scholarly journals K Channel Subconductance Levels Result from Heteromeric Pore Conformations

2005 ◽  
Vol 126 (2) ◽  
pp. 87-103 ◽  
Author(s):  
Mark L. Chapman ◽  
Antonius M.J. VanDongen
Keyword(s):  
Single Channel ◽  
K Channel ◽  
K Channels ◽  
Multiple Sensors ◽  
Channel Opening ◽  
Single Channel Analysis ◽  
Binary Character ◽  
A Subunit ◽  
Subconductance States ◽  
Voltage Sensing Domain

Voltage-gated K channels assemble from four identical subunits symmetrically arranged around a central permeation pathway. Each subunit harbors a voltage-sensing domain. The sigmoidal nature of the activation kinetics suggests that multiple sensors need to undergo a conformational change before the channel can open. Following activation, individual K channels alternate stochastically between two main permeation states, open and closed. This binary character of single channel behavior suggests the presence of a structure in the permeation pathway that can exist in only two conformations. However, single channel analysis of drk1 (Kv2.1) K channels demonstrated the existence of four additional, intermediate conductance levels. These short-lived subconductance levels are visited when the channel gate moves between the closed and fully open state. We have proposed that these sublevels arise from transient heteromeric pore conformations, in which some, but not all, subunits are in the “open” state. A minimal model based on this hypothesis relates specific subconductance states with the number of activated subunits (Chapman et al., 1997). To stringently test this hypothesis, we constructed a tandem dimer that links two K channel subunits with different activation thresholds. Activation of this dimer by strong depolarizations resulted in the characteristic binary open–close behavior. However, depolarizations to membrane potentials in between the activation thresholds of the two parents elicited highly unusual single channel gating, displaying frequent visits to two subconductance levels. The voltage dependence and kinetics of the small and large sublevels associate them with the activation of one and two subunits, respectively. The data therefore support the hypothesis that subconductance levels result from heteromeric pore conformations. In this model, both sensor movement and channel opening have a subunit basis and these processes are allosterically coupled.

scholarly journals A Gastropod Toxin Selectively Slows Early Transitions in the Shaker K Channel's Activation Pathway

2004 ◽  
Vol 123 (6) ◽  
pp. 685-696 ◽  
Author(s):  
Jon T. Sack ◽  
Richard W. Aldrich ◽  
William F. Gilly
Keyword(s):  
Single Channel ◽  
K Channel ◽  
Channel Activity ◽  
Gating Currents ◽  
K Channels ◽  
Voltage Sensors ◽  
Essentially Normal ◽  
Channel Opening ◽  
Single Channel Activity ◽  
Kinetics Of

A toxin from a marine gastropod's defensive mucus, a disulfide-linked dimer of 6-bromo-2-mercaptotryptamine (BrMT), was found to inhibit voltage-gated potassium channels by a novel mechanism. Voltage-clamp experiments with Shaker K channels reveal that externally applied BrMT slows channel opening but not closing. BrMT slows K channel activation in a graded fashion: channels activate progressively slower as the concentration of BrMT is increased. Analysis of single-channel activity indicates that once a channel opens, the unitary conductance and bursting behavior are essentially normal in BrMT. Paralleling its effects against channel opening, BrMT greatly slows the kinetics of ON, but not OFF, gating currents. BrMT was found to slow early activation transitions but not the final opening transition of the Shaker ILT mutant, and can be used to pharmacologically distinguish early from late gating steps. This novel toxin thus inhibits activation of Shaker K channels by specifically slowing early movement of their voltage sensors, thereby hindering channel opening. A model of BrMT action is developed that suggests BrMT rapidly binds to and stabilizes resting channel conformations.

scholarly journals Trapping of a Methanesulfonanilide by Closure of the Herg Potassium Channel Activation Gate

2000 ◽  
Vol 115 (3) ◽  
pp. 229-240 ◽  
Author(s):  
John S. Mitcheson ◽  
Jun Chen ◽  
Michael C. Sanguinetti
Keyword(s):  
Single Channel ◽  
K Channel ◽  
Membrane Hyperpolarization ◽  
K Channels ◽  
Large Size ◽  
Channel Opening ◽  
Activated State ◽  
Activation Gate ◽  
Herg Channels ◽  
Positively Charged

Deactivation of voltage-gated potassium (K+) channels can slow or prevent the recovery from block by charged organic compounds, a phenomenon attributed to trapping of the compound within the inner vestibule by closure of the activation gate. Unbinding and exit from the channel vestibule of a positively charged organic compound should be favored by membrane hyperpolarization if not impeded by the closed gate. MK-499, a methanesulfonanilide compound, is a potent blocker (IC50 = 32 nM) of HERG K+ channels. This bulky compound (7 × 20 Å) is positively charged at physiological pH. Recovery from block of HERG channels by MK-499 and other methanesulfonanilides is extremely slow (Carmeliet 1992; Ficker et al. 1998), suggesting a trapping mechanism. We used a mutant HERG (D540K) channel expressed in Xenopus oocytes to test the trapping hypothesis. D540K HERG has the unusual property of opening in response to hyperpolarization, in addition to relatively normal gating and channel opening in response to depolarization (Sanguinetti and Xu 1999). The hyperpolarization-activated state of HERG was characterized by long bursts of single channel reopening. Channel reopening allowed recovery from block by 2 μM MK-499 to occur with time constants of 10.5 and 52.7 s at −160 mV. In contrast, wild-type HERG channels opened only briefly after membrane hyperpolarization, and thus did not permit recovery from block by MK-499. These findings provide direct evidence that the mechanism of slow recovery from HERG channel block by methanesulfonanilides is due to trapping of the compound in the inner vestibule by closure of the activation gate. The ability of HERG channels to trap MK-499, despite its large size, suggests that the vestibule of this channel is larger than the well studied Shaker K+ channel.

scholarly journals Membrane-delimited Inhibition of Maxi-K Channel Activity by the Intermediate Conductance Ca2+-activated K Channel

2006 ◽  
Vol 127 (2) ◽  
pp. 159-169 ◽  
Author(s):  
Jill Thompson ◽  
Ted Begenisich
Keyword(s):  
Single Channel ◽  
Expression System ◽  
Chemical Activation ◽  
K Channel ◽  
Channel Activity ◽  
Single Family ◽  
Open Probability ◽  
K Channels ◽  
Channel Proteins ◽  
K Activity

The complexity of mammalian physiology requires a diverse array of ion channel proteins. This diversity extends even to a single family of channels. For example, the family of Ca2+-activated K channels contains three structural subfamilies characterized by small, intermediate, and large single channel conductances. Many cells and tissues, including neurons, vascular smooth muscle, endothelial cells, macrophages, and salivary glands express more than a single class of these channels, raising questions about their specific physiological roles. We demonstrate here a novel interaction between two types of Ca2+-activated K channels: maxi-K channels, encoded by the KCa1.1 gene, and IK1 channels (KCa3.1). In both native parotid acinar cells and in a heterologous expression system, activation of IK1 channels inhibits maxi-K activity. This interaction was independent of the mode of activation of the IK1 channels: direct application of Ca2+, muscarinic receptor stimulation, or by direct chemical activation of the IK1 channels. The IK1-induced inhibition of maxi-K activity occurred in small, cell-free membrane patches and was due to a reduction in the maxi-K channel open probability and not to a change in the single channel current level. These data suggest that IK1 channels inhibit maxi-K channel activity via a direct, membrane-delimited interaction between the channel proteins. A quantitative analysis indicates that each maxi-K channel may be surrounded by four IK1 channels and will be inhibited if any one of these IK1 channels opens. This novel, regulated inhibition of maxi-K channels by activation of IK1 adds to the complexity of the properties of these Ca2+-activated K channels and likely contributes to the diversity of their functional roles.

Basolateral potassium channels in renal proximal tubule

1987 ◽  
Vol 253 (3) ◽  
pp. F476-F487 ◽  
Author(s):  
H. Sackin ◽  
L. G. Palmer
Keyword(s):  
Single Channel ◽  
Basolateral Membrane ◽  
K Channel ◽  
Channel Conductance ◽  
Single Channel Conductance ◽  
K Channels ◽  
Open Time ◽  
Excised Patches ◽  
The Mean ◽  
Inside Out

Potassium (K+) channels in the basolateral membrane of unperfused Necturus proximal tubules were studied in both cell-attached and excised patches, after removal of the tubule basement membrane by manual dissection without collagenase. Two different K+ channels were identified on the basis of their kinetics: a short open-time K+ channel, with a mean open time less than 1 ms, and a long open-time K+ channel with a mean open time greater than 20 ms. The short open-time channel occurred more frequently than the longer channel, especially in excised patches. For inside-out excised patches with Cl- replaced by gluconate, the current-voltage relation of the short open-time K+ channel was linear over +/- 60 mV, with a K+-Na+ selectivity of 12 +/- 2 (n = 12), as calculated from the reversal potential with oppositely directed Na+ and K+ gradients. With K-Ringer in the patch pipette and Na-Ringer in the bath, the conductance of the short open-time channel was 47 +/- 2 pS (n = 15) for cell-attached patches, 26 +/- 2 pS (n = 15) for patches excised (inside out) into Na-Ringer, and 36 +/- 6 pS (n = 3) for excised patches with K-Ringer on both sides. These different conductances can be partially explained by a dependence of single-channel conductance on the K+ concentration on the interior side of the membrane. In experiments with a constant K+ gradient across excised patches, large changes in Na+ at the interior side of the membrane produced no change in single-channel conductance, arguing against a direct block of the K+ channel by Na+. Finally, the activity of the short open-time channel was voltage gated, where the mean number of open channels decreased as a linear function of basolateral membrane depolarization for potentials between -60 and 0 mV. Depolarization from -60 to -40 mV decreased the mean number of open K+ channels by 28 +/- 8% (n = 6).

scholarly journals Potassium channel types in arterial chemoreceptor cells and their selective modulation by oxygen.

1992 ◽  
Vol 100 (3) ◽  
pp. 401-426 ◽  
Author(s):  
M D Ganfornina ◽  
J López-Barneo
Keyword(s):  
Time Course ◽  
Single Channel ◽  
K Channel ◽  
Open Probability ◽  
K Channels ◽  
Glomus Cells ◽  
Control Value ◽  
Voltage Dependent ◽  
K Current ◽  
Chemoreceptor Cells

Single K+ channel currents were recorded in excised membrane patches from dispersed chemoreceptor cells of the rabbit carotid body under conditions that abolish current flow through Na+ and Ca2+ channels. We have found three classes of voltage-gated K+ channels that differ in their single-channel conductance (gamma), dependence on internal Ca2+ (Ca2+i), and sensitivity to changes in O2 tension (PO2). Ca(2+)-activated K+ channels (KCa channels) with gamma approximately 210 pS in symmetrical K+ solutions were observed when [Ca2+]i was greater than 0.1 microM. Small conductance channels with gamma = 16 pS were not affected by [Ca2+]i and they exhibited slow activation and inactivation time courses. In these two channel types open probability (P(open)) was unaffected when exposed to normoxic (PO2 = 140 mmHg) or hypoxic (PO2 approximately 5-10 mmHg) external solutions. A third channel type (referred to as KO2 channel), having an intermediate gamma(approximately 40 pS), was the most frequently recorded. KO2 channels are steeply voltage dependent and not affected by [Ca2+]i, they inactivate almost completely in less than 500 ms, and their P(open) reversibly decreases upon exposure to low PO2. The effect of low PO2 is voltage dependent, being more pronounced at moderately depolarized voltages. At 0 mV, for example, P(open) diminishes to approximately 40% of the control value. The time course of ensemble current averages of KO2 channels is remarkably similar to that of the O2-sensitive K+ current. In addition, ensemble average and macroscopic K+ currents are affected similarly by low PO2. These observations strongly suggest that KO2 channels are the main contributors to the macroscopic K+ current of glomus cells. The reversible inhibition of KO2 channel activity by low PO2 does not desensitize and is not related to the presence of F-, ATP, and GTP-gamma-S at the internal face of the membrane. These results indicate that KO2 channels confer upon glomus cells their unique chemoreceptor properties and that the O2-K+ channel interaction occurs either directly or through an O2 sensor intrinsic to the plasma membrane closely associated with the channel molecule.

Alpha-subunit of Gk activates atrial K+ channels of chick, rat, and guinea pig

1988 ◽  
Vol 254 (6) ◽  
pp. H1200-H1205 ◽  
Author(s):  
G. E. Kirsch ◽  
A. Yatani ◽  
J. Codina ◽  
L. Birnbaumer ◽  
A. M. Brown
Keyword(s):  
Guinea Pig ◽  
Single Channel ◽  
Neonatal Rat ◽  
Alpha Subunit ◽  
K Channel ◽  
Muscarinic Agonist ◽  
Guanine Nucleotide Binding Protein ◽  
K Channels ◽  
Open Time ◽  
Guanine Nucleotide Binding

A specific guanine nucleotide-binding protein, Gk, is the link by which muscarinic receptors activate atrial potassium channels (Science Wash. DC 235: 207-211, 1987). In adult guinea pigs, the alpha-subunit at picomolar concentrations mediates the holo-G protein effect (Science Wash. DC 236: 442-445, 1987), but in chick embryo it has been reported that the beta gamma-dimer at nanomolar concentrations rather than the alpha-subunit is the effective mediator (Nature Lond. 325: 321-326, 1987). This difference might have a phylogenetic or ontogenetic basis, and the present experiments tested these possibilities. Preactivated alpha k derived from human red blood cell Gk, when applied to the intracellular surface of inside-out membrane patches from the atria of embryonic chick, neonatal rat, and adult guinea pig activated single K+ channel currents. In each case, the alpha k-activated channels had the same single-channel conductance and mean open time as the muscarinic agonist-activated channels. Half-maximal activation was achieved at alpha k-concentrations of 2.4-13.8 pM. Hence, alpha k-activation of these K+ channels is independent of differences in age or species. The detergent 3-[3-cholamidopropyl)-dimethyammoniol]-1-propanesulfonate (CHAPS), which was used by Logothetis et al. (Nature Lond. 325: 321-326, 1987) at 184 microM to suspend the hydrophobic beta gamma-dimers, activated the same currents. We conclude that the effects of the beta gamma-dimer on these K+ channels is unknown and that as we had proposed earlier (Science Wash. DC 236: 442-445, 1987) it is the alpha-subunit that mediates the Gk effect.

Ca2+-activated K channels of canine colonic myocytes

1989 ◽  
Vol 257 (3) ◽  
pp. C470-C480 ◽  
Author(s):  
A. Carl ◽  
K. M. Sanders
Keyword(s):  
Proximal Colon ◽  
K Channel ◽  
Potential Range ◽  
Single Channels ◽  
Patch Clamp Technique ◽  
K Channels ◽  
Circular Smooth Muscle ◽  
Channel Opening ◽  
Close Time

K channels in enzymatically dispersed circular smooth muscle cells from the canine proximal colon were studied with the patch-clamp technique. The most prominent channel in cell-attached and excised, inside-out patches was a K channel, which had slope conductances of approximately 100 pS at a holding potential of 0 mV in a physiological K+ gradient and approximately 200 pS in symmetrical 140 mM K+ solutions. The relative permeabilities of the channel for monovalent cations were 1.0 K+:0.5 Rb+: less than 0.07 Li+:less than 0.07 Na+. The channels were activated by potential and intracellular Ca2+. At Ca2+ concentrations less than 10(-7) M, channel openings were rare except at very positive potentials. At Ca2+ concentrations between 10(-7) and 10(-6) M the probability of channel opening increased steeply, and the voltage for channel activation shifted to a negative potential range, which cells experience during electrical slow wave events in situ. The effect of Ca2+ on the open-state probability of single channels was mainly due to a decrease in mean close time. Channels were blocked by 1 mM tetraethylammonium applied to the outside of the patch but up to 10 mM tetraethylammonium applied to the inside of the patch, and 4-aminopyridine applied to either side did not block the channel. The data suggest that this channel mediates a current important in the termination of electrical slow waves, which are the primary excitable event in colonic circular muscles.

Internal and external TEA block in single cloned K+ channels

1991 ◽  
Vol 261 (4) ◽  
pp. C583-C590 ◽  
Author(s):  
G. E. Kirsch ◽  
M. Taglialatela ◽  
A. M. Brown
Keyword(s):  
Single Channel ◽  
Channel Structure ◽  
K Channel ◽  
Cdna Clones ◽  
K Channels ◽  
Open Time ◽  
Voltage Dependent ◽  
Internal Site ◽  
Channel Open Time ◽  
Single Channel Currents

Tetraethylammonium (TEA) has been used recently to probe natural and mutational variants of voltage-dependent K+ channels encoded by cDNA clones. Its usefulness as a probe of channel structure prompted us to examine the molecular mechanism by which TEA blocks single-channel currents in Xenopus oocytes expressing the rat brain K+ channel, RCK2. TEA at the intracellular surface of membrane patches decreased channel open time and increased the duration of closed intervals. Tetrapentylammonium had similar but more potent effects. Extracellular application of TEA caused an apparent reduction of single-channel amplitude. Block was slower at the high-affinity internal site than at the low-affinity external site. Internal TEA selectively blocks open K+ channels, and the voltage dependence of the block indicates that the binding site lies within the membrane electric field at a point 25% of the distance from the cytoplasmic margin. External TEA also interacts with the open channel but is less sensitive to membrane potential. The results indicate that the internal and external TEA binding sites define the inner and outer margins of the aqueous pore.

Different properties of the atrial G protein-gated K+ channels activated by extracellular ATP and adenosine

1995 ◽  
Vol 269 (4) ◽  
pp. H1349-H1358 ◽  
Author(s):  
C. Fu ◽  
A. Pleumsamran ◽  
U. Oh ◽  
D. Kim
Keyword(s):  
G Protein ◽  
Single Channel ◽  
Extracellular Atp ◽  
K Channel ◽  
Affinity Constant ◽  
Channel Activity ◽  
K Channels ◽  
Atrial Cells ◽  
K Current ◽  
Inside Out

Extracellular ATP (ATPo) and adenosine activate G protein-gated inwardly rectifying K+ currents in atrial cells. Earlier studies have suggested that the two agonists may use separate pathways to activate the K+ current. Therefore, we examined whether the K+ channels activated by the two agonists have different properties under identical ionic conditions. In cell-attached patches, K+ channels activated by 100 microM ATP in the pipette had a single-channel conductance and mean open time of 32.0 +/- 0.2 pS and 0.5 +/- 0.1 ms, respectively, compared with 31.3 +/- 0.3 pS and 0.9 +/- 0.1 ms for the K+ channels activated by adenosine (140 mM KCl). With ATPo as the agonist, the K+ channel activity in cell-attached patches was approximately threefold lower than that in inside-out patches with 100 microM GTP in the bath. Applying ATP to the cytoplasmic side of the membrane (ATPi) produced a biphasic concentration-dependent effect on channel activity: an increase at low [mean affinity constant (K0.5) = 190 microM] and a decrease at high (K0.5 = 1.3 mM) concentrations. In contrast, with adenosine as the agonist, K+ channel activity in cell-attached patches was approximately fourfold greater than that in inside-out patches with 100 microM GTP in the bath. In inside-out patches, ATPi only augmented the K+ channel activity (K0.5 = 32 microM). These results show that although both ATPo and adenosine activate kinetically similar K+ channels in atrial cells, the channels are regulated differently by intracellular nucleotides.

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