory glucose-stimulated electrical activity, which is correlated with pulsatile release of insulin, results from the interaction between the beta-cell and intraislet hormones and neurotransmitters. Our data also provide evidence for a close interplay between ion channels in the plasma membrane and InsP3-induced mobilization of intracellular Ca2+ in an excitable cell.     

Detection of K+ and Cl-channels from calf cardiac sarcolemma in planar lipid bilayer membranes

The ionic currents underlying the cardiac action potential are believed to be much more complex than those in nerve. During the cardiac action potential, various membrane channels control the flow of K+, Na+, Ca2+ and Cl- across the sarcolemma of cardiac muscle cells. Thus, it has become increasingly clear that a detailed knowledge of the mechanisms that activate (or inactivate) heart channels is required to understand cardiac excitability. We report here the use of planar lipid bilayer techniques to detect and characterize K+ and Cl- channels in purified heart sarcolemma membrane vesicles. We have identified four different types of channel on the basis of their selectivity, conductance and gating kinetics. We present in some detail the properties of a K+ channel and a Cl- channel. We have tentatively identified the K+ channel with the ix type of current found in Purkinje, myocardial ventricular and atrial fibres. The chloride channel might be related to the transient chloride current found in Purkinje fibres.     

Cyclic GMP-sensitive conductance in outer segment membrane of catfish cones

A cyclic GMP-sensitive conductance has recently been observed with patch-clamp recording in excised inside-out patches of plasma membrane from frog and toad rod outer segments. This conductance has properties suggesting that it is probably the light-sensitive conductance involved in visual transduction. We now report a similar conductance in the outer segment membrane of catfish cones. Cyclic GMP showed positive cooperativity in opening this conductance, with a Hill coefficient of 1.6-3.0 and a half-saturating cGMP concentration of 35-70 microM. Cyclic AMP at 1 mM, or changing Ca concentration (in the presence of Mg), had little effect on the conductance. In physiological solutions the cGMP-induced current had a reversal potential near +10 mV; the current amplitude increased roughly exponentially with membrane potential in both depolarizing and hyperpolarizing directions. Our results suggest that cGMP is also the internal transmitter for phototransduction in cones.     

G-protein beta gamma-subunits activate the cardiac muscarinic K+-channel via phospholipase A2

Muscarinic receptors of cardiac pacemaker and atrial cells are linked to a potassium channel (IK.ACh) by a pertussis toxin-sensitive GTP-binding protein. The dissociation of G-proteins leads to the generation of two potential transducing elements, alpha-GTP and beta gamma. IK.ACh is activated by G-protein alpha- and beta gamma-subunits applied to the intracellular surface of inside-out patches of membrane. beta gamma has been shown to activate the membrane-bound enzyme phospholipase A2 in retinal rods. Arachidonic acid, which is produced from the action of phospholipase A2 on phospholipids, is metabolized to compounds which may act as second messengers regulating ion channels in Aplysia. Muscarinic receptor activation leads to the generation of arachidonic acid in some cell lines. We therefore tested the hypothesis that beta gamma activates IK.ACh by stimulation of phospholipase A2. When patches were first incubated with antibody that blocks phospholipase A2 activity, or with the lipoxygenase inhibitor, nordihydroguaiaretic acid, beta gamma failed to activate IK.ACh. Arachidonic acid and several of its metabolites derived from the 5-lipoxygenase pathway, activated the channel. Blockade of the cyclooxygenase pathway did not inhibit arachidonic acid-induced channel activation. We conclude that the beta gamma-subunit of G-proteins activates IK.ACh by stimulating the production of lipoxygenase-derived second messengers.     

Potassium channels in the nodal membrane of rat myelinated fibres

Following some preliminary reports, mammalian fibres from rabbit and rat have recently been successfully studied in detail by means of the voltage clamp. The early transient or sodium conductance system was found to be similar to that in frog and squid axons. However, the delayed conductance or potassium currents were found to be negligible. Only after chemical and osmotic manipulations, which were said to expose channels buried under the myelin, did Chiu and Ritchie find delayed currents in rabbit fibres. If confirmed, this would mean that the membrane conductance system of mammalian fibres is so different from that of invertebrate and amphibian axon models as to make the data base gathered from amphibian myelinated fibres (frog and toad) and invertebrate giant axons (squid and myxicola) irrelevant to human nd other mammalian fibres. However, we show here that it is possible to find in the normal nodal membrane of rat myelinated fibres potassium currents that flow through channels which are similar in many respects to those found in the frog node of squid axons.     

Arachidonic acid metabolites as intracellular modulators of the G protein-gated cardiac K+ channel

Arachidonic acid is released from cell membranes in response to receptor-dependent as well as receptor-independent stimulation in various cells, including cardiac myocytes. Arachidonic acid is converted to prostaglandins by cyclooxygenase and to leukotrienes by 5-lipoxygenase, metabolites which are very biologically active and modulate cellular functions such as platelet aggregation, smooth muscle contraction and neural excitation. The molecular mechanisms underlying their modulations are, however, still badly understood. Here, we report that the 5-lipoxygenase metabolites of arachidonic acid activate the pertussis toxin-sensitive G protein-gated muscarinic K+ channel (IK.ACh): arachidonic acid activation of IK.ACh was prevented by the lipoxygenase inhibitors, nordihydroguaiaretic acid and AA-861; leukotriene A4 and C4 activated IK.ACh. The activation occurred in pertussis toxin-treated atrial cells and ceased when inside-out patches were formed but the patches were still susceptible to stimulation by GTP and to inhibition by GDP-beta-S. These results indicate that arachidonic acid metabolites may stimulate the G-protein in a receptor-independent way.     

Chloride conductance regulated by cyclic AMP-dependent protein kinase in cardiac myocytes

In heart cells, cyclic AMP-dependent protein kinase (PKA) regulates calcium- and potassium-ion current by phosphorylating the ion channels or closely associated regulatory proteins. We report here that isoprenaline induced large chloride-ion currents in voltage-clamped, internally-dialysed myocytes from guinea-pig ventricles. The Cl- current could be activated by intracellular dialysis with cAMP or the catalytic subunit of PKA, indicating regulation by phosphorylation. In approximately symmetrical solutions of high Cl- concentration, the macroscopic cardiac Cl- current showed little rectification, unlike the single-channel current in PKA-regulated Cl- channels of airway epithelial cells. But, like epithelial Cl- -channel currents, the cardiac Cl- current was sensitive to the distilbene,4,4'-dinitrostilbene-2,2'-disulphonic acid (DNDS). In the absence of kinase activation, cardiac sarcolemmal Cl- conductance was negligible. During beta-adrenergic stimulation of the heart, this novel Cl- conductance should accelerate action-potential repolarization and so protect impulse propagation in the face of the possibly arrhythmogenic increases in heart rate and in calcium entry into the cells.     

Removal of phospho-head groups of membrane lipids immobilizes voltage sensors of K+ channels

A fundamental question about the gating mechanism of voltage-activated K+ (Kv) channels is how five positively charged voltage-sensing residues in the fourth transmembrane segment are energetically stabilized, because they operate in a low-dielectric cell membrane. The simplest solution would be to pair them with negative charges. However, too few negatively charged channel residues are positioned for such a role. Recent studies suggest that some of the channel's positively charged residues are exposed to cell membrane phospholipids and interact with their head groups. A key question nevertheless remains: is the phospho-head of membrane lipids necessary for the proper function of the voltage sensor itself? Here we show that a given type of Kv channel may interact with several species of phospholipid and that enzymatic removal of their phospho-head creates an insuperable energy barrier for the positively charged voltage sensor to move through the initial gating step(s), thus immobilizing it, and also raises the energy barrier for the downstream step(s).     

Noradrenaline and beta-adrenoceptor agonists increase activity of voltage-dependent calcium channels in hippocampal neurons

The predominance of unconventional transmitter release sites at noradrenaline-containing synapses and the diffuse projections of noradrenaline-containing fibres originating in locus coeruleus have led to speculation that noradrenaline may act as a neuromodulator in the central nervous system. Evidence suggests that it has a modulatory function in the plasticity of the developing nervous system, in controlling behavioural states of an organism, and in learning and memory. Recently, Hopkins and Johnston demonstrated that noradrenaline enhances the magnitude, duration and probability of induction of long-term potentiation (LTP) at mossy fibre synapses in the hippocampal formation, and LTP is widely believed to be a cellular substrate for aspects of memory. To investigate the membrane effects of noradrenaline on central neurons, we used a newly developed preparation in which patch-clamp techniques can be applied to exposed adult cortical neurons. We report here that noradrenaline produces an enhancement in the activity of voltage-dependent calcium channels in granule cells of the hippocampal dentate gyrus. This action appears to be mediated by beta-adrenoceptors and can be mimicked by cyclic AMP.     

Ankyrin binding to (Na+ + K+)ATPase and implications for the organization of membrane domains in polarized cells

The interaction between membrane proteins and cytoplasmic structural proteins is thought to be one mechanism for maintaining the spatial order of proteins within functional domains on the plasma membrane. Such interactions have been characterized extensively in the human erythrocyte, where a dense, cytoplasmic matrix of proteins comprised mainly of spectrin and actin, is attached through a linker protein, ankyrin, to the anion transporter (Band 3). In several nonerythroid cell types, including neurons, exocrine cells and polarized epithelial cells homologues of ankyrin and spectrin (fodrin) are localized in specific membrane domains. Although these results suggest a functional linkage between ankyrin and fodrin and integral membrane proteins in the maintenance of membrane domains in nonerythroid cells, there has been little direct evidence of specific molecular interactions. Using a direct biological and chemical approach, we show here that ankyrin binds to the ubiquitous (Na+ + K+)ATPase, which has an asymmetrical distribution in polarized cells.