Protein kinase injection reduces voltage-dependent potassium currents

Intracellular iontophoretic injection of the catalytic subunit of cyclic adenosine monophosphate-dependent protein kinase increased input resistance and decreased a delayed voltage-dependent K+ current of the type B photoreceptor in the nudibranch Hermissenda crassicornis to a greater extent than an early, rapidly inactivating K+ current (IA). This injection also enhanced the long-lasting depolarization of type B cells after a light step. These findings suggest the involvement of cyclic adenosine monophosphate-dependent phosphorylation in the differential regulation of photoreceptor K+ currents particularly during illumination. On the other hand, conditioning-induced changes in IA may also be regulated by a different type of phosphorylation (for example, Ca2+-dependent).     

Mechanism of voltage gating in potassium channels

The mechanism of ion channel voltage gating-how channels open and close in response to voltage changes-has been debated since Hodgkin and Huxley's seminal discovery that the crux of nerve conduction is ion flow across cellular membranes. Using all-atom molecular dynamics simulations, we show how a voltage-gated potassium channel (KV) switches between activated and deactivated states. On deactivation, pore hydrophobic collapse rapidly halts ion flow. Subsequent voltage-sensing domain (VSD) relaxation, including inward, 15-angstrom S4-helix motion, completes the transition. On activation, outward S4 motion tightens the VSD-pore linker, perturbing linker-S6-helix packing. Fluctuations allow water, then potassium ions, to reenter the pore; linker-S6 repacking stabilizes the open pore. We propose a mechanistic model for the sodium/potassium/calcium voltage-gated ion channel superfamily that reconciles apparently conflicting experimental data.     

IgG from patients with Lambert-Eaton syndrome blocks voltage-dependent calcium channels

Lambert-Eaton syndrome, an autoimmune disorder frequently associated with small-cell carcinoma of the lung, is characterized by impaired evoked release of acetylcholine from the motor nerve terminal. Immunoglobulin G (IgG) antibodies from patients with the syndrome, applied to bovine adrenal chromaffin cells, reduced the voltage-dependent calcium channel currents by about 40 percent. When calcium was administered directly into the cytoplasm, however, the IgG-treated cells exhibited normal exocytotic secretion, as assayed by membrane capacitance measurement. Measurement with the fluorescent calcium indicator fura-2 indicated that the IgG treatment reduced potassium-stimulated increase in free intracellular calcium concentration. The pathogenic IgG modified neither kinetics of calcium channel activation nor elementary channel activity, suggesting that a reduction in the number of functional calcium channels underlies the IgG-induced effect. Therefore, Lambert-Eaton syndrome IgG reacts with voltage-dependent calcium channels and blocks their function, a phenomenon that can account for the presynaptic impairment characteristic of this disorder.     

1990: annus mirabilis of potassium channels

Voltage-gated potassium channels make up a large molecular family of integral membrane proteins that are fundamentally involved in the generation of bioelectric signals such as nerve impulses. These proteins span the cell membrane, forming potassium-selective pores that are rapidly switched open or closed by changes in membrane voltage. After the cloning of the first potassium channel over 3 years ago, recombinant DNA manipulation of potassium channel genes is now leading to a molecular understanding of potassium channel behavior. During the past year, functional domains responsible for channel gating and potassium selectivity have been identified, and detailed structural pictures underlying these functions are beginning to emerge.     

Occult Drosophila calcium channels and twinning of calcium and voltage-activated potassium channels

In the membrane of the flight muscle cells of developing Drosophila a large calcium-sensitive potassium current, IKc, was found. It was present before the development of voltage-activated potassium channels and seems to be the first potassium current to develop in the membrane. Also present in these early cells were large numbers of occult (hidden) calcium channels, which remained inactive until the end of pupal development. These inactive calcium channels could be made to function by injecting adenosine triphosphate or ethyleneglycol tetraacetic acid into the early cells. IKc has kinetic properties resembling the later developing voltage-sensitive current IKv, and is distinct from the fast, transient calcium-dependent outward current IAc, which appears much later in development. IAc closely resembles the voltage-sensitive current IAv, also present in these cells. Thus, both of the voltage-sensitive potassium channel types, IAv and IKv, have similar calcium-sensitive counterparts, IAc and IKc, that are present in the same cells.     

Structure of the cytoplasmic beta subunit-T1 assembly of voltage-dependent K+ channels

The structure of the cytoplasmic assembly of voltage-dependent K+ channels was solved by x-ray crystallography at 2.1 angstrom resolution. The assembly includes the cytoplasmic (T1) domain of the integral membrane alpha subunit together with the oxidoreductase beta subunit in a fourfold symmetric T1(4)beta4 complex. An electrophysiological assay showed that this complex is oriented with four T1 domains facing the transmembrane pore and four beta subunits facing the cytoplasm. The transmembrane pore communicates with the cytoplasm through lateral, negatively charged openings above the T1(4)beta4 complex. The inactivation peptides of voltage-dependent K(+) channels reach their site of action by entering these openings.     

Protective role of ATP-sensitive potassium channels in hypoxia-induced generalized seizure

Adenosine triphosphate (ATP)-sensitive potassium (K(ATP)) channels are activated by various metabolic stresses, including hypoxia. The substantia nigra pars reticulata (SNr), the area with the highest expression of K(ATP) channels in the brain, plays a pivotal role in the control of seizures. Mutant mice lacking the Kir6.2 subunit of K(ATP) channels [knockout (KO) mice] were susceptible to generalized seizures after brief hypoxia. In normal mice, SNr neuron activity was inactivated during hypoxia by the opening of the postsynaptic K(ATP) channels, whereas in KO mice, the activity of these neurons was enhanced. K(ATP) channels exert a depressant effect on SNr neuronal activity during hypoxia and may be involved in the nigral protection mechanism against generalized seizures.     

Molecular basis of gating charge immobilization in Shaker potassium channels

Voltage-dependent ion channels respond to changes in the membrane potential by means of charged voltage sensors intrinsic to the channel protein. Changes in transmembrane potential cause movement of these charged residues, which results in conformational changes in the channel. Movements of the charged sensors can be detected as currents known as gating currents. Measurement of the gating currents of the Drosophila Shaker potassium channel indicates that the charge on the voltage sensor of the channels is progressively immobilized by prolonged depolarizations. The charge is not immobilized in a mutant of the channel that lacks inactivation. These results show that the region of the molecule responsible for inactivation interacts, directly or indirectly, with the voltage sensor to prevent the return of the charge to its original position. The gating transitions between closed states of the channel appear not to be independent, suggesting that the channel subunits interact during activation.     

Cyclic AMP-modulated potassium channels in murine B cells and their precursors

A voltage-dependent potassium current (the delayed rectifier) has been found in murine B cells and their precursors with the whole-cell patch-clamp technique. The type of channel involved in the generation of this current appears to be present throughout all stages of pre-B-cell differentiation, since it is detected in pre-B cell lines infected with Abelson murine leukemia virus; these cell lines represent various phases of B-cell development. Thus, the presence of this channel is not obviously correlated with B-cell differentiation. Although blocked by Co2+, the channel, or channels, does not appear to be activated by Ca2+ entry. It is, however, inactivated by high intracellular Ca2+ concentrations. In addition, elevation of intracellular adenosine 3', 5'-monophosphate induces at all potentials a rapid decrease in the peak potassium conductance and increased rates of activation and inactivation. Therefore, potassium channels can be physiologically modulated by second messengers in lymphocytes.     

Single-channel and genetic analyses reveal two distinct A-type potassium channels in Drosophila

Whole-cell and single-channel voltage-clamp techniques were used to identify and characterize the channels underlying the fast transient potassium current (A current) in cultured myotubes and neurons of Drosophila. The myotube (A1) and neuronal (A2) channels are distinct, differing in conductance, voltage dependence, and gating kinetics. The myotube currents have a faster and more voltage-dependent macroscopic inactivation rate, a larger steady-state component, and a less negative steady-state inactivation curve than the neuronal currents. The myotube channels have a conductance of 12 to 16 picosiemens, whereas the neuronal channels have a conductance of 5 to 8 picosiemens. In addition, the myotube channel is affected by Shaker mutations, whereas the neuronal channel is not. Together, these data suggest that the two channels are separate molecular structures, the expression of which is controlled, at least in part, by different genes.     

Hypoxic dilation of coronary arteries is mediated by ATP-sensitive potassium channels

The function of the heart depends critically on an adequate oxygen supply through the coronary arteries. Coronary arteries dilate when the intravascular oxygen tension decreases. Hypoxic vasodilation in isolated, perfused guinea pig hearts can be prevented by glibenclamide, a blocker of adenosine triphosphate (ATP)-sensitive potassium channels, and can be mimicked by cromakalim, which opens ATP-sensitive potassium channels. Opening of potassium channels in coronary smooth muscle cells and the subsequent drop in intracellular calcium is probably the major cause of hypoxic and ischemic vasodilation in the mammalian heart.     

Arachidonic acid and other fatty acids directly activate potassium channels in smooth muscle cells

Arachidonic acid, as well as fatty acids that are not substrates for cyclooxygenase and lipoxygenase enzymes, activated a specific type of potassium channel in freshly dissociated smooth muscle cells. Activation occurred in excised membrane patches in the absence of calcium and all nucleotides. Therefore signal transduction pathways that require such soluble factors, including the NADPH-dependent cytochrome P450 pathway, do not mediate the response. Thus, fatty acids directly activate potassium channels and so may constitute a class of signal molecules that regulate ion channels.     

Restoration of inactivation in mutants of Shaker potassium channels by a peptide derived from ShB

Site-directed mutagenesis experiments have suggested a model for the inactivation mechanism of Shaker potassium channels from Drosophila melanogaster. In this model, the first 20 amino acids form a cytoplasmic domain that interacts with the open channel to cause inactivation. The model was tested by the internal application of a synthetic peptide, with the sequence of the first 20 residues of the ShB alternatively spliced variant, to noninactivating mutant channels expressed in Xenopus oocytes. The peptide restored inactivation in a concentration-dependent manner. Like normal inactivation, peptide-induced inactivation was not noticeably voltage-dependent. Trypsin-treated peptide and peptides with sequences derived from the first 20 residues of noninactivating mutants did not restore inactivation. These results support the proposal that inactivation occurs by a cytoplasmic domain that occludes the ion-conducting pore of the channel.     

A conserved domain in axonal targeting of Kv1 (Shaker) voltage-gated potassium channels

Axonal voltage-gated potassium (Kv1) channels regulate action-potential invasion and hence transmitter release. Although evolutionarily conserved, what mediates their axonal targeting is not known. We found that Kv1 axonal targeting required its T1 tetramerization domain. When fused to unpolarized CD4 or dendritic transferrin receptor, T1 promoted their axonal surface expression. Moreover, T1 mutations eliminating Kvbeta association compromised axonal targeting, but not surface expression, of CD4-T1 fusion proteins. Thus, proper association of Kvbeta with the Kv1 T1 domain is essential for axonal targeting.     

A component of calcium-activated potassium channels encoded by the Drosophila slo locus.

Calcium-activated potassium channels mediate many biologically important functions in electrically excitable cells. Despite recent progress in the molecular analysis of voltage-activated K+ channels, Ca(2+)-activated K+ channels have not been similarly characterized. The Drosophila slowpoke (slo) locus, mutations of which specifically abolish a Ca(2+)-activated K+ current in muscles and neurons, provides an opportunity for molecular characterization of these channels. Genomic and complementary DNA clones from the slo locus were isolated and sequenced. The polypeptide predicted by slo is similar to voltage-activated K+ channel polypeptides in discrete domains known to be essential for function. Thus, these results indicate that slo encodes a structural component of Ca(2+)-activated K+ channels.     

Mutant potassium channels with altered binding of charybdotoxin, a pore-blocking peptide inhibitor

The inhibition by charybdotoxin of A-type potassium channels expressed in Xenopus oocytes was studied for several splicing variants of the Drosophila Shaker gene and for several site-directed mutants of this channel. Charybdotoxin blocking affinity is lowered by a factor of 3.5 upon replacing glutamate-422 with glutamine, and by a factor of about 12 upon substituting lysine in this position. Replacement of glutamate-422 by aspartate had no effect on toxin affinity. Thus, the glutamate residue at position 422 of this potassium channel is near or in the externally facing mouth of the potassium conduction pathway, and the positively charged toxin is electrostatically focused toward its blocking site by the negative potential set up by glutamate-422.     

Direct activation of mammalian atrial muscarinic potassium channels by GTP regulatory protein Gk

The mammalian heart rate is regulated by the vagus nerve, which acts via muscarinic acetylcholine receptors to cause hyperpolarization of atrial pacemaker cells. The hyperpolarization is produced by the opening of potassium channels and involves an intermediary guanosine triphosphate-binding regulatory (G) protein. Potassium channels in isolated, inside-out patches of membranes from atrial cells now are shown to be activated by a purified pertussis toxin-sensitive G protein of subunit composition alpha beta gamma, with an alpha subunit of 40,000 daltons. Thus, mammalian atrial muscarinic potassium channels are activated directly by a G protein, not indirectly through a cascade of intermediary events. The G protein regulating these channels is identified as a potent Gk; it is active at 0.2 to 1 pM. Thus, proteins other than enzymes can be under control of receptor coupling G proteins.     

Newly identified brain potassium channels gated by the guanine nucleotide binding protein Go

Potassium channels in neurons are linked by guanine nucleotide binding (G) proteins to numerous neurotransmitter receptors. The ability of Go, the predominant G protein in the brain, to stimulate potassium channels was tested in cell-free membrane patches of hippocampal pyramidal neurons. Four distinct types of potassium channels, which were otherwise quiescent, were activated by both isolated brain G0 and recombinant Go alpha. Hence brain Go can couple diverse brain potassium channels to neurotransmitter receptors.     

The alpha subunit of the GTP binding protein Gk opens atrial potassium channels

Guanine nucleotide binding (G) proteins (subunit composition alpha beta gamma) dissociate on activation with guanosine triphosphate (GTP) analogs and magnesium to give alpha-guanine nucleotide complexes and free beta gamma subunits. Whether the opening of potassium channels by the recently described Gk in isolated membrane patches from mammalian atrial myocytes was mediated by the alpha k subunit or beta gamma dimer was tested. The alpha k subunit was found to be active, while the beta gamma dimer was inactive in stimulating potassium channel activity. Thus, Gk resembles Gs, the stimulatory regulatory component of adenylyl cyclase, and transducin, the regulatory component of the visual system, in that it regulates its effector function--the activity of the ligand-gated potassium channel--through its guanine nucleotide binding subunit.