KATP channels gated by intracellular nucleotides and phospholipids.

The KATP channel is a heterooctamer composed of two different subunits, four inwardly rectifying K+ channel subunits, either Kir6. 1 or Kir6.2, and four sulfonylurea receptors (SUR), which belong to the family of ABC transporters. This unusual molecular architecture is related to the complex gating behaviour of these channels. Intracellular ATP inhibits KATP channels by binding to the Kir6.x subunits, whereas Mg-ADP increases channel activity by a hydrolysis reaction at the SUR. This ATP/ADP dependence allows KATP channels to link metabolism to excitability, which is important for many physiological functions, such as insulin secretion and cell protection during periods of ischemic stress. Recent work has uncovered a new class of regulatory molecules for KATP channel gating. Membrane phospholipids such as phosphoinositol 4, 5-bisphosphate and phosphatidylinositiol 4-monophosphate were found to interact with KATP channels resulting in increased open probability and markedly reduced ATP sensitivity. The membrane concentration of these phospholipids is regulated by a set of enzymes comprising phospholipases, phospholipid phosphatases and phospholipid kinases providing a possible mechanism for control of cell excitability through signal transduction pathways that modulate activity of these enzymes. This review discusses the mechanisms and molecular determinants that underlie gating of KATP channel by nucleotides and phospholipids and their physiological implications.     

Saturation of anti cyclic nucleotides antibodies by endogenous cyclic nucleotides.

Rabbits immunized against cyclic AMP or cyclic GMP produce antibodies which are fully saturated by their respective endogenous cyclic nucleotides. This was proved a) in comparing radioimmunological measurements of cyclic nucleotides in antiserum and the binding site concentration determined by equilibrium dialysis, b) in showing the ineffectiveness of serum phosphodiesterase to hydrolyze the cyclic AMP present in the anti-cyclic AMP antiserum. Immunological and radioimmunological implications of this phenomenon are discussed.     

Calmodulin binding to Arabidopsis cyclic nucleotide gated ion channels

Recently we have reported that the αC-helix in the cyclic nucleotide binding domain (CNBD) is required for channel regulation and function of cyclic nucleotide gated ion channels (CNGCs) in Arabidopsis. A mutation at arginine 557 to cysteine (R557C) in the αC-helix of the CNBD caused an alteration in channel regulation. Protein sequence alignments revealed that R557 is located in a region that is important for calmodulin (CaM) binding. It has been hypothesized that CaM negatively regulates plant CNGCs similar to their counter parts in animals. However, only a handful of studies has been published so far and we still do not have much information about the regulation of CNGCs by CaM. Here, we conducted in silico binding prediction of CaM and Arabidopsis CNGC12 (AtCNGC12) to further study the role of R557. Our analysis revealed that R557 forms salt bridges with both D79 and E83 in AtCaM1. Interestingly, a mutation of R557 to C causes the loss of these salt bridges. Our data further suggests that this alteration in CaM binding causes the observed altered channel regulation and that R557 plays an important role in CaM binding.Key words: calmodulin, CaM, cyclic nucleotide gated ion channels, CNGC, environmental effect, defense responses, temperatureWe recently reported about the role of the αC-helix in the cyclic nucleotide binding domain (CNBD) for channel regulation and function of cyclic nucleotide gated ion channels (CNGCs) in Arabidopsis.¹ CNGCs were first discovered in vertebrate retinal photoreceptors and olfactory sensory neurons.^2,3 They are composed of a cytoplasmic N-terminus, six membrane spanning regions (S1–S6), a pore domain located between S5 and S6 and a cytoplasmic C-terminus and share similarities with the voltage-gated outward rectifying K⁺-selective ion channel (Shaker) proteins.² However, CNGCs are ligand gated and opened by the direct binding of cyclic nucleotides monophosphates (cNMPs), such as cAMP and cGMP (cNMPs).⁴ The cytoplasmic C-terminus contains a CNBD that is connected to the pore domain by a C-linker region.⁵ The function and regulation of CNGCs has been extensively studied in retinal photoreceptor and olfactory sensory neurons and it has been reported that their channel activity is moderated by Ca²⁺/calmodulin (CaM).⁶ A variety of Ca²⁺ permeable channels are known to be regulated by Ca²⁺/CaM. In many cases, CaM downregulates their activity, thereby creating negative feedback regulation for Ca²⁺ entry.⁷ CNGCs are also considered to follow this mode of regulation. Some animal CNGCs possess a CaM binding domain in the cytoplasmic N-terminus. For example, it has been reported that CaM binds to a short segment in the N-terminal region of the A2 subunit of CNGCs of olfactory sensory neurons in a Ca²⁺ dependent manner.^8,9The first plant CNGC, HvCBT1 (Hordeum vulgare calmodulin (CaM)-binding transporter), was identified as a CaM-binding protein in barley.¹⁰ Interestingly, in contrast to animal CNGCs, the CaM binding domain in HvCBT1 was shown to be located at the cytoplasmic C-terminal region.¹⁰ Subsequently, several CNGCs were identified from Arabidopsis and Nicotiana tabacum^11,12 and some of these CNGCs have also been shown to possess the CaM binding domain in the C-terminal region that partially overlaps with the CNBD.^11–14 This difference in the location of the CaM binding domain between animal and plant CNGCs implies that different mechanisms for CNGC regulation may have evolved in animals and plants.The Arabidopsis mutant constitutive expresser of PR genes 22 (cpr22), which contains a novel chimeric CNGC, AtCNGC11/12, shows environmentally sensitive constitutive defense responses, such as heightened salicylic acid accumulation and hypersensitive response-like spontaneous programmed cell death.^15,16 It has been reported that the expression of AtCNGC11/12 and its channel activity is attributable for the cpr22 phenotype.^17,18 A genetic screen for mutants that suppress cpr22-conferred phenotypes identified over 20 novel mutant alleles in AtCNGC11/12.^1,18 These intragenic mutants are excellent tools to study the structure-function relationship of plant CNGCs. One of these mutants, suppressor S58, possesses a single amino acid substitution, arginine 557 to cysteine (R557C), in the αC-helix of the CNBD. The suppressor S58 lost all cpr22 related phenotypes, such as spontaneous cell death formation, under ambient temperature conditions. However, these phenotypes were recovered at 16°C, suggesting that the stability of channel function is affected by temperature.¹ Interestingly, this temperature sensitivity was also observed in the original mutant, cpr22.¹⁹ All salicylic acid-dependent phenotypes of cpr22 are enhanced under low temperature and low humidity conditions. Furthermore, this type of environmental sensitivity has been reported not only for cpr22, but also for various other pathogen resistance mutants as well as for defense responses in wild type plants.¹⁶ Therefore, it is possible that the basis of the temperature sensitivity observed in S58 may be related to a general environmental sensitivity in defense responses.Characterization of S58 and functional complementation using heterologous expression analyses suggested that R557 in the αC-helix of the CNBD is important for channel regulation, but not for basic channel function.¹ To further investigate this, we aimed to elucidate the molecular mechanism by which R557C (S58 mutation) alters channel regulation to determine the functional role of R557. Since R557 is located in the CNBD, it is possible that R557C alters cNMP binding resulting in disruption of channel opening. In animal systems, it has been reported that cNMPs bind within the pocket formed by the αC-helix and the β-barrel that is composed of the eight β sheets in the CNBD.^20,21 The αC-helix was suggested to function as the lid of this pocket that stabilizes cNMP binding by forming hydrophobic interactions with the bound cNMP.²¹ However, it is unlikely that R557 interacts directly with the bound cNMP because of its hydrophilic nature.Interestingly, a 19–20 amino acid sequence of the αC-helix was suggested to function as CaM binding domain in AtCNGC1 and AtCNGC2 by Köhler and Neuhaus using yeast two hybrid analysis.¹⁴ Arazi et al.¹³ biochemically demonstrated that a 23 amino acid sequence that overlaps with this 19–20 amino acids is the CaM binding domain in the tobacco CNGC, NtCBP4. Furthermore, they reported that the 4 additional amino acids (W R T/S W) which are located just outside of the 19–20 amino acid sequence are crucial for efficient binding to CaM. Sequence alignment revealed that R557 is located in this crucial sequence (W R T/S W).¹ Thus, it can be hypothesized that the R557C mutation causes a modification in the binding affinity to CaM resulting in an alteration in channel regulation. Therefore, we generated in silico models of CaM binding with the αC-helix of the CNBD in AtCNGC12 (identical to AtCNGC11/12) and AtCNGC12:R557C (S58). We first modeled Arabidopsis CaM1 (AtCaM1) based on the crystal structure of a potato CaM, PCM6 (PDB# 1RFJ).²² There are seven different CaM genes in Arabidopsis that encode two sets of identical isoforms (CaM1 and CaM4; CaM2, CaM3 and CaM5) and two additional distinct isoforms (CaM6 and CaM7).^23,24 All of them share high similarity with PCM6. Using the AtCaM1 model, we modeled possible interactions between AtCaM1 and the αC-helix of the CNBD in AtCNGC12 or AtCNGC12:R557C. As shown in Figure 1 (center part), a hydrophobic pocket of CaM that is necessary for binding with target proteins²⁵ was seen in AtCaM1 creating a binding pocket for the αC-helix of the CNBD in AtCNGC12. In this model, R557 creates salt bridges with both D79 and E83 of AtCaM1 (indicated by a box) and these salt bridges appear to play a role for binding to CaM. This type of salt bridges have been reported to be crucial for CaM binding with several different target proteins.²⁶ Interestingly, these salt bridges are no longer seen in AtCNGC12:R557C (Fig. 1, right part, indicated by a box); indicating that the mutation may cause an affinity change in CaM binding. Such an affinity change will likely cause an alteration in channel regulation. Since CaM is likely a negative regulator of CNGCs, this alteration in CaM affinity does not provide a simple mechanism for the R557C mutation. However, the change in CaM binding affinity may cause complex regulatory changes in CNGCs. Thus, we are currently conducting various biochemical analyses to validate this hypothesis.Open in a separate windowFigure 1Computational structural modeling of CaM binding with AtCNGC12 and AtCNGC12:R557C. Modeling of the tertiary structure of AtCaM1, and the αC-helix of AtCNGC12 and AtCNGC12:R557 was conducted using the crystallized structures of the potato CaM, PCM 6 (PDB# 1RFJ)²² and the cytoplasmic C-terminus of the invertebrate CNGC, SpIH (Flynn et al. 2007, PDB# 2PTM),²⁷ respectively, as templates. The protein fold recognition server (Phyre)²⁸ was used to model these proteins. The binding modeling was performed using an algorithm for molecular docking (PatchDock).²⁹ All the images were generated using PyMOL.³⁰ CaM is colored in cyan and the αC-helix is shown in magenta. Left part: overall binding model between AtCaM1 and AtCNGC12, Center part: close up of the boxed area of the left part in AtCNGC12, Right part: the same area in AtCNGC12:R557C. M73, M52 and M37 of AtCaM1 create a hydrophobic pocket together with F562 and I564 of AtCNGC12, which is a typical binding configuration between CaM and target proteins. R557 creates salt bridges with both D79 and E83 (center part). These salt bridges are no longer seen between AtCaM1 and AtCNGC12:R557C (right part).Although plants CNGCs have only recently been revealed to mediate multiple stress responses and also play important roles in some developmental pathways, studies that aim to elucidate their structural and regulatory properties are still very much in their infancy. Our current study will certainly contribute to a better understanding of the structure-function relationship and regulation of plant CNGCs.     

Gap junction channels exhibit connexin-specific permeability to cyclic nucleotides

Gap junction channels exhibit connexin dependent biophysical properties, including selective intercellular passage of larger solutes, such as second messengers and siRNA. Here, we report the determination of cyclic nucleotide (cAMP) permeability through gap junction channels composed of Cx43, Cx40, or Cx26 using simultaneous measurements of junctional conductance and intercellular transfer of cAMP. For cAMP detection the recipient cells were transfected with a reporter gene, the cyclic nucleotide-modulated channel from sea urchin sperm (SpIH). cAMP was introduced via patch pipette into the cell of the pair that did not express SpIH. SpIH-derived currents (I(h)) were recorded from the other cell of a pair that expressed SpIH. cAMP diffusion through gap junction channels to the neighboring SpIH-transfected cell resulted in a five to sixfold increase in I(h) current over time. Cyclic AMP transfer was observed for homotypic Cx43 channels over a wide range of conductances. However, homotypic Cx40 and homotypic Cx26 exhibited reduced cAMP permeability in comparison to Cx43. The cAMP/K(+) permeability ratios were 0.18, 0.027, and 0.018 for Cx43, Cx26, and Cx40, respectively. Cx43 channels were approximately 10 to 7 times more permeable to cAMP than Cx40 or Cx26 (Cx43 > Cx26 > or = Cx40), suggesting that these channels have distinctly different selectivity for negatively charged larger solutes involved in metabolic/biochemical coupling. These data suggest that Cx43 permeability to cAMP results in a rapid delivery of cAMP from cell to cell in sufficient quantity before degradation by phosphodiesterase to trigger relevant intracellular responses. The data also suggest that the reduced permeability of Cx26 and Cx40 might compromise their ability to deliver cAMP rapidly enough to cause functional changes in a recipient cell.     

Yeast hygromycin sensitivity as a functional assay of cyclic nucleotide gated cation channels.

Cyclic nucleotide gated cation channels (CNGCs) are a large (20 genes in Arabidopsis thaliana) family of plant ligand gated (i.e. cyclic nucleotides activate currents) ion channels, however, little is known about their functional properties. One reason for this is the recalcitrance of plant CNGC expression in heterologous systems amenable to patch clamp studies. Here, we show results demonstrating the efficacy of using growth of a K+ uptake-deficient yeast (trk1,2) as a functional assay of CNGCs as inwardly-conducting cell membrane cation (K+) transporters. Prior work demonstrated that trk1,2 is hypersensitive to the antibiotic hygromycin (hyg) and that expression of an inwardly conducting K+ transporter suppresses hyg hypersensitivity. We find that increasing [hyg] in solid YPD medium inhibits trk1,2 growth around a filter disk saturated with 3 M K+. Northern analysis indicated that message is transcribed in trk1,2 transformed with the CNGC coding sequences. Confocal imaging of yeast expressing CNGC-fluorescent fusion proteins indicated channel targeting to the cell membrane. Trk1,2 expressing several plant CNGCs grown in the presence of hyg demonstrated (a) greater growth than trk1,2 transformed with empty plasmid, and (b) enhanced growth when cAMP was added to the medium. Alternatively, cAMP inhibited growth of yeast transformed with either the empty plasmid, or the plant K+ channel KAT1; this channel is not a CNGC. Growth of trk1,2 was dependent on filter disk [K+]; suggesting that complementation of hyg hypersensitivity due to presence of a functional plant CNGC was dependent on K+ movement into the cytosol. We conclude that plant CNGC functional characterization can be facilitated by this assay system.     

Single-channel recording of ligand-gated ion channels

Electrophysiological recording of single-channel currents is the most direct method available for obtaining detailed and precise information about the kinetic behavior of ion channels. A wide variety of cell types can be used for single-channel recording, but to obtain the highest resolution of the briefest channel opening and closing events, low-noise recordings, coupled with a minimal filtering frequency, are required. Here, we present a protocol designed to help those with some electrophysiological expertise who wish to explore the properties of native and recombinant single ligand-gated ion channels. We have focused on the practical aspects of recording single GABA channels from cell-attached and outside-out patches and also introduced some of the preliminary considerations that are necessary for the analysis of single-channel data, including an introduction to single-channel analysis software.     

Regulation and function of cyclic nucleotides.

Recent work has greatly expanded our knowledge of the structure, regulation and diversity of enzymes involved in the synthesis and degradation of cyclic nucleotides. This review focuses on recent work that provides insight into the structure and function of the cyclases and phosphodiesterases that regulate cyclic nucleotide metabolism. Particular emphasis is given to the roles played by multiple isoforms of each enzyme system.     

Single-channel properties of a volume-sensitive anion conductance. Current activation occurs by abrupt switching of closed channels to an open state

Swelling-induced loss of organic osmolytes from cells is mediated by an outwardly rectified, volume-sensitive anion channel termed VSOAC (Volume-Sensitive Organic osmolyte/Anion Channel). Similar swelling- activated anion channels have been described in numerous cell types. The unitary conductance and gating kinetics of VSOAC have been uncertain, however. Stationary noise analysis and single-channel measurements have produced estimates for the unitary conductance of swelling-activated, outwardly rectified anion channels that vary by > 15-fold. We used a combination of stationary and nonstationary noise analyses and single-channel measurements to estimate the unitary properties of VSOAC. Current noise was analyzed initially by assuming that graded changes in macroscopic current were due to graded changes in channel open probability. Stationary noise analysis predicts that the unitary conductance of VSOAC is approximately 1 pS at 0 mV. In sharp contrast, nonstationary noise analysis demonstrates that VSOAC is a 40-50 pS channel at +120 mV (approximately 15 pS at 0 mV). Measurement of single-channel events in whole-cell currents and outside- out membrane patches confirmed the nonstationary noise analysis results. The discrepancy between stationary and nonstationary noise analyses and single-channel measurements indicates that swelling- induced current activation is not mediated by a graded increase in channel open probability as assumed initially. Instead, activation of VSOAC appears to involve an abrupt switching of single channels from an OFF state, where channel open probability is zero, to an ON state, where open probability is near unity.     

Phosphodiesterase of cyclic nucleotides

Problems are considered on form multiplicity, purification and molecular weight of cyclic nucleotides phosphodiesterase. A supposition is made that the molecular weight of the catalytic subunit of the enzyme for most studied objects is about 60000. The catalytic subunit may form di- and trimers and be associated with regulatory proteins of different type. The problem of phosphodiesterase regulation is analyzed on the basis of potentialities of the equilibrium shift between the protein subunits of the enzyme; the role of cyclic nucleotides as well as of triphosphonucleotides are shown to influence the regulation of the enzymic activity. In some cases the mechanism of changes in the activity of phosphodiesterase bound with the receptor is shown to be similar to that for adenylate cyclase. In particular, the role of GTP and one of the protein subunits of phosphodiesterase in this process is stated.     

Antibody-dependent lymphocyte mediated cytotoxicity mechanism and modulation by cyclic nucleotides.

The injury to antibody coated thymocytes by the “K cell” among nonsensitized rat splenocytes was modulated by altering the cellular level of cAMP and cGMP. Preincubation of the attacking cell population with 1 μg/ml cholera toxin caused an elevation of cAMP levels of 1–18 pM per 10⁷ cells and was associated with a proportionate reduction in cytotoxicity. Agents which are known to raise cAMP levels including the Prostaglandins (PG) E₁ (10^?7–10^?5 M), PGF_2α (10^?5–10^?3 M), PGA₁ (10^?9–10^?5M), and theophylline (10^?3 M) also produced marked suppression of antibody dependent lymphocyte mediated cytotoxicity. Direct elevation of cellular levels of cGMP by the analog 8-bromo cGMP (5 × 10^?6M) led to augmentation of cytotoxicity. Removal by EDTA and MgEDTA of calcium and magnesium ions from the culture media markedly inhibited cytotoxicity.     

Regulation of slow calcium channels of myocardial cells and vascular smooth muscle cells by cyclic nucleotides and phosphorylation

The slow Ca²⁺ channels (L-type) of the heart are stimulated by cAMP. Elevation of cAMP produces a very rapid increase in number of slow channels available for voltage activation during excitation. The probability of a Ca²⁺ channel opening and the mean open time of the channel are increased. Therefore, any agent that increases the cAMP level of the myocardial cell will tend to potentiate I_Ca, Ca²⁺ influx, and contraction. The action of cAMP is mediated by PK-A and phosphorylation of the slow Ca²⁺ channel protein or an associated regulatory protein (stimulatory type). The myocardial slow Ca²⁺ channels are also rogulated by cGMP, in a manner that is opposite orantagonistic to that of cAMP. We have demonstrated this at both the macroscople level (whole-cell voltage clamp) and the single-channel level. The effect of cGMP is mediated by PK-G and phosphorylation of a protein, as for example, a regulatory protein (inhibitory-type) associated with the Ca²⁺ channel. Introduction of PK-G intracellularly causes a relatively rapid inhibition of I_Ca(L) in both chick and rat heart cells. Such inhibition occurs for both the basal and stimulated I_Ca(L). In addition, the cGMP/PK-G system was reported to stimulate a phosphatase that dephosphorylates the Ca²⁺ channel. In addition to the slower indirect pathway—exerted via cAMP/PK-A—there is a faster more-direct pathway for I_Ca(L) stimulation by the -adrenergic receptor. This latter pathway involves direct modulation of the channel activity by the alpha subunit (_s*) of the G_s-protein. In vascular smooth muscle cells the two pathways (direct and indirect) also appear to be present, although the indirect pathway producesinhibition of I_Ca(L). PK-C and calmodulin-PK also may play roles in regulation of the myocardial slow Ca²⁺ channels. Both of these protein kinases stimulate the activity of these channels. Thus, it appears that the slow Ca²⁺ channel is a complex structure, including perhaps several associated regulatory proteins, which can be regulated by a number of factors intrinsic and extrinsic to the cell, and thereby control can be exercised over the force of contraction of the heart.This review-type article was prepared by modifying an article published in a book by Sperelakiset al., 1994.     

Effect of cyclic nucleotides on calcium dependent K+ channels, in human erythrocytes

K+ efflux has been analyzed in human erythrocytes incubated in a K+ free medium containing ouabain, bumetanide, CaCl2, and the Ca2+ ionophore A23187. In these conditions, a K+ efflux, which is exponentially dependent on the concentration of A23187 present in the medium, has been observed. This flux is almost completely abolished by either quinine or EGTA, so that, the above K+ efflux has been considered Ca2+ dependent. The effects of cAMP, and cGMP, have been tested on this flux. Ca2+ dependent K+ efflux decreases in presence of millimolar concentrations of cAMP in the medium. The addition of methyl-isobutyl-xanthine to the incubation medium containing cAMP enhances the inhibitory effect of this compound. cGMP also inhibits the Ca2+ dependent K+ efflux. Our results suggest that cyclic nucleotides may modulate the activation of Ca2+ dependent K+ channels in human erythrocytes.     

Single-channel properties and pH sensitivity of two-pore domain K+ channels of the TALK family

The two-pore K2P channel family comprises TASK, TREK, TWIK, TRESK, TALK, and THIK subfamilies, and TALK-1, TALK-2, and TASK-2 are functional members of the TALK subfamily. Here we report for the first time the single-channel properties of TALK-2 and its pHo sensitivity, and compare them to those of TALK-1 and TASK-2. In transfected COS-7 cells, the three TALK K2P channels could be identified easily by their differences in single-channel conductance and gating kinetics. The single-channel conductances of TALK-1, TALK-2, and TASK-2 in symmetrical 150 mM KCl were 21, 33, and 70 pS (-60 mV), respectively. TALK-2 was sensitive mainly to the alkaline range (pH 7-10), whereas TALK-1 and TASK-2 were sensitive to a wider pHo range (6-10). The effect of pH changes was mainly on the opening frequency. Thus, members of the TALK family expressed in native tissues may be identified based on their single-channel kinetics and pHo sensitivity.     

Inhibition of the olfactory cyclic nucleotide gated ion channel by intracellular calcium.

When olfactory receptor neurons are exposed to sustained application of odours, the elicited ionic current is transient. This adaptation-like effect appears to require the influx of Ca2+ through the odour-sensitive conductance; in the absence of extracellular Ca2+ the current remains sustained. Odour transduction proceeds through a G-protein-based second messenger system, resulting finally in the direct activation of an ion channel by cyclic AMP. This channel is one possible site for a negative feedback loop using Ca2+ as a messenger. In recordings of single cyclic AMP gated channels from olfactory receptor neurons, the open probability of the channel in saturating cAMP concentrations was dependent on the concentration of intracellular Ca2+. It could be reduced from 0.6 in 100 nm Ca2+ to 0.09 in 3 microM Ca2+. However, as neither the single channel conductance nor the mean open time were affected by Ca+ concentration, this does not appear to be a mechanism of simple channel block. Rather, these results suggest that intracellular Ca2+ acts allosterically to stabilize a closed state of the channel.     

Voltage gated ionic channels in rat cultured astrocytes, reactive astrocytes and an astrocyte-oligodendrocyte progenitor cell

Astrocytes (both type 1 and type 2), cultured from the central nervous system of newborn or 7 day old rats show voltage gated sodium and potassium channels that are activated when the membrane is depolarized to greater than -40 mV. The sodium channels in these cells have an h-infinity curve similar to that of nodal membranes but the activation (peak current-voltage) curves are shifted along the voltage axis by about +30 mV. These sodium currents are blocked only by high concentrations of tetrodotoxin. The voltage activated potassium currents in both types of astrocyte show at least two components; an inactivating component that is suppressed at holding potentials of greater than -40 mV and a persistent, non-inactivating current. Several types of single channel currents were observed in outside-out membrane patches from type 2 astrocytes. One type of potassium channel showed inactivation on depolarization and may contribute to the whole-cell inactivating current. In contrast, oligodendrocytes showed no obvious voltage gated membrane channels. The properties of the type 2 astrocyte-oligodendrocyte progenitor cell were investigated in two ways: 1) by examination of cells just beginning to differentiate along the "electrically silent" oligodendrocyte pathway or 2) by recording from progenitor cells cultured for 24 hours in the presence of cycloheximide to block the appearance of new membrane channels. In both cases, voltage gated inward (sodium) and outward (potassium) currents were noted. The outward current response showed both an inactivating and a non-inactivating component. Similar voltage activated inward and outward membrane currents were noted in reactive astrocytes freshly isolated (3-6 hours) from lesioned areas of adult rat brains.(ABSTRACT TRUNCATED AT 250 WORDS)     

Multiple cyclic nucleotide‐gated channels coordinate calcium oscillations and polar growth of root hairs

Calcium gradients underlie polarization in eukaryotic cells. In plants, a tip‐focused Ca²⁺‐gradient is fundamental for rapid and unidirectional cell expansion during epidermal root hair development. Here we report that three members of the cyclic nucleotide‐gated channel family are required to maintain cytosolic Ca²⁺ oscillations and the normal growth of root hairs. CNGC6, CNGC9 and CNGC14 were expressed in root hairs, with CNGC9 displaying the highest root hair specificity. In individual channel mutants, morphological defects including root hair swelling and branching, as well as bursting, were observed. The developmental phenotypes were amplified in the three cngc double mutant combinations. Finally, cngc6/9/14 triple mutants only developed bulging trichoblasts and could not form normal root hair protrusions because they burst after the transition to the rapid growth phase. Prior to developmental defects, single and double mutants showed increasingly disturbed patterns of Ca²⁺ oscillations. We conclude that CNGC6, CNGC9 and CNGC14 fulfill partially but not fully redundant functions in generating and maintaining tip‐focused Ca²⁺ oscillations, which are fundamental for proper root hair growth and polarity. Furthermore, the results suggest that these calmodulin‐binding and Ca²⁺‐permeable channels organize a robust tip‐focused oscillatory calcium gradient, which is not essential for root hair initiation but is required to control the integrity of the root hair after the transition to the rapid growth phase. Our findings also show that root hairs possess a large ability to compensate calcium‐signaling defects, and add new players to the regulatory network, which coordinates cell wall properties and cell expansion during polar root hair growth.     

Modification of radiosensitivity of mammalian cells by cyclic nucleotides

Some $\text{in vitro}$ and $\text{in vivo}$ studies suggest that adesosine 3′,5′-cyclic monophosphate (cyclic AMP) may be one of the important factors in determining the radiosensitivity of certain mammalian cells; however, the role of guanosine 3',5'-cyclic monophosphate (cyclic GMP) in radiosensitivity of mammalian cells is completely unknown. Recent data also suggest that the mechanism of radiation protection afforded by moderate hypoxia and SH-containing compounds may involve an alteration in the intracellular level of cyclic AMP. At least one $\text{in vivo}$ study shows that cyclic AMP protects hair follicles and gut epithelial cells against radiation damage; however, it does not protect lymphosarcoma and breast carcinoma in mice. If a similar phenomenon is found in humans, an elevation of the intracellular level of cyclic AMP during radiation exposure may improve the effectiveness of radiation therapy in those cases where the radiation damage of normal tissue becomes the limiting factor for a continuation of the therapy program. More $\text{in vitro}$ and $\text{in vivo}$ studies on normal and cancer cells are needed to substantiate the role of cyclic nucleotides in radiosensitivity.