Double-gating mechanism and diversity of an adenosine triphosphate (ATP)-sensitive K+ channel in neurons acutely dissociated from rat neocortex

Classically, ion channels are classified into 2 groups: chemical-sensitive (ligand-gated) and voltage sensitive channels. Single ATP-sensitive K⁺ (K-ATP) channel currents were recorded in acutely dissociated rat neocortical neurons using patch clamp technique. A type of K-ATP channel has been found to be gated not only by intracellular ATP, but also by membrane potential (V_m), and proved to be a novel mechanism underlying the gating of ion channels, namely bi-gating mechanism. The results also show that the K-ATP channels possess heterogeneity and diversity. These types of K-ATP channels have been identified in 40.12% of all patches, which are different in activation-threshold and voltage-sensitivity. The present experiment studied the type-3 K-ATP channel with a unitary conductance of about 80 pS in detail (n = 15). Taking account of all the available data, a variety of K-ATP channels are suggested to exist in body, and one type of them is bi-gated by both chemical substances and membrane potentials. This property of the K-ATP channels may be related to their pathophysiological function.     

Double-gating mechanism and diversity of an adenosine triphosphate (ATP)-sensitive K+ channel in neurons acutely dissociated from rat neocortex

Classically, ion channels are classified into 2 groups: chemical-sensitive (ligand-gated) and voltage sensitive channels. Single ATP-sensitive K⁺ (K-ATP) channel currents were recorded in acutely dissociated rat neocortical neurons using patch clamp technique. A type of K-ATP channel has been found to be gated not only by intracellular ATP, but also by membrane potential (V_m), and proved to be a novel mechanism underlying the gating of ion channels, namely bi-gating mechanism. The results also show that the K-ATP channels possess heterogeneity and diversity. These types of K-ATP channels have been identified in 40.12% of all patches, which are different in activation-threshold and voltage-sensitivity. The present experiment studied the type-3 K-ATP channel with a unitary conductance of about 80 pS in detail (n = 15). Taking account of all the available data, a variety of K-ATP channels are suggested to exist in body, and one type of them is bi-gated by both chemical substances and membrane potentials. This property of the K-ATP channels may be related to their pathophysiological function. Project supported by the National Natural Science Foundation of China and Natural Science Foundation of Guangdong Province     

Action potential duration and activation of ATP-sensitive potassium current in isolated guinea-pig ventricular myocytes

It is difficult to associate the ATP-sensitive potassium (K-ATP) channel of cardiac muscle with hypoxia/ischemia induced action potential shortening because this occurs before intracellular ATP falls to levels associated in vitro with channel opening. This leaves the cardiac K-ATP channel without any obvious physiological function. We have quantitatively examined the relationship between action potential duration and K-ATP channel activity in enzymatically isolated ventricular myocytes of the guinea-pig. In whole-cell voltage-clamp recording experiments when the K-ATP channel opener SR 44866 (2-10 microM) stimulated an outward membrane current greater than 50 pA at 0 mV membrane potential (the equivalent of 30 open K-ATP channels or 1% of the cell K-ATP channel population) action potential duration was reduced by more than 50%. In the majority of cell-attached membrane patch recordings metabolic inhibition stimulated K-ATP channel open probability of 1-2% which continued for long periods (7-25 min) before cell contracture and coincident major K-ATP channel activation (open probability 65%). Our quantitative analysis thus shows that physiologically relevant activity of K-ATP channels in cardiac muscle is confined to a very small percentage of the possible cell K-ATP current and thus intracellular ATP would not have to fall very far before the opening of K-ATP channels would influence cardiac excitability.     

Molecular physiology of neuronal K-ATP channels (review).

ATP sensitive potassium (K-ATP) channels are widely expressed in many cell types including neurons. K-ATP channels are heteromeric membrane proteins that consist of two very different subunits: the pore-forming, two-transmembrane spanning potassium channel subunit (Kir6) and the regulatory, 17 transmembrane spanning sulphonylurea receptor (SUR). This ensemble--joined together in a 4:4 stoichiometry--endows this channel with a unique combination of functional properties. The open probability of K-ATP channels directly depends on the intracellular ATP/ADP levels allowing the channels to directly couple the metabolic state of a cell to its electrical activity. Here, recent progress on the molecular composition and functional diversity of neuronal K-ATP channels is reviewed. One is particular concerned with single-cell mRNA expression studies that give insight to the coexpression patterns of Kir6 and SUR isoforms in identified neurons. In addition, the physiological roles of neuronal K-ATP channels in glucose sensing and adapting neuronal activity to metabolic demands are discussed, as well as their emerging pathophysiological functions in acute brain ischemia and chronic neurodegenerative diseases.     

Alternative sulfonylurea receptor expression defines metabolic sensitivity of K-ATP channels in dopaminergic midbrain neurons

ATP-sensitive potassium (K-ATP) channels couple the metabolic state to cellular excitability in various tissues. Several isoforms of the K-ATP channel subunits, the sulfonylurea receptor (SUR) and inwardly rectifying K channel (Kir6.X), have been cloned, but the molecular composition and functional diversity of native neuronal K-ATP channels remain unresolved. We combined functional analysis of K-ATP channels with expression profiling of K-ATP subunits at the level of single substantia nigra (SN) neurons in mouse brain slices using an RT-multiplex PCR protocol. In contrast to GABAergic neurons, single dopaminergic SN neurons displayed alternative co-expression of either SUR1, SUR2B or both SUR isoforms with Kir6.2. Dopaminergic SN neurons expressed alternative K-ATP channel species distinguished by significant differences in sulfonylurea affinity and metabolic sensitivity. In single dopaminergic SN neurons, co-expression of SUR1 + Kir6.2, but not of SUR2B + Kir6.2, correlated with functional K-ATP channels highly sensitive to metabolic inhibition. In contrast to wild-type, surviving dopaminergic SN neurons of homozygous weaver mouse exclusively expressed SUR1 + Kir6.2 during the active period of dopaminergic neurodegeneration. Therefore, alternative expression of K-ATP channel subunits defines the differential response to metabolic stress and constitutes a novel candidate mechanism for the differential vulnerability of dopaminergic neurons in response to respiratory chain dysfunction in Parkinson's disease.     

Book reviews

ATP sensitive potassium (K-ATP) channels are widely expressed in many cell types including neurons. K-ATP channels are heteromeric membrane proteins that consist of two very different subunits: the pore-forming, two-transmembrane spanning potassium channel subunit (Kir6) and the regulatory, 17 transmembrane spanning sulphonylurea receptor (SUR). This ensemble―joined together in a 4:4 stoichiometry―endows this channel with a unique combination of functional properties. The open probability of K-ATP channels directly depends on the intracellular ATP/ADP levels allowing the channels to directly couple the metabolic state of a cell to its electrical activity. Here, recent progress on the molecular composition and functional diversity of neuronal K-ATP channels is reviewed. One is particular concerned with single-cell mRNA expression studies that give insight to the coexpression patterns of Kir6 and SUR isoforms in identified neurons. In addition, the physiological roles of neuronal K-ATP channels in glucose sensing and adapting neuronal activity to metabolic demands are discussed, as well as their emerging pathophysiological functions in acute brain ischemia and chronic neurodegenerative diseases.     

Hypothalamic ependymal-glial cells express the glucose transporter GLUT2, a protein involved in glucose sensing

The GLUT2 glucose transporter and the K-ATP-sensitive potassium channels have been implicated as an integral part of the glucose-sensing mechanism in the pancreatic islet beta cells. The expression of GLUT2 and K-ATP channels in the hypothalamic region suggest that they are also involved in a sensing mechanism in this area. The hypothalamic glial cells, known as tanycytes alpha and beta, are specialized ependymal cells that bridge the cerebrospinal fluid and the portal blood of the median eminence. We used immunocytochemistry, in situ hybridization and transport analyses to demonstrate the glucose transporters expressed in tanycytes. Confocal microscopy using specific antibodies against GLUT1 and GLUT2 indicated that both transporters are expressed in alpha and beta tanycytes. In addition, primary cultures of mouse hypothalamic tanycytes were found to express both GLUT1 and GLUT2 transporters. Transport studies, including 2-deoxy-glucose and fructose uptake in the presence or absence of inhibitors, indicated that these transporters are functional in cultured tanycytes. Finally, our analyses indicated that tanycytes express the K-ATP channel subunit Kir6.1 in vitro. As the expression of GLUT2 and K-ATP channel is linked to glucose-sensing mechanisms in pancreatic beta cells, we postulate that tanycytes may be responsible, at least in part, for a mechanism that allows the hypothalamus to detect changes in glucose concentrations.     

Phosphorylation of Ion channels

The introduction of highly specific reagents such as enzymes and inhibitors directly into living cells has proven to be a powerful tool in studying the modulation of cellular activity by protein phosphorylation. The use of exogenous kinases can be thought of as a pharmacological approach: this demonstrates that phosphorylation can produce modulation, but does not address the question of whether the cell actually uses this mechanism under normal physiological conditions. The complementary approach, the introduction of highly specific inhibitors such as R subunit or PKI, does ask whether endogenous kinase activity is necessary for a given physiological response. Together these two approaches have provided rather compelling evidence that cAMP-dependent and calcium/phospholipid-dependent protein phosphorylations can regulate membrane excitability. In several cases single-channel analysis has allowed the demonstration that an ion channel itself or something very close to the channel is the phosphorylation target, and it seems reasonable to assume that this will also be the case for many if not all of the other systems described above. Have any general principles emerged from the results to date? Certainly it seems clear that protein phosphorylation regulates not one but many classes of ion channels. As summarized in the Table, different channels can be modulated in different cells, some channels are activated while others are inhibited, and in some cells more than one channel is subject to modulation by phosphorylation. The list in the Table is probably not yet complete, and indeed it is not inconceivable that all ion channels can under appropriate conditions be regulated by phosphorylation. What aspect of channel function is altered by phosphorylation? The total membrane current, I, carried by a particular species of ion channel is given by Npi, where N is the number of active channels in the membrane, p is the probability that an individual channel will be open, and i is the single-channel current. In principle a change in I, the quantity measured in whole cell experiments, could be caused by a change in any one (or more) of the parameters, N, p or i (see Fig. 1). In the two cases in which single-channel measurements have allowed this question to be investigated, changes in N (Shuster et al., 1985) and p (Ewald et al., 1985) have been observed. Here again it seems unlikely that any one mechanism operates in all cases, and it would not be surprising to find that phosphorylation of some other channel results in a change in i.(ABSTRACT TRUNCATED AT 400 WORDS)     

Channel regulation by extracellular redox protein

The most widely studied stimuli for ion channel activation are changes in membrane voltage and binding of a chemical ligand in a pocket of the channel protein. While modulation by redox potential has also been appreciated our study shows previously unrecognised channel activation via electron donation from the extracellular redox protein thioredoxin (TRX).(1) The ion channel type involved is a member of the Transient Receptor Potential (TRP) family. Activation by TRX led us to consider the relevance of TRP channels to the inflammatory condition of rheumatoid arthritis, where functions of ion channels are relatively unknown and TRX concentrations are high. TRP channel activation was found to be inhibitory for secretion of matrix metalloproteinases, suggesting activation by TRX may have a protective role against disease. Here we expand on our original article and discuss the potential wider implications of the findings in terms of concepts for channel activation and relevance to other ion channel types and systems.     

Signal-transduction pathways that regulate smooth muscle function. II. Receptor-ion channel coupling mechanisms in gastrointestinal smooth muscle

Regulation of membrane ion channels by second messengers is an important mechanism by which gastrointestinal smooth muscle excitability is controlled. Receptor-mediated phosphorylation of Ca(2+) channels has been known for some time; however, recent findings indicate that these channels may also modulate intracellular signaling. The plasmalemma ion channels may also function as a point of convergence between different receptor types. In this review, the molecular mechanisms that link channel function and signal transduction are discussed. Emerging evidence also indicates altered second-messenger modulation of the Ca(2+) channel in the pathophysiology of smooth muscle dysmotility.     

Cytosolic adenylate kinases regulate K-ATP channel activity in human beta-cells

The role of adenylate kinase (AK) as a determinant of K-ATP channel activity in human pancreatic β-cells was investigated. We have identified that two cytosolic isoforms of AK, AK1 and AK5 are expressed in human islets and INS-1 cells. Elevated concentrations of glucose inhibit AK1 expression and AK1 immunoprecipitates with the Kir6.2 subunit of K-ATP. AK activation by ATP + AMP stimulates K-ATP channel activity and this stimulation is abolished by AK inhibitors. We propose that glucose stimulation of β-cells inhibits AK through glycolysis and also through the elevation of diadenosine polyphosphate levels. Glucose-dependent inhibition of AK increases the ATP/ADP ratio in the microenvironment of the K-ATP channel promoting channel closure and insulin secretion. The down-regulation of AK1 expression by hyperglycemia may contribute to the defective coupling of glucose metabolism to K-ATP channel activity in type 2 diabetes.     

Genistein can modulate channel function by a phosphorylation-independent mechanism: importance of hydrophobic mismatch and bilayer mechanics

Genistein, a generic tyrosine kinase inhibitor, has been used extensively as a tool to investigate the possible regulation of membrane function by tyrosine phosphorylation. Genistein, in micromolar concentrations, alters the function of numerous ion channels and other membrane proteins, but only in few cases has it been demonstrated that the changes in membrane protein (ion channel) function are due to changes in a protein's phosphorylation status. The major common denominator characterizing proteins that are modulated by genistein seems to be that they are imbedded into, and span, the bilayer component of the plasma membrane. We therefore explored whether genistein could alter ion channel function by a bilayer-mediated mechanism and examined genistein's effect on gramicidin A (gA) channels in planar phospholipid bilayers. gA channels form by transmembrane dimerization of two nonconducting subunits, and genistein potentiates gA channel activity by increasing the appearance rate and prolonging the lifetime of bilayer-spanning gA dimers. That is, genistein shifts the equilibrium between nonconducting monomers and conducting dimers in favor of the bilayer-spanning dimers; the changes in channel activity therefore cannot be due to changes in bilayer fluidity. To obtain further insights into the mechanism underlying this modulation of gA channel function, we examined the effects of genistein on channels formed by gA analogues that differ in amino acid sequence. For a given channel length, the effects of genistein on gA dimerization do not depend on the specific sequence, or the chirality, of the channel-forming gA analogues. In contrast, when we change the channel length (by decreasing or increasing the number of amino acid residues in the sequence), or the bilayer thickness (by changing methylene groups in the acyl chains), the magnitude of genistein's effect increases with increasing hydrophobic mismatch between the channel length and the bilayer thickness. These results strongly suggest that genistein alters bilayer mechanical properties, which in turn modulates channel function. This bilayer-mediated mechanism is likely to apply to other pharmacological reagents and membrane proteins.     

Zipper transition in an alpha-helix: a mechanism for gating of voltage-sensitive ion channels in a biological membrane

It is suggested that the gating currents which control the ion channels in a biological membrane are comprised of positive charges crossing the membrane along chains of hydrogen bonds. These chains are the sets of hydrogen bonds which hold alpha-helical protein segments in their rigid conformations. The passage of a positive charge in one direction along such a chain will convert hydrogen bonds from the usual rigid N--C = O...H--N form to a flaccid N = C--O--H...N form. This "zipper" transition can be reversed by the passage of the positive charge along the return route. A flaccid protein rod can clog an ion channel and thereby close it. When all of the protein rods framing an ion channel are in the rigid conformation, the channel is open. This mechanism is used to explain some of the observed characteristics of calcium ion channels and sodium ion channels.     

New insights concerning the glucose-dependent insulin secretagogue action of glucagon-like peptide-1 in pancreatic beta-cells.

The GLP-1 receptor is a Class B heptahelical G-protein-coupled receptor that stimulates cAMP production in pancreatic beta-cells. GLP-1 utilizes this receptor to activate two distinct classes of cAMP-binding proteins: protein kinase A (PKA) and the Epac family of cAMP-regulated guanine nucleotide exchange factors (cAMPGEFs). Actions of GLP-1 mediated by PKA and Epac include the recruitment and priming of secretory granules, thereby increasing the number of granules available for Ca(2+)-dependent exocytosis. Simultaneously, GLP-1 promotes Ca(2+) influx and mobilizes an intracellular source of Ca(2+). GLP-1 sensitizes intracellular Ca(2+) release channels (ryanodine and IP (3) receptors) to stimulatory effects of Ca(2+), thereby promoting Ca(2+)-induced Ca(2+) release (CICR). In the model presented here, CICR activates mitochondrial dehydrogenases, thereby upregulating glucose-dependent production of ATP. The resultant increase in cytosolic [ATP]/[ADP] concentration ratio leads to closure of ATP-sensitive K(+) channels (K-ATP), membrane depolarization, and influx of Ca(2+) through voltage-dependent Ca(2+) channels (VDCCs). Ca(2+) influx stimulates exocytosis of secretory granules by promoting their fusion with the plasma membrane. Under conditions where Ca(2+) release channels are sensitized by GLP-1, Ca(2+) influx also stimulates CICR, generating an additional round of ATP production and K-ATP channel closure. In the absence of glucose, no "fuel" is available to support ATP production, and GLP-1 fails to stimulate insulin secretion. This new "feed-forward" hypothesis of beta-cell stimulus-secretion coupling may provide a mechanistic explanation as to how GLP-1 exerts a beneficial blood glucose-lowering effect in type 2 diabetic subjects.     

Channel Regulation by Extracellular Redox Protein

The most widely studied stimuli for ion channel activation are changes in membrane voltage and binding of a chemical ligand in a pocket of the channel protein. While modulation by redox potential has also been appreciated our study shows previously unrecognised channel activation via electron donation from the extracellular redox protein thioredoxin (TRX)1. The ion channel type involved is a member of the Transient Receptor Potential (TRP) family. Activation by TRX led us to consider the relevance of TRP channels to the inflammatory condition of rheumatoid arthritis, where functions of ion channels are relatively unknown and TRX concentrations are high. TRP channel activation was found to be inhibitory for secretion of matrix metalloproteinases, suggesting activation by TRX may have a protective role against disease. Here we expand on our original article and discuss the potential wider implications of the findings in terms of concepts for channel activation and relevance to other ion channel types and systems.     

Recent advances in the characterization of epithelial ionic channels

Physiologists have long recognized the importance of channels in the functioning of neurons and excitable membranes. This brief review has been an attempt to illustrate how channel properties are also essential to an understanding of epithelial transport physiology. Among their more important functions, channels influence membrane potentials and serve as conduits for ion movements. As the need to understand the molecular basis for ion transport continues to develop, it is crucial to be able to distinguish between different channel properties. For example, apparent voltage-dependent properties can arise because of a voltage-dependent gating process, or alternatively, because of a rectification of channel conductance. Voltage-dependent effects can also be only indirect, mediated by changes in cell volume, intracellular ion levels, the levels of secondary intracellular messengers such as Ca2+ (perhaps through voltage-dependent membrane Ca2+ channels), or possibly even by morphological changes. An important area for future research is to differentiate mechanisms which modulate the activity of open channels. For example, a decrease in channel number, a reduction in open-channel conductance or a decline in the probability of channel opening can all underlie changes in macroscopic permeability. The factors which mediate hormonal activation of epithelial channels particularly need to be understood. Specifically, the mechanisms of aldosterone and anti-diuretic hormone activation of apical membrane Na+ channels need to be identified. In conclusion, we are witnessing a new era in epithelial electrophysiology which promises to resolve many issues concerning the cellular regulation of ion transport and open new, unanticipated avenues of inquiry.     

PKC regulation of ion channels: The involvement of PIP2

Ion channels are integral membrane proteins whose gating has been increasingly shown to depend on the presence of the low-abundance membrane phospholipid, phosphatidylinositol (4,5) bisphosphate. The expression and function of ion channels is tightly regulated via protein phosphorylation by specific kinases, including various PKC isoforms. Several channels have further been shown to be regulated by PKC through altered surface expression, probability of channel opening, shifts in voltage dependence of their activation, or changes in inactivation or desensitization. In this review, we survey the impact of phosphorylation of various ion channels by PKC isoforms and examine the dependence of phosphorylated ion channels on phosphatidylinositol (4,5) bisphosphate as a mechanistic endpoint to control channel gating.     

Cystic fibrosis affects chloride and sodium channels in human airway epithelia

Abnormalities of epithelial function in cystic fibrosis (CF) have been linked to defects in cell membrane permeability to chloride or sodium ions. Recently, a class of chloride channels in airway epithelial cells have been reported to lack their usual sensitivity to phosphorylation via cAMP-dependent protein kinase, suggesting that CF could be due to a single genetic defect in these channels. We have examined single chloride and sodium channels in control and CF human nasal epithelia using the patch-clamp technique. The most common chloride channel was not the one previously associated with CF, but it was also abnormal in CF cells. In addition, the number of sodium channels was unusually high in CF. These findings suggest a wider disturbance of ion channel properties in CF than would be produced by a defect in a single type of channel.     

Single-channel recordings from cultured human retinal pigment epithelial cells

We have applied patch-clamp techniques to on-cell and excised-membrane patches from human retinal pigment epithelial cells in tissue culture. Single-channel currents from at least four ion channel types were observed: three or more potassium-selective channels with single-channel slope conductances near 100, 45, and 25 pS as measured in on-cell patches with physiological saline in the pipette, and a relatively nonselective channel with subconductance states, which has a main-state conductance of approximately 300 pS at physiological ion concentrations. The permeability ratios, PK/PNa, measured in excised patches were 21 for the 100-pS channels, 3 for the 25-pS channels, and 0.8 for the 300-pS nonselective channel. The 45-pS channels appeared to be of at least two types, with PK/PNa's of approximately 41 for one type and 3 for the other. The potassium-selective channels were spontaneously active at all potentials examined. The average open time for these channels ranged from a few milliseconds to many tens of milliseconds. No consistent trend relating potassium-selective channel kinetics to membrane potential was apparent, which suggests that channel activity was not regulated by the membrane potential. In contrast to the potassium-selective channels, the activity of the nonselective channel was voltage dependent: the open probability of this channel declined to low values at large positive or negative membrane potentials and was maximal near zero. Single-channel conductances observed at several symmetrical KCl concentrations have been fitted with Michaelis-Menten curves in order to estimate maximum channel conductances and ion-binding constants for the different channel types. The channels we have recorded are probably responsible for the previously observed potassium permeability of the retinal pigment epithelium apical membrane.     

Interactions of n-3 fatty acids with ion channels in excitable tissues

In summary, we have shown that the conventional explanation for the site of action of a ligand which alters the conductance of a membrane ion channel is that the ligand interacts or binds with the ion channel protein, changing its conductance, is inadequate to explain the primary site of action of the antiarrhythmic n-3 PUFAs. We have shown that when a neutral asparagine is replaced by a positively charged lysine in the N406 amino acid site in the alpha-subunit of the human cardiac sodium channel, the n-3 fatty acids lose their inhibitory action on the sodium current. The inadequacy of this finding to explain the primary site of action of the n-3 PUFAs is demonstrated by the inhibitory effect on all other cardiac ion channels, so far tested. We show that ion channels, which share no amino acid homology with the PUFAs, have their conductance also reduced in the presence of the PUFAs, Thus a more general conceptual framework or paradigm is needed to account for the broad action of the PUFAs on diverse different ion channels lacking amino acid homology. We have been testing the membrane tension hypothesis of Andersen and associates. According to this hypothesis, the fatty acids are not acting directly on the ion channel protein but accumulating in the phospholipid membrane in immediate juxtaposition to the site in the membrane where the ion channel protein penetrates the membrane phospholipid bilayer. This alters membrane tensions exerted by the phospholipid membrane on the ion channel, which in turn causes conformational changes in the ion channel, altering the conductance of the ion channel. Our preliminary data seem to support this membrane tension hypothesis.