K(ATP) channel regulators: balanced diets include carbohydrates, proteins, and fats.
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Acyl Coenzyme A↗
Biomedical subjects
C G Nichols
Publications and source records attributed to C G Nichols.
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 K(ATP) channel were examined. 2-Aminoethyl methanethiosulfonate (MTSEA(+)) failed to modify Cd(2+)-insensitive control-Kir6.2 channels, but rapidly and irreversibly modified Kir6.2[L164C] (L164C) channels. Although a single Cd(2+) 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 Cd(2+)-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 Cd(2+) 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 A, but this widening is readily accomplished by minimal M2 helix motion and side-chain rearrangement.
Amino Acid Sequence↗
Inward rectifiers in the heart: an update on I(K1).
The cardiac inward rectifier potassium current (I(K1)), present in all ventricular and atrial myocytes, has been suggested to play a major role in repolarization of the action potential and stabilization of the resting potential. The molecular basis is now ascribed to members of the Kir2 sub-family of inward rectifier K channel genes, and the availability of recombinant expression systems has led to elucidation of the mechanism of inward rectification, as well as additional regulatory mechanisms involving intracellular pH and phosphorylation. In vivo manipulation of the genes encoding I(K1)and regulatory proteins now promise to provide new insights to the role of this conductance in the heart. This review details recent advances and considers the prospects for further elucidation of the role of this conductance in cardiac electrical activity.
Action Potentials↗
ATP interaction with the open state of the K(ATP) channel.
The mechanism of ATP-sensitive potassium (K(ATP)) channel closure by ATP is unclear, and various kinetic models in which ATP binds to open or to closed states have previously been presented. Effects of phosphatidylinositol bisphosphate (PIP2) and multiple Kir6.2 mutations on ATP inhibition and open probability in the absence of ATP are explainable in kinetic models where ATP stabilizes a closed state and interaction with an open state is not required. Evidence that ATP can in fact interact with the open state of the channel is presented here. The mutant Kir6.2[L164C] is very sensitive to Cd2+ block, but very insensitive to ATP, with no significant inhibition in 1 mM ATP. However, 1 mM ATP fully protects the channel from Cd2+ block. Allosteric kinetic models in which the channel can be in either open or closed states with or without ATP bound are considered. Such models predict a pedestal in the ATP inhibition, i.e., a maximal amount of inhibition at saturating ATP concentrations. This pedestal is predicted to occur at >50 mM ATP in the L164C mutant, but at >1 mM in the double mutant L164C/R176A. As predicted, ATP inhibits Kir6.2[L164C/R176A] to a maximum of approximately 40%, with a clear plateau beyond 2 mM. These results indicate that ATP acts as an allosteric ligand, interacting with both open and closed states of the channel.
Adenosine Triphosphate↗
Sarcolemmal K(ATP) channels in the heart: molecular mechanisms brought to light, but physiologic consequences still in the dark.
ATP-sensitive potassium (K(ATP)) channels are inhibited by intracellular ATP and thus couple the metabolic state of the cell to its electrical activity. Tremendous progress has been made in the identification of the molecular basis of K(ATP) channel function and regulation. The answer to one key question, however, has proven elusive: What are the precise conditions for, and functional consequences of, sarcolemmal K(ATP) activation in physiologic and pathophysiologic states? Here we consider recent studies of the molecular basis of cardiac K(ATP) channel activity and the role of these channels in cardiac function during ischemia.
Adenosine Triphosphate↗
A novel method for measurement of submembrane ATP concentration.
There has been considerable debate as to whether adenosine triphosphate (ATP) is compartmentalized within cells and, in particular, whether the ATP concentration directly beneath the plasma membrane, experienced by membrane proteins, is the same as that of the bulk cytoplasm. This issue has been difficult to address because there is no indicator of cytosolic ATP, such as those available for Ca(2+), capable of resolving the submembrane ATP concentration ([ATP](sm)) in real time within a single cell. We show here that mutant ATP-sensitive K(+) channels can be used to measure [ATP](sm) by comparing the increase in current amplitude on patch excision with the ATP dose-response curve. In Xenopus oocytes, [ATP](sm) was 4.6 +/- 0.3 mm (n = 29) under resting conditions, slightly higher than that measured for the bulk cytoplasm (2.3 mm). In mammalian (COSm6) cells, [ATP](sm) was slightly lower and averaged 1.4 +/- 0.1 mm (n = 66). Metabolic poisoning (10 min of 3 mm azide) produced a significant fall in [ATP](sm) in both types of cells: to 1.2 +/- 0.1 mm (n = 24) in oocytes and 0.8 +/- 0.11 mm for COSm6 cells. We conclude that [ATP](sm) lies in the low millimolar range and that there is no gradient between bulk cytosolic and submembrane [ATP].
Adenosine Triphosphate↗
Targeted overactivity of beta cell K(ATP) channels induces profound neonatal diabetes.
A paradigm for control of insulin secretion is that glucose metabolism elevates cytoplasmic [ATP]/[ADP] in beta cells, closing K(ATP) channels and causing depolarization, Ca2+ entry, and insulin release. Decreased responsiveness of K(ATP) channels to elevated [ATP]/[ADP] should therefore lead to decreased insulin secretion and diabetes. To test this critical prediction, we generated transgenic mice expressing beta cell K(ATP) channels with reduced ATP sensitivity. Animals develop severe hyperglycemia, hypoinsulinemia, and ketoacidosis within 2 days and typically die within 5. Nevertheless, islet morphology, insulin localization, and alpha and beta cell distributions were normal (before day 3), pointing to reduced insulin secretion as causal. The data indicate that normal K(ATP) channel activity is critical for maintenance of euglycemia and that overactivity can cause diabetes by inhibiting insulin secretion.
3-Hydroxybutyric Acid↗
Modulation of nucleotide sensitivity of ATP-sensitive potassium channels by phosphatidylinositol-4-phosphate 5-kinase.
ATP-sensitive potassium channels (K(ATP) channels) regulate cell excitability in response to metabolic changes. K(ATP) channels are formed as a complex of a sulfonylurea receptor (SURx), a member of the ATP-binding cassette protein family, and an inward rectifier K(+) channel subunit (Kir6.x). Membrane phospholipids, in particular phosphatidylinositol (PI) 4,5-bisphosphate (PIP(2)), activate K(ATP) channels and antagonize ATP inhibition of K(ATP) channels when applied to inside-out membrane patches. To examine the physiological relevance of this regulatory mechanism, we manipulated membrane PIP(2) levels by expressing either the wild-type or an inactive form of PI-4-phosphate 5-kinase (PIP5K) in COSm6 cells and examined the ATP sensitivity of coexpressed K(ATP) channels. Channels from cells expressing the wild-type PIP5K have a 6-fold lower ATP sensitivity (K(1/2), the half maximal inhibitory concentration, approximately 60 microM) than the sensitivities from control cells (K(1/2) approximately 10 microM). An inactive form of the PIP5K had little effect on the K(1/2) of wild-type channels but increased the ATP-sensitivity of a mutant K(ATP) channel that has an intrinsically lower ATP sensitivity (from K(1/2) approximately 450 microM to K(1/2) approximately 100 microM), suggesting a decrease in membrane PIP(2) levels as a consequence of a dominant-negative effect of the inactive PIP5K. These results show that PIP5K activity, which regulates PIP(2) and PI-3,4,5-P(3) levels, is a significant determinant of the physiological nucleotide sensitivity of K(ATP) channels.
Adenosine Triphosphate↗
Structure and dynamics of the pore of inwardly rectifying K(ATP) channels.
Inwardly rectifying K(+) currents are generated by a complex of four Kir (Kir1-6) subunits. Pore properties are conferred by the second transmembrane domain (M2) of each subunit. Using cadmium ions as a cysteine-interacting probe, we examined the accessibility of substituted cysteines in M2 of the Kir6.2 subunit of inwardly rectifying K(ATP) channels. The ability of Cd(2+) ions to inhibit channels was used as the estimate of accessibility. The distribution of Cd(2+) accessibility is consistent with an alpha-helical structure of M2. The apparent surface of reactivity is broad, and the most reactive residues correspond to the solvent-accessible residues in the bacterial KcsA channel crystal structure. In several mutants, single channel measurements indicated that inhibition occurred by a single transition from the open state to a zero-conductance state. Analysis of currents expressed from mixtures of control and L164C mutant subunits indicated that at least three cysteines are required for coordination of the Cd(2+) ion. Application of phosphatidylinositol 4,5-diphosphate to inside-out membrane patches stabilized the open state of all mutants and also reduced cadmium sensitivity. Moreover, the Cd(2+) sensitivity of several mutants was greatly reduced in the presence of inhibitory ATP concentrations. Taken together, these results are consistent with state-dependent accessibility of single Cd(2+) ions to coordination sites within a relatively narrow inner vestibule.
Adenosine Triphosphate↗
Modulation of potassium channels in the hearts of transgenic and mutant mice with altered polyamine biosynthesis.
Inward rectification of cardiac I(K1)channels was modulated by genetic manipulation of the naturally occurring polyamines. Ornithine decarboxylase (ODC) was overexpressed in mouse heart under control of the cardiac alpha -myosin heavy chain promoter (alpha MHC). In ODC transgenic hearts, putrescine and cadaverine levels were highly elevated ( identical with 35-fold for putrescine), spermidine was increased 3.6-fold, but spermine was essentially unchanged. I(K1)density was reduced by identical with 38%, although the voltage-dependence of rectification was essentially unchanged. Interestingly, the fast component of transient outward (I(to,f)) current was increased, but the total outward current amplitude was unchanged. I(K1)and I(to)currents were also studied in myocytes from mutant Gyro (Gy) mice in which the spermine synthase gene is disrupted, leading to a complete loss of spermine. I(K1)current densities were not altered in Gy myocytes, but the steepness of rectification was reduced indicating a role for spermine in controlling rectification. Intracellular dialysis of myocytes with putrescine, spermidine and spermine caused reduction, no change and increase of the steepness of rectification, respectively. Taken together with kinetic analysis of I(K1)activation these results are consistent with spermine being a major rectifying factor at potentials positive to E(K), spermidine dominating at potentials around and negative to E(K), and putrescine playing no significant role in rectification in the mouse heart.
Animals↗
The kinetic and physical basis of K(ATP) channel gating: toward a unified molecular understanding.
K(ATP) channels can be formed from Kir6.2 subunits with or without SUR1. The open-state stability of K(ATP) channels can be increased or reduced by mutations throughout the Kir6.2 subunit, and is increased by application of PIP(2) to the cytoplasmic membrane. Increase of open-state stability is manifested as an increase in the channel open probability in the absence of ATP (Po(zero)) and a correlated decrease in sensitivity to inhibition by ATP. Single channel lifetime analyses were performed on wild-type and I154C mutant channels expressed with, and without, SUR1. Channel kinetics include a single, invariant, open duration; an invariant, brief, closed duration; and longer closed events consisting of a "mixture of exponentials," which are prolonged in ATP and shortened after PIP(2) treatment. The steady-state and kinetic data cannot be accounted for by assuming that ATP binds to the channel and causes a gate to close. Rather, we show that they can be explained by models that assume the following regarding the gating behavior: 1) the channel undergoes ATP-insensitive transitions from the open state to a short closed state (C(f)) and to a longer-lived closed state (C(0)); 2) the C(0) state is destabilized in the presence of SUR1; and 3) ATP can access this C(0) state, stabilizing it and thereby inhibiting macroscopic currents. The effect of PIP(2) and mutations that stabilize the open state is then to shift the equilibrium of the "critical transition" from the open state to the ATP-accessible C(0) state toward the O state, reducing accessibility of the C(0) state, and hence reducing ATP sensitivity.
ATP-Binding Cassette Transporters↗
Structural determinants of PIP(2) regulation of inward rectifier K(ATP) channels.
Phosphatidylinositol 4,5-bisphosphate (PIP(2)) activates K(ATP) and other inward rectifier (Kir) channels. To determine residues important for PIP(2) regulation, we have systematically mutated each positive charge in the COOH terminus of Kir6.2 to alanine. The effects of these mutations on channel function were examined using (86)Rb efflux assays on intact cells and inside-out patch-clamp methods. Both methods identify essentially the same basic residues in two narrow regions (176-222 and 301-314) in the COOH terminus that are important for the maintenance of channel function and interaction with PIP(2). Only one residue (R201A) simultaneously affected ATP and PIP(2) sensitivity, which is consistent with the notion that these ligands, while functionally competitive, are unlikely to bind to identical sites. Strikingly, none of 13 basic residues in the terminal portion (residues 315-390) of the COOH terminus affected channel function when neutralized. The data help to define the structural requirements for PIP(2) sensitivity of K(ATP) channels. Moreover, the regions and residues defined in this study parallel those uncovered in recent studies of PIP(2) sensitivity in other inward rectifier channels, indicating a common structural basis for PIP(2) regulation.
Adenosine Triphosphate↗
ATP inhibition of KATP channels: control of nucleotide sensitivity by the N-terminal domain of the Kir6.2 subunit.
1. To gain insight into the role of the cytoplasmic regions of the Kir6.2 subunit in regulating channel activity, we have expressed the sulphonylurea receptor SUR1 with Kir6.2 subunits containing systematic truncations of the N- and C-termini. Up to 30 amino acids could be truncated from the N-terminus, and up to 36 amino acids from the C-terminus without loss of functional channels in co-expression with SUR1. Furthermore, Kir6.2DeltaC25 and Kir6. 2DeltaC36 subunits expressed functional channels in the absence of SUR1. 2. In co-expression with SUR1, N-terminal truncations increased Ki,ATP ([ATP] causing half-maximal inhibition of channel activity) by as much as 10-fold, accompanied by an increase in the ATP-insensitive open probability, whereas the C-terminal truncations did not affect the ATP sensitivity of co-expressed channels. 3. A mutation in the near C-terminal region, K185Q, reduced ATP sensitivity of co-expressed channels by approximately 30-fold, and on the Kir6.2DeltaN2-30 background, this mutation decreased ATP sensitivity of co-expressed channels by approximately 400-fold. 4. Each of these mutations also reduced the sensitivity to inhibition by ADP, AMP and adenosine tetraphosphate. 5. The results can be quantitatively explained by assuming that the N-terminal deletions stabilize the ATP-independent open state, whereas the Kir6.2K185Q mutation may alter the stability of ATP binding. These two effects are energetically additive, causing the large reduction of ATP sensitivity in the double mutant channels.
ATP-Binding Cassette Transporters↗
Expression of a functional Kir4 family inward rectifier K+ channel from a gene cloned from mouse liver.
1. A low stringency polymerase chain reaction (PCR) homology screening procedure was used to probe a mouse liver cDNA library to identify novel inward rectifier K+ channel genes. A single gene (mLV1) was identified that exhibited extensive sequence homology with previously cloned inward rectifier K+ channel genes. The mLV1 gene showed greatest sequence identity with genes belonging to the Kir4 subfamily. The amino acid sequence of mLV1 was 96 % identical to a Kir channel cloned from human kidney (hKir4.2), and approximately 60 % identical to the Kir4.1 channel cloned from human and rat, so that mLV1 was classified as mKir4.2. 2. Xenopus oocytes injected with cRNA encoding mKir4.2 displayed a large inwardly rectifying K+ current, while control oocytes injected with H2O displayed no similar K+ current. The current was blocked by Ba2+ and Cs+ in a voltage-dependent fashion and displayed inward rectification that was intermediate between that of the strong inward rectifier Kir2.1 and the weak inward rectifier Kir1.1. The current was weakly blocked by TEA in a voltage-independent fashion. 3. mKir4.2 current was subject to modulation by several distinct mechanisms. Intracellular acidification decreased mKir4.2 current in a reversible fashion, while activation of protein kinase C decreased mKir4.2 current in a manner that was not rapidly reversible. Incubation of oocytes in elevated [K+] produced a slowly developing enhancement of current. 4. Oocytes co-injected with cRNA for mKir4.2 and Kir5.1, a protein that does not form functional homomeric channels, displayed membrane currents with properties distinct from those expressing mKir4.2 alone. Co-injected oocytes displayed larger currents than mKir4.2, with novel kinetic properties and an increased sensitivity to Ba2+ block at negative potentials, suggesting that mKir4.2 forms functional heteromultimeric channels with Kir5.1, as has been shown for Kir4.1 5. These results demonstrate for the first time that a Kir4.2 channel gene product forms functional channels in Xenopus oocytes, that these Kir channels display novel properties, and that Kir4.2 subunits may be responsible for physiological modulation of functional Kir channels.
Amino Acid Sequence↗
Nucleotides and insulin secretion.
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Adenosine Triphosphate↗
Sulfonylurea and K(+)-channel opener sensitivity of K(ATP) channels. Functional coupling of Kir6.2 and SUR1 subunits.
The sensitivity of K(ATP) channels to high-affinity block by sulfonylureas and to stimulation by K(+) channel openers and MgADP (PCOs) is conferred by the regulatory sulfonylurea receptor (SUR) subunit, whereas ATP inhibits the channel through interaction with the inward rectifier (Kir6.2) subunit. Phosphatidylinositol 4, 5-bisphosphate (PIP(2)) profoundly antagonized ATP inhibition of K(ATP) channels expressed from cloned Kir6.2+SUR1 subunits, but also abolished high affinity tolbutamide sensitivity. By stabilizing the open state of the channel, PIP(2) drives the channel away from closed state(s) that are preferentially affected by high affinity tolbutamide binding, thereby producing an apparent loss of high affinity tolbutamide inhibition. Mutant K(ATP) channels (Kir6. 2[DeltaN30] or Kir6.2[L164A], coexpressed with SUR1) also displayed an "uncoupled" phenotype with no high affinity tolbutamide block and with intrinsically higher open state stability. Conversely, Kir6. 2[R176A]+SUR1 channels, which have an intrinsically lower open state stability, displayed a greater high affinity fraction of tolbutamide block. In addition to antagonizing high-affinity block by tolbutamide, PIP(2) also altered the stimulatory action of the PCOs, diazoxide and MgADP. With time after PIP(2) application, PCO stimulation first increased, and then subsequently decreased, probably reflecting a common pathway for activation of the channel by stimulatory PCOs and PIP(2). The net effect of increasing open state stability, either by PIP(2) or mutagenesis, is an apparent "uncoupling" of the Kir6.2 subunit from the regulatory input of SUR1, an action that can be partially reversed by screening negative charges on the membrane with poly-L-lysine.
ATP-Binding Cassette Transporters↗
Membrane phospholipid control of nucleotide sensitivity of KATP channels.
Adenosine triphosphate (ATP)-sensitive potassium (KATP) channels couple cell metabolism to electrical activity. Phosphatidylinositol phosphates (PIPs) profoundly antagonized ATP inhibition of KATP channels when applied to inside-out membrane patches. It is proposed that membrane-incorporated PIPs can bind to positive charges in the cytoplasmic region of the channel's Kir6.2 subunit, stabilizing the open state of the channel and antagonizing the inhibitory effect of ATP. The tremendous effect of PIPs on ATP sensitivity suggests that in vivo alterations of membrane PIP levels will have substantial effects on KATP channel activity and hence on the gain of metabolism-excitation coupling.
ATP-Binding Cassette Transporters↗
The gamma subunit of the Na,K-ATPase induces cation channel activity.
The gamma subunit of the Na,K-ATPase is a hydrophobic protein of approximately 10 kDa. The gamma subunit was expressed in Sf-9 insect cells and Xenopus oocytes to ascertain its role in Na,K-ATPase function. Immunoblotting has shown that the gamma subunit is expressed in Sf-9 cells infected with recombinant baculovirus containing the cDNA for the human gamma subunit. Confocal microscopy demonstrates that the gamma subunit can be delivered to the plasma membrane of Sf-9 cells independently of the other Na,K-ATPase subunits and that gamma colocalizes with alpha1 when these proteins are coexpressed. When Sf-9 cells were coinfected with alpha1 and gamma, antibodies to the gamma subunit were able to coimmunoprecipitate the alpha1 subunit, suggesting that gamma is able to associate with alpha1. The gamma subunit is a member of a family of single-pass transmembrane proteins that induces ion fluxes in Xenopus oocytes. Evidence that the gamma subunit is a functional component was supported by experiments showing gamma-induced cation channel activity when expressed in oocytes and increases in Na+ and K+ uptake when expressed in Sf-9 cells.
Amino Acid Sequence↗