Flexibility of the Kir6.2 inward rectifier K+ channel pore

Interactions of sulfhydryl reagents with introduced cysteines in the pore-forming (Kir6.2) subunits of the KATP channel were examined. 2-Aminoethyl methanethiosulfonate (MTSEA+) failed to modify Cd2+-insensitive control-Kir6.2 channels, but rapidly and irreversibly modified Kir6.2[L164C] (L164C) channels. Although a single Cd2+ ion is coordinated by L164C, four MTSEA+ “hits” can occur, each sequentially reducing the single-channel current. A dimeric fusion of control-Kir6.2 and L164C subunits generates Cd2+-insensitive channels, confirming that at least three cysteines are required for coordination, but MTSEA+ modification of the dimer occurs in two hits. L164C channels were not modified by bromotrimethyl ammoniumbimane (qBBr+), even though qBBr+ caused voltage-dependent block (as opposed to modification) that was comparable to that of MTSEA+ or 3-(triethylammonium)propyl methanethiosulfonate (MTSPTrEA+), implying that qBBr+ can also enter the inner cavity but does not modify L164C residues. The Kir channel pore structure was modeled by homology with the KcsA crystal structure. A stable conformation optimally places the four L164C side chains for coordination of a single Cd2+ ion. Modification of these cysteines by up to four MTSEA+ (or three MTSPTrEA+, or two qBBr+) does not require widening of the cavity to accommodate the derivatives within it. However, like the KcsA crystal structure, the energy-minimized model shows a narrowing at the inner entrance, and in the Kir6.2 model this narrowing excludes all ions. To allow entry of ions as large as MTSPTrEA+ or qBBr+, the entrance must widen to >8 Å, but this widening is readily accomplished by minimal M2 helix motion and side-chain rearrangement.

DEG/ENaC ion channels involved in sensory transduction are modulated by cold temperature

Several DEG/ENaC cation channel subunits are expressed in the tongue and in cutaneous sensory neurons, where they are postulated to function as receptors for salt and sour taste and for touch. Because these tissues are exposed to large temperature variations, we examined how temperature affects DEG/ENaC channel function. We found that cold temperature markedly increased the constitutively active Na+ currents generated by epithelial Na+ channels (ENaC). Half-maximal stimulation occurred at 25°C. Cold temperature did not induce current from other DEG/ENaC family members (BNC1, ASIC, and DRASIC). However, when these channels were activated by acid, cold temperature potentiated the currents by slowing the rate of desensitization. Potentiation was abolished by a “Deg” mutation that alters channel gating. Temperature changes in the physiologic range had prominent effects on current in cells heterologously expressing acid-gated DEG/ENaC channels, as well as in dorsal root ganglion sensory neurons. The finding that cold temperature modulates DEG/ENaC channel function may provide a molecular explanation for the widely recognized ability of temperature to modify taste sensation and mechanosensation.

Putting ion channels to work: Mechanoelectrical transduction, adaptation, and amplification by hair cells

As in other excitable cells, the ion channels of sensory receptors produce electrical signals that constitute the cellular response to stimulation. In photoreceptors, olfactory neurons, and some gustatory receptors, these channels essentially report the results of antecedent events in a cascade of chemical reactions. The mechanoelectrical transduction channels of hair cells, by contrast, are coupled directly to the stimulus. As a consequence, the mechanical properties of these channels shape our hearing process from the outset of transduction. Channel gating introduces nonlinearities prominent enough to be measured and even heard. Channels provide a feedback signal that controls the transducer's adaptation to large stimuli. Finally, transduction channels participate in an amplificatory process that sensitizes and sharpens hearing.

Interaction of Cryptochrome 1, Phytochrome, and Ion Fluxes in Blue-Light-Induced Shrinking of Arabidopsis Hypocotyl Protoplasts1

Protoplasts isolated from red-light-adapted Arabidopsis hypocotyls and incubated under red light exhibited rapid and transient shrinking within a period of 20 min in response to a blue-light pulse and following the onset of continuous blue light. Long-persisting shrinkage was also observed during continuous stimulation. Protoplasts from a hy4 mutant and the phytochrome-deficient phyA/phyB double mutant of Arabidopsis showed little response, whereas those from phyA and phyB mutants showed a partial response. It is concluded that the shrinking response itself is mediated by the HY4 gene product, cryptochrome 1, whereas the blue-light responsiveness is strictly controlled by phytochromes A and B, with a greater contribution by phytochrome B. It is shown further that the far-red-absorbing form of phytochrome (Pfr) was not required during or after, but was required before blue-light perception. Furthermore, a component that directly determines the blue-light responsiveness was generated by Pfr after a lag of 15 min over a 15-min period and decayed with similar kinetics after removal of Pfr by far-red light. The anion-channel blocker 5-nitro-2-(3-phenylpropylamino)-benzoic acid prevented the shrinking response. This result, together with those in the literature and the kinetic features of shrinking, suggests that anion channels are activated first, and outward-rectifying cation channels are subsequently activated, resulting in continued net effluxes of Cl− and K+. The postshrinking volume recovery is achieved by K+ and Cl− influxes, with contribution by the proton motive force. External Ca2+ has no role in shrinking and the recovery. The gradual swelling of protoplasts that prevails under background red light is shown to be a phytochrome-mediated response in which phytochrome A contributes more than phytochrome B.

Mutations in copper-zinc superoxide dismutase that cause amyotrophic lateral sclerosis alter the zinc binding site and the redox behavior of the protein.

A series of mutant human and yeast copper-zinc superoxide dismutases has been prepared, with mutations corresponding to those found in familial amyotrophic lateral sclerosis (ALS; also known as Lou Gehrig's disease). These proteins have been characterized with respect to their metal-binding characteristics and their redox reactivities. Replacement of Zn2+ ion in the zinc sites of several of these proteins with either Cu2+ or Co2+ gave metal-substituted derivatives with spectroscopic properties different from those of the analogous derivative of the wild-type proteins, indicating that the geometries of binding of these metal ions to the zinc site were affected by the mutations. Several of the ALS-associated mutant copper-zinc superoxide dismutases were also found to be reduced by ascorbate at significantly greater rate than the wild-type proteins. We conclude that similar alterations in the properties of the zinc binding site can be caused by mutations scattered throughout the protein structure. This finding may help to explain what is perhaps the most perplexing question in copper-zinc superoxide dismutase-associated familial ALS-i.e., how such a diverse set of mutations can result in the same gain of function that causes the disease.

Immunolocalization of ion transport proteins in human autosomal dominant polycystic kidney epithelial cells.

The kidneys of patients with autosomal dominant polycystic kidney disease become massively enlarged due to the progressive expansion of myriad fluid-filled cysts. The epithelial cells that line the cyst walls are responsible for secreting the cyst fluid, but the mechanism through which this secretion occurs is not well established. Recent studies suggest that renal cyst epithelial cells actively secrete Cl across their apical membranes, which in turn drives the transepithelial movement of Na and water. The characteristics of this secretory flux suggest that it is dependent upon the participation of an apical cystic fibrosis transmembrane conductance regulator (CFTR)-like Cl channel and basolateral Na,K-ATPase. To test this hypothesis, we have immunolocalized the CFTR and Na,K-ATPase proteins in intact cysts and in cyst epithelial cells cultured in vitro on permeable filter supports. In both settings, cyst epithelial cells were found to possess Na,K-ATPase exclusively at their basolateral surfaces; apical labeling was not detected. The CFTR protein was present at the apical surfaces of cyst epithelial cells that had been stimulated to secrete through incubation in forskolin. CFTR was detected in intracellular structures in cultured cyst epithelial cells that had not received the forskolin treatment. These results demonstrate that the renal epithelial cells that line cysts in autosomal dominant polycystic kidney disease express transport systems with the appropriate polarity to mediate active Cl and fluid secretion.

Norepinephrine transporters have channel modes of conduction.

Neurotransmitter transporters couple to existing ion gradients to achieve reuptake of transmitter into presynaptic terminals. For coupled cotransport, substrates and ions cross the membrane in fixed stoichiometry. This is in contrast to ion channels, which carry an arbitrary number of ions depending on the channel open time. Members of the gamma-aminobutyric acid transporter gene family presumably function with fixed stoichiometry in which a set number of ions cotransport with one transmitter molecule. Here we report channel-like events from a presumably fixed stoichiometry [norepinephrine (NE)+, Na+, and Cl-], human NE (hNET) in the gamma-aminobutyric acid transporter gene family. These events are stimulated by NE and by guanethidine, an hNET substrate, and they are blocked by cocaine and the antidepressant desipramine. Voltage-clamp data combined with NE uptake data from these same cells indicate that hNETs have two functional modes of conduction: a classical transporter mode (T-mode) and a novel channel mode (C-mode). Both T-mode and C-mode are gated by the same substrates and antagonized by the same blockers. T-mode is putatively electrogenic because the transmitter and cotransported ions sum to one net charge. However, C-mode carries virtually all of the transmitter-induced current, even though it occurs with low probability. This is because each C-mode opening transports hundreds of charges per event. The existence of a channel mode of conduction in a previously established fixed-stoichiometry transporter suggests the appearance of an aqueous pore through the transporter protein during the transport cycle and may have significance for transporter regulation.

Primary structure and functional expression of a cGMP-gated potassium channel.

Cyclic nucleotides modulate potassium (K) channel activity in many cells and are thought to act indirectly by inducing channel protein phosphorylation. Herein we report the isolation from rabbit of a gene encoding a K channel (Kcn1) that is specifically activated by cGMP and not by cAMP. Analysis of the deduced amino acid sequence (725 amino acids) indicates that, in addition to a core region that is highly homologous to Shaker K channels, Kcn1 also contains a cysteine-rich region similar to that of ligand-gated ion channels and a cyclic nucleotide-binding region. Northern blot analysis detects gene expression in kidney, aorta, and brain. Kcn1 represents a class of K channels that may be specifically regulated by cGMP and could play an important role in mediating the effects of substances, such as nitric oxide, that increase intracellular cGMP.

Protein kinase A-independent modulation of ion channels in the brain by cyclic AMP.

Ion channels underlying the electrical activity of neurons can be regulated by neurotransmitters via two basic mechanisms: ligand binding and covalent modification. Whereas neurotransmitters often act by binding directly to ion channels, the intracellular messenger cyclic AMP is thought usually to act indirectly, by activating protein kinase A, which in turn can phosphorylate channel proteins. Here we show that cyclic AMP, and transmitters acting via cyclic AMP, can act in a protein kinase A-independent manner in the brain. In hippocampal pyramidal cells, cyclic AMP and norepinephrine were found to cause a depolarization by enhancing the hyperpolarization-activated mixed cation current, IQ (also called Ih). This effect persisted even after protein kinase A activity was blocked, thus strongly suggesting a kinase-independent action of cyclic AMP. The modulation of this current by ascending monoaminergic fibers from the brainstem is likely to be a widespread mechanism, participating in the state control of the brain during arousal and attention.

TRPC1, a human homolog of a Drosophila store-operated channel.

In many vertebrate and invertebrate cells, inositol 1,4,5-trisphospate production induces a biphasic Ca2+ signal. Mobilization of Ca2+ from internal stores drives the initial burst. The second phase, referred to as store-operated Ca2+ entry (formerly capacitative Ca2+ entry), occurs when depletion of intracellular Ca2+ pools activates a non-voltage-sensitive plasma membrane Ca2+ conductance. Despite the prevalence of store-operated Ca2+ entry, no vertebrate channel responsible for store-operated Ca2+ entry has been reported. trp (transient receptor potential), a Drosophila gene required in phototransduction, encodes the only known candidate for such a channel throughout phylogeny. In this report, we describe the molecular characterization of a human homolog of trp, TRPC1. TRPC1 (transient receptor potential channel-related protein 1) was 40% identical to Drosophila TRP over most of the protein and lacked the charged residues in the S4 transmembrane region proposed to be required for the voltage sensor in many voltage-gated ion channels. TRPC1 was expressed at the highest levels in the fetal brain and in the adult heart, brain, testis, and ovaries. Evidence is also presented that TRPC1 represents the archetype of a family of related human proteins.

Probing the transmembrane topology of cyclic nucleotide-gated ion channels with a gene fusion approach.

Cyclic nucleotide-gated (CNG) cation channels contain two short sequence motifs--a residual voltage-sensor (S4) and a pore-forming (P) segment--that are reminiscent of similar segments in voltage-activated Shaker-type K+ channels. It has been tacitly assumed that CNG channels and this K+ channel subfamily share a common overall topology, characterized by a hydrophobic domain comprising six membrane-spanning segments. We have systematically investigated the topology of CNG channels from bovine rod photoreceptor and Drosophila melanogaster by a gene fusion approach using the bacterial reporter enzymes alkaline phosphatase and beta-galactosidase, which are active only in the periplasm and only in the cytoplasm, respectively. Enzymatic activity was determined after expression of fusion constructs in Escherichia coli. CNG channels were found to have six membrane-spanning segments, suggesting that CNG and Shaker-type K+ channels, albeit distant relatives within a gene superfamily of ion channels, share a common topology.

A mutation in the epithelial sodium channel causing Liddle disease increases channel activity in the Xenopus laevis oocyte expression system.

We have studied the functional consequences of a mutation in the epithelial Na+ channel that causes a heritable form of salt-sensitive hypertension, Liddle disease. This mutation, identified in the original kindred described by Liddle, introduces a premature stop codon in the channel beta subunit, resulting in a deletion of almost all of the C terminus of the encoded protein. Coexpression of the mutant beta subunit with wild-type alpha and gamma subunits in Xenopus laevis oocytes resulted in an approximately 3-fold increase in the macroscopic amiloride-sensitive Na+ current (INa) compared with the wild-type channel. This change in INa reflected an increase in the overall channel activity characterized by a higher number of active channels in membrane patches. The truncation mutation in the beta subunit of epithelial Na+ channel did not alter the biophysical and pharmacological properties of the channel--including unitary conductance, ion selectivity, or sensitivity to amiloride block. These results provide direct physiological evidence that Liddle disease is related to constitutive channel hyperactivity in the cell membrane. Deletions of the C-terminal end of the beta and gamma subunits of rat epithelial Na+ channel were functionally equivalent in increasing INa, suggesting that the cytoplasmic domain of the gamma subunit might be another molecular target for mutations responsible for salt-sensitive forms of hypertension.

Citrate modulates the regulation by Zn2+ of N-methyl-D-aspartate receptor-mediated channel current and neurotransmitter release.

The effect of the two metal-ion chelators EDTA and citrate on the action of N-methyl-D-aspartate (NMDA) receptors was investigated by use of cultured mouse cerebellar granule neurons and Xenopus oocytes, respectively, to monitor either NMDA-evoked transmitter release or membrane currents. Transmitter release from the glutamatergic neurons was determined by superfusion of the cells after preloading with the glutamate analogue D-[3H]aspartate. The oocytes were injected with mRNA isolated from mouse cerebellum and, after incubation to allow translation to occur, currents mediated by NMDA were recorded electrophysiologically by voltage clamp at a holding potential of -80 mV. It was found that citrate as well as EDTA could attenuate the inhibitory action of Zn2+ on NMDA receptor-mediated transmitter release from the neurons and membrane currents in the oocytes. These effects were specifically related to the NMDA receptor, since the NMDA receptor antagonist MK-801 abolished the action and no effects of Zn2+ and its chelators were observed when kainate was used to selectively activate non-NMDA receptors. Since it was additionally demonstrated that citrate (and EDTA) preferentially chelated Zn2+ rather than Ca2+, the present findings strongly suggest that endogenous citrate released specifically from astrocytes into the extracellular space in the brain may function as a modulator of NMDA receptor activity. This is yet another example of astrocytic influence on neuronal activity.

Development of a K(+)-channel probe and its use for identification of an intracellular plant membrane K+ channel.

Polyclonal antibodies were generated against a 9-amino acid, synthetic peptide corresponding to the selectivity filter in the pore region of K(+)-channel proteins. The sequence of amino acids in the ion-conducting pore region of K+ channels is the only highly conserved region of members of this protein family. The objectives of the present work were (i) to determine whether the anti-channel pore peptide antibody was immunoreactive with known K(+)-channel proteins and (ii) to demonstrate the usefulness of the antibody by employing it to identify a newly discovered K(+)-channel protein. Anti-channel pore peptide was immunoreactive with various K(+)-channel subtypes native to a number of different species. Immunoblot analysis demonstrated affinity of the antibody for the drk1, maxi-K, and KAT1 K(+)-channel proteins. Studies also suggested that the anti-channel pore peptide antibody did not immunoreact with membrane proteins other than K+ channels. The anti-channel pore peptide antibody was used to establish the identity of a 62-kDa chloroplast inner envelope polypeptide as a putative component of a K(+)-channel protein. It was concluded that an antibody generated against the conserved pore region/selectivity filter of K+ channels has broad but selective affinity for this class of proteins. This K(+)-channel probe may be a useful tool for identification of K(+)-channel proteins in native membranes.

Electrical investigation of p-type 4H-SiC heavily doped by ion implantation

In the last decades, an increasing interest in the research field of wide bandgap semiconductors was observed, mostly due to the progressive approaching of silicon-based devices to their theoretical limits. 4H-SiC is an example among these, and is a mature compound for applications. The main advantages offered 4H-SiC in comparison with silicon are an higher breakdown field, an higher thermal conductivity, a higher operating temperature, very high hardness and melting point, biocompatibility, but also low switching losses in high frequencies applications and lower on-resistances in unipolar devices. Then, 4H-SiC power devices offer great performance improvement; moreover, they can work in hostile environments where silicon power devices cannot function. Ion implantation technology is a key process in the fabrication of almost all kinds of SiC devices, owing to the advantage of a spatially selective doping. This work is dedicated to the electrical investigation of several differently-processed 4H-SiC ion- implanted samples, mainly through Hall effect and space charge spectroscopy experiments. It was also developed the automatic control (Labview) of several experiments. In the work, the effectiveness of high temperature post-implant thermal treatments (up to 2000°C) were studied and compared considering: (i) different methods, (ii) different temperatures and (iii) different duration of the annealing process. Preliminary p + /n and Schottky junctions were also investigated as simple test devices. 1) Heavy doping by ion implantation of single off-axis 4H-SiC layers The electrical investigation is one of the most important characterization of ion-implanted samples, which must be submitted to mandatory post-implant thermal treatment in order to both (i) recover the lattice after ion bombardment, and (ii) address the implanted impurities into lattice sites so that they can effectively act as dopants. Electrical investigation can give fundamental information on the efficiency of the electrical impurity activation. To understand the results of the research it should be noted that: (a) To realize good ohmic contacts it is necessary to obtain spatially defined highly doped regions, which must have conductivity as low as possible. (b) It has been shown that the electrical activation efficiency and the electrical conductivity increase with the annealing temperature increasing. (c) To maximize the layer conductivity, temperatures around 1700°C are generally used and implantation density high till to 10 21 cm -3 . In this work, an original approach, different from (c), is explored by the using very high annealing temperature, around 2000°C, on samples of Al + -implant concentration of the order of 10 20 cm -3 . Several Al + -implanted 4H-SiC samples, resulting of p-type conductivity, were investigated, with a nominal density varying in the range of about 1-5∙10 20 cm -3 and subjected to two different high temperature thermal treatments. One annealing method uses a radiofrequency heated furnace till to 1950°C (Conventional Annealing, CA), the other exploits a microwave field, providing a fast heating rate up to 2000°C (Micro-Wave Annealing, MWA). In this contest, mainly ion implanted p-type samples were investigated, both off-axis and on-axis <0001> semi-insulating 4H-SiC. Concerning p-type off-axis samples, a high electrical activation of implanted Al (50-70%) and a compensation ratio below 10% were estimated. In the work, the main sample processing parameters have been varied, as the implant temperature, CA annealing duration, and heating/cooling rates, and the best values assessed. MWA method leads to higher hole density and lower mobility than CA in equivalent ion implanted layers, resulting in lower resistivity, probably related to the 50°C higher annealing temperature. An optimal duration of the CA treatment was estimated in about 12-13 minutes. A RT resistivity on the lowest reported in literature for this kind of samples, has been obtained. 2) Low resistivity data: variable range hopping Notwithstanding the heavy p-type doping levels, the carrier density remained less than the critical one required for a semiconductor to metal transition. However, the high carrier densities obtained was enough to trigger a low temperature impurity band (IB) conduction. In the heaviest doped samples, such a conduction mechanism persists till to RT, without significantly prejudice the mobility values. This feature can have an interesting technological fall, because it guarantee a nearly temperature- independent carrier density, it being not affected by freeze-out effects. The usual transport mechanism occurring in the IB conduction is the nearest neighbor hopping: such a regime is effectively consistent with the resistivity temperature behavior of the lowest doped samples. In the heavier doped samples, however, a trend of the resistivity data compatible with a variable range hopping (VRH) conduction has been pointed out, here highlighted for the first time in p-type 4H-SiC. Even more: in the heaviest doped samples, and in particular, in those annealed by MWA, the temperature dependence of the resistivity data is consistent with a reduced dimensionality (2D) of the VRH conduction. In these samples, TEM investigation pointed out faulted dislocation loops in the basal plane, whose average spacing along the c-axis is comparable with the optimal length of the hops in the VRH transport. This result suggested the assignment of such a peculiar behavior to a kind of spatial confinement into a plane of the carrier hops. 3) Test device the p + -n junction In the last part of the work, the electrical properties of 4H-SiC diodes were also studied. In this case, a heavy Al + ion implantation was realized on n-type epilayers, according to the technological process applied for final devices. Good rectification properties was shown from these preliminary devices in their current-voltage characteristics. Admittance spectroscopy and deep level transient spectroscopy measurements showed the presence of electrically active defects other than the dopants ones, induced in the active region of the diodes by ion implantation. A critical comparison with the literature of these defects was performed. Preliminary to such an investigation, it was assessed the experimental set up for the admittance spectroscopy and current-voltage investigation and the automatic control of these measurements.