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C G Nichols

Publications and source records attributed to C G Nichols.

At least 19 recordsLinked to original sources
PubMed2007

Downregulation of Kir4.1 inward rectifying potassium channel subunits by RNAi impairs potassium transfer and glutamate uptake by cultured cortical astrocytes.

Glial cell-mediated potassium and glutamate homeostases play important roles in the regulation of neuronal excitability. Diminished potassium and glutamate buffering capabilities of astrocytes result in hyperexcitability of neurons and abnormal synaptic transmission. The role of the different K+ channels in maintaining the membrane potential and buffering capabilities of cortical astrocytes has not yet been definitively determined due to the lack of specific K+ channel blockers. The purpose of the present study was to assess the role of the inward-rectifying K+ channel subunit Kir4.1 on potassium fluxes, glutamate uptake and membrane potential in cultured rat cortical astrocytes using RNAi, whole-cell patch clamp and a colorimetric assay. The membrane potentials of control cortical astrocytes had a bimodal distribution with peaks at -68 and -41 mV. This distribution became unimodal after knockdown of Kir4.1, with the mean membrane potential being shifted in the depolarizing direction (peak at -45 mV). The ability of Kir4.1-suppressed cells to mediate transmembrane potassium flow, as measured by the current response to voltage ramps or sequential application of different extracellular [K+], was dramatically impaired. In addition, glutamate uptake was inhibited by knock-down of Kir4.1-containing channels by RNA interference as well as by blockade of Kir channels with barium (100 microM). Together, these data indicate that Kir4.1 channels are primarily responsible for significant hyperpolarization of cortical astrocytes and are likely to play a major role in potassium buffering. Significant inhibition of glutamate clearance in astrocytes with knock-down of Kir4.1 highlights the role of membrane hyperpolarization in this process.

Animals↗
PubMed2006

Expression of Kir2.1 and Kir6.2 transgenes under the control of the alpha-MHC promoter in the sinoatrial and atrioventricular nodes in transgenic mice.

Kir2.1 and Kir6.2 are ion channel subunits partly responsible for the background inward rectifier and ATP-sensitive K(+) currents (I(K1) and I(KATP)) in the heart. Very little is known about how the distribution of ion channel subunits is controlled. In this study, we have investigated the expression (at protein and mRNA levels) of GFP-tagged Kir2.1 and Kir6.2 transgenes under the control of the alpha-MHC promoter in the sinoatrial node (SAN), atrioventricular node (AVN), His bundle and working myocardium of transgenic mice. After dissection, serial 10-microm cryosections were cut. Histological staining was carried out to identify tissues, confocal microscopy was carried out to map the distribution of the GFP-tagged ion channel subunits and in situ hybridization was carried out to map the distribution of corresponding mRNAs. We demonstrate heterologous expression of the ion channel subunits in the working myocardium, but not necessarily in the SAN, AVN or His bundle; the distribution of the subunits does not correspond to the expected distribution of alpha-MHC. Both protein and mRNA expression does, however, correspond to the expected distributions of native Kir6.2 and Kir2.1 in the SAN, AVN, His bundle and working myocardium. The data demonstrate novel transcriptional and/or post-transcriptional control of ion channel subunit expression and raise important questions about the control of regional expression of ion channels.

Animals↗
PubMed2006

Hyperinsulinism in mice with heterozygous loss of K(ATP) channels.

AIMS/HYPOTHESIS: ATP-sensitive K(+) (K(ATP)) channels couple glucose metabolism to insulin secretion in pancreatic beta cells. In humans, loss-of-function mutations of beta cell K(ATP) subunits (SUR1, encoded by the gene ABCC8, or Kir6.2, encoded by the gene KCNJ11) cause congenital hyperinsulinaemia. Mice with dominant-negative reduction of beta cell K(ATP) (Kir6.2[AAA]) exhibit hyperinsulinism, whereas mice with zero K(ATP) (Kir6.2(-/-)) show transient hyperinsulinaemia as neonates, but are glucose-intolerant as adults. Thus, we propose that partial loss of beta cell K(ATP) in vivo causes insulin hypersecretion, but complete absence may cause insulin secretory failure. MATERIALS AND METHODS: Heterozygous Kir6.2(+/-) and SUR1(+/-) animals were generated by backcrossing from knockout animals. Glucose tolerance in intact animals was determined following i.p. loading. Glucose-stimulated insulin secretion (GSIS), islet K(ATP) conductance and glucose dependence of intracellular Ca(2+) were assessed in isolated islets. RESULTS: In both of the mechanistically distinct models of reduced K(ATP) (Kir6.2(+/-) and SUR1(+/-)), K(ATP) density is reduced by approximately 60%. While both Kir6.2(-/-) and SUR1(-/-) mice are glucose-intolerant and have reduced glucose-stimulated insulin secretion, heterozygous Kir6.2(+/-) and SUR1(+/-) mice show enhanced glucose tolerance and increased GSIS, paralleled by a left-shift in glucose dependence of intracellular Ca(2+) oscillations. CONCLUSIONS/INTERPRETATION: The results confirm that incomplete loss of beta cell K(ATP) in vivo underlies a hyperinsulinaemic phenotype, whereas complete loss of K(ATP) underlies eventual secretory failure.

ATP-Binding Cassette Transporters↗
PubMed2006

The mitochondria and insulin release: Nnt just a passing relationship.

Nicotinamide nucleotide transhydrogenase (Nnt) detoxifies reactive oxygen species (ROS), byproducts of mitochondrial metabolism that, when accumulated, can decrease mitochondrial ATP production. In this issue of Cell Metabolism, demonstrate that Nnt in pancreatic beta cells is important for insulin release. Their compelling data highlight the critical roles for ATP generation and subsequent closure of KATP channels for insulin secretion.

Animals↗
PubMed2005

Molecular basis of inward rectification: structural features of the blocker defined by extended polyamine analogs.

Polyamines cause inward rectification of Kir K(+) channels by blocking deep within the channel pore. We investigated structural constraints of polyamine block of strongly rectifying mutant K(ATP) channels (Kir6.2[L164C,N160D,C166S] + SUR1). We studied three groups of polyamine analogs: 1) conformationally restricted linear tetra-amines with a cycloalkyl or alkene group between the second and third amines (CGC-11047, CGC-11093, CGC-11099, and CGC-11098), 2) conformationally restricted linear deca-amines with a cycloalkyl or alkene group between the fifth and sixth amines (CGC-11150, CGC-11179, and CGC-11241), and 3) cyclic tetra-amines (CGC-11174, CGC-11197, CGC-11199, and CGC-11254). All linear analogs cause a voltage-dependent block similar to that of spermine, but slightly weaker (at 1 microM, V(1/2) for spermine block = -10 +/- 1 mV, Z = 2.9 +/- 0.1, n = 19; V(1/2) for analogs varies from polyamine -7 to +10 mV, Z = 2.6-3.9). These data indicate tolerance for conformational restriction and an upper limit to the voltage dependence of the blocking process. There was no voltage-dependent block by the cyclic compounds; instead, they induce irreversible rundown of the current. Structural models of Kir channels suggest that a narrow entry at the top of the cytoplasmic pore may exclude cyclic analogs from the inner cavity, thereby explaining the structure-activity relationship that we observe.

Animals↗
PubMed2003

Polyamine flux in Xenopus oocytes through hemi-gap junctional channels.

Diverse polyamine transport systems have been described in different cells, but the molecular entities that mediate polyamine influx and efflux remain incompletely defined. We have previously demonstrated that spermidine efflux from oocytes is a simple electrodiffusive process, inhibitable by external Ca2+, consistent with permeation through a membrane cation channel. Hemi-gap junctional channels in Xenopus oocytes are formed from connexin 38 (Cx38), and produce a calcium-sensitive (Ic) current that is inhibited by external Ca2+. Spermidine efflux is also calcium sensitive, and removal of external calcium increases both Ic currents and spermidine efflux in Xenopus oocytes. Injection of Cx38 cRNA or Cx38 antisense oligonucleotides (to increase or decrease, respectively, Cx38 expression) also increases or decreases spermidine efflux in parallel. Spermidine efflux has a large voltage-dependent component, which is abolished with injection of Cx38 antisense oligonucleotides. In addition, spermidine uptake is significantly increased in Cx38 cRNA-injected oocytes in the absence of external calcium. The data indicate that hemi-gap junctional channels provide the Ca2+-inhibited pathway for electrodiffusive efflux of polyamines from oocytes, and it is likely that hemi-gap junctional channels provide Ca2+ and metabolism-sensitive polyamine permeation pathways in other cells.

Animals↗
PubMed2002

Hyperinsulinism induced by targeted suppression of beta cell KATP channels.

ATP-sensitive K+ (K(ATP)) channels couple cell metabolism to electrical activity. To probe the role of K(ATP) in glucose-induced insulin secretion, we have generated transgenic mice expressing a dominant-negative, GFP-tagged K(ATP) channel subunit in which residues 132-134 (Gly-Tyr-Gly) in the selectivity filter were replaced by Ala-Ala-Ala, under control of the insulin promoter. Transgene expression was confirmed by both beta cell-specific green fluorescence and complete suppression of channel activity in those cells ( approximately 70%) that did fluoresce. Transgenic mice developed normally with no increased mortality and displayed normal body weight, blood glucose levels, and islet architecture. However, hyperinsulinism was evident in adult mice as (i) a disproportionately high level of circulating serum insulin for a given glucose concentration ( approximately 2-fold increase in blood insulin), (ii) enhanced glucose-induced insulin release from isolated islets, and (iii) mild yet significant enhancement in glucose tolerance. Enhanced glucose-induced insulin secretion results from both increased glucose sensitivity and increased release at saturating glucose concentration. The results suggest that incomplete suppression of K(ATP) channel activity can give rise to a maintained hyperinsulinism.

Adenosine Triphosphate↗
PubMed2002

Structural basis of inward rectifying potassium channel gating.

The last 10 years have seen rapid advances in the understanding of potassium channel function. Since the first inward rectifying (Kir) channels were cloned in 1994, the structural basis of channel function has been significantly elucidated, and determination of the crystal structure of a bacterial K channel (KcsA) in 1998 provided an atomic resolution of the permeation pathway. This review considers recent experimental studies aimed at uncovering the structural basis of Kir channel activity, and the applicability of comparative models based on KcsA to illuminate Kir channel pore structure and opening and closing processes.

Humans↗
PubMed2002

The role of NH2-terminal positive charges in the activity of inward rectifier KATP channels.

Approximately half of the NH(2) terminus of inward rectifier (Kir) channels can be deleted without significant change in channel function, but activity is lost when more than approximately 30 conserved residues before the first membrane spanning domain (M1) are removed. Systematic replacement of the positive charges in the NH(2) terminus of Kir6.2 with alanine reveals several residues that affect channel function when neutralized. Certain mutations (R4A, R5A, R16A, R27A, R39A, K47A, R50A, R54A, K67A) change open probability, whereas an overlapping set of mutants (R16A, R27A, K39A, K47A, R50A, R54A, K67A) change ATP sensitivity. Further analysis of the latter set differentiates mutations that alter ATP sensitivity as a consequence of altered open state stability (R16A, K39A, K67A) from those that may affect ATP binding directly (K47A, R50A, R54A). The data help to define the structural determinants of Kir channel function, and suggest possible structural motifs within the NH(2) terminus, as well as the relationship of the NH(2) terminus with the extended cytoplasmic COOH terminus of the channel.

Amino Acid Sequence↗
PubMed2002

K(ATP) channels and insulin secretion disorders.

ATP-sensitive potassium (K(ATP)) channels are inhibited by intracellular ATP and activated by ADP. Nutrient oxidation in beta-cells leads to a rise in [ATP]-to-[ADP] ratios, which in turn leads to reduced K(ATP) channel activity, depolarization, voltage-dependent Ca(2+) channel activation, Ca(2+) entry, and exocytosis. Persistent hyperinsulinemic hypoglycemia of infancy (HI) is a genetic disorder characterized by dysregulated insulin secretion and, although rare, causes severe mental retardation and epilepsy if left untreated. The last five or six years have seen rapid advance in understanding the molecular basis of K(ATP) channel activity and the molecular genetics of HI. In the majority of cases for which a genotype has been uncovered, causal HI mutations are found in one or the other of the two genes, SUR1 and Kir6.2, that encode the K(ATP) channel. This article will review studies that have defined the link between channel activity and defective insulin release and will consider implications for future understanding of the mechanisms of control of insulin secretion in normal and diseased states.

Adenosine Triphosphate↗
PubMed2002

Diabetes and insulin secretion: whither KATP?

The critical involvement of ATP-sensitive potassium (KATP) channels in insulin secretion is confirmed both by the demonstration that mutations that reduce KATP channel activity underlie many if not most cases of persistent hyperinsulinemia, and by the ability of sulfonylureas, which inhibit KATP channels, to enhance insulin secretion in type II diabetics. By extrapolation, we contend that mutations that increase beta-cell KATP channel activity should inhibit glucose-dependent insulin secretion and underlie, or at least predispose to, a diabetic phenotype. In transgenic animal models, this prediction seems to be borne out. Although earlier genetic studies failed to demonstrate a linkage between KATP mutations and diabetes in humans, recent studies indicate significant association of KATP channel gene mutations or polymorphisms and type II diabetes. We suggest that further efforts to understand the involvement of KATP channels in diabetes are warranted.

Adenosine Triphosphate↗
PubMed2002

Contractility and ischemic response of hearts from transgenic mice with altered sarcolemmal K(ATP) channels.

The functional significance of ATP-sensitive K(+) (K(ATP)) channels is controversial. In the present study, transgenic mice expressing a mutant Kir6.2, with reduced ATP sensitivity, were used to examine the role of sarcolemmal K(ATP) in normal cardiac function and after an ischemic or metabolic challenge. We found left ventricular developed pressure (LVDP) was 15-20% higher in hearts from transgenics in the absence of cardiac hypertrophy. beta-Adrenergic stimulation caused a positive inotropic response from nontransgenic hearts that was not observed in transgenic hearts. Decreasing extracellular Ca(2+) decreased LVDP in hearts from nontransgenics but not in those from transgenics. These data suggest an increase in intracellular [Ca(2+)] in transgenic hearts. Additional studies have demonstrated hearts from nontransgenics and transgenics have a similar postischemic LVDP. However, ischemic preconditioning does not improve postischemic recovery in transgenics. Transgenic hearts also demonstrate a poor recovery after metabolic inhibition. These data are consistent with the hypothesis that sarcolemmal K(ATP) channels are required for development of normal myocardial function, and perturbations of K(ATP) channels lead to hearts that respond poorly to ischemic or metabolic challenges.

Adenosine Triphosphate↗
PubMed2001

Tolerance for ATP-insensitive K(ATP) channels in transgenic mice.

To examine the role of sarcolemmal K(ATP) channels in cardiac function, we generated transgenic mice expressing GFP-tagged Kir6.2 subunits with reduced ATP sensitivity under control of the cardiac alpha-myosin heavy chain promoter. Four founder mice were isolated, and both founders and progeny were all apparently normal and fertile. Electrocardiograms from conscious animals also appeared normal, although mean 24-hour heart rate was approximately 10% lower in transgenic animals compared with littermate controls. In excised membrane patches, K(ATP) channels were very insensitive to inhibitory ATP: mean K(1/2) ([ATP] causing half-maximal inhibition) was 2.7 mmol/L in high-expressing line 4 myocytes, compared with 51 micromol/L in littermate control myocytes. Counterintuitively, K(ATP) channel density was approximately 4-fold lower in transgenic membrane patches than in control. This reduction of total K(ATP) conductance was confirmed in whole-cell voltage-clamp conditions, in which K(ATP) was activated by metabolic inhibition. K(ATP) conductance was not obvious after break-in of either control or transgenic myocytes, and there was no action potential shortening in transgenic myocytes. In marked contrast to the effects of expression of similar transgenes in pancreatic beta-cells, these experiments demonstrate a profound tolerance for reduced ATP sensitivity of cardiac K(ATP) channels and highlight differential effects of channel activity in the electrical activity of the 2 tissues.

Action Potentials↗
PubMed2001

Genetic manipulation of cardiac K(+) channel function in mice: what have we learned, and where do we go from here?

In the mammalian myocardium, potassium (K(+)) channels control resting potentials, action potential waveforms, automaticity, and refractory periods and, in most cardiac cells, multiple types of K(+) channels that subserve these functions are expressed. Molecular cloning has revealed the presence of a large number of K(+) channel pore forming (alpha) and accessory (beta) subunits in the heart, and considerable progress has been made recently in defining the relationships between expressed K(+) channel subunits and functional cardiac K(+) channels. To date, more than 20 mouse models with altered K(+) channel expression/functioning have been generated using dominant-negative transgenic and targeted gene deletion approaches. In several instances, the genetic manipulation of K(+) channel subunit expression has revealed the role of specific K(+) channel subunit subfamilies or individual K(+) channel subunit genes in the generation of myocardial K(+) channels. In other cases, however, the phenotypic consequences have been unexpected. This review summarizes what has been learned from the in situ genetic manipulation of cardiac K(+) channel functioning in the mouse, discusses the limitations of the models developed to date, and explores the likely directions of future research.

Action Potentials↗
PubMed2001

MDR1 P-glycoprotein reduces influx of substrates without affecting membrane potential.

MDR1 (multidrug resistance) P-glycoprotein (Pgp; ABCB1) decreases intracellular concentrations of structurally diverse drugs. Although Pgp is generally thought to be an efflux transporter, the mechanism of action remains elusive. To determine whether Pgp confers drug resistance through changes in transmembrane potential (E(m)) or ion conductance, we studied electrical currents and drug transport in Pgp-negative MCF-7 cells and MCF-7/MDR1 stable transfectants that were established and maintained without chemotherapeutic drugs. Although E(m) and total membrane conductance did not differ between MCF-7 and MCF-7/MDR1 cells, Pgp reduced unidirectional influx and steady-state cellular content of Tc-Sestamibi, a substrate for MDR1 Pgp, without affecting unidirectional efflux of substrate from cells. Depolarization of membrane potentials with various concentrations of extracellular K(+) in the presence of valinomycin did not inhibit the ability of Pgp to reduce intracellular concentration of Tc-Sestamibi, strongly suggesting that the drug transport activity of MDR1 Pgp is independent of changes in E(m) or total ion conductance. Tetraphenyl borate, a lipophilic anion, enhanced unidirectional influx of Tc-Sestamibi to a greater extent in MCF-7/MDR1 cells than in control cells, suggesting that Pgp may, directly or indirectly, increase the positive dipole potential within the plasma membrane bilayer. Overall, these data demonstrate that changes in E(m) or macroscopic conductance are not coupled with function of Pgp in multidrug resistance. The dominant effect of MDR1 Pgp in this system is reduction of drug influx, possibly through an increase in intramembranous dipole potential.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed2001

Heterologous expression of the Na(+),K(+)-ATPase gamma subunit in Xenopus oocytes induces an endogenous, voltage-gated large diameter pore.

1. The gamma subunit is a specific component of the plasmalemmal Na(+),K(+)-ATPase. Like structurally related single-spanning membrane proteins such as cardiac phospholemman, Mat-8 and renal CHIF, large ion conductances are activated when gamma subunits are expressed in Xenopus oocytes. 2. Here we report critical properties of the gamma-activated conductance. The gamma-activated conductance showed non-selective cationic and anionic permeation, and extremely slow kinetics, with an activation time constant > 1 s following steps to -100 mV. 3. The gamma-activated conductance was inhibited by extracellular divalent ions including Ba(2+) (K(i) = 0.7 mM) and Ca(2+) (K(i) = 0.4 mM). 4. 2-Deoxyglucose (MW approximately 180), inulin (MW approximately 5000) and spermidine (MW approximately 148) efflux could occur through the gamma-activated conductance pathway, indicating a large pore diameter. In contrast, dextran-70 (MW approximately 70 000) did not pass through the gamma-activated channel, indicating an upper limit to the pore size of approximately 50 A (5 nm). 5. Similar conductances that are permeable to large molecules were activated by extreme hyperpolarization (> -150 mV) of uninjected oocytes. 6. We conclude that the Na(+),K(+)-ATPase gamma subunits activate Ca(2+)- and voltage-gated, non-selective, large diameter pores that are intrinsically present within the oocyte membrane.

Animals↗
PubMed2001

Dynamic sensitivity of ATP-sensitive K(+) channels to ATP.

ATP and MgADP regulate K(ATP) channel activity and hence potentially couple cellular metabolism to membrane electrical activity in various cell types. Using recombinant K(ATP) channels that lack sensitivity to MgADP, expressed in COSm6 cells, we demonstrate that similar on-cell activity can be observed with widely varying apparent submembrane [ATP] ([ATP](sub)). Metabolic inhibition leads to a biphasic change in the channel activity; activity first increases, presumably in response to a fast decrease in [ATP](sub), and then declines. The secondary decrease in channel activity reflects a marked increase in ATP sensitivity and is correlated with a fall in polyphosphoinositides (PPIs), including phosphatidylinositol 4,5-bisphosphate, probed using equilibrium labeling of cells with [(3)H]myo-inositol. Both ATP sensitivity and PPIs rapidly recover following removal of metabolic inhibition, and in both cases recovery is blocked by wortmannin. These data are consistent with metabolism having a dual effect on K(ATP) channel activity: rapid activation of channels because of relief of ATP inhibition and much slower reduction of channel activity mediated by a fall in PPIs. These two mechanisms constitute a feedback system that will tend to render K(ATP) channel activity transiently responsive to a change in [ATP](sub) over a wide range of steady state concentrations.

Adenosine Diphosphate↗