scholarly journals Potassium blocks barium permeation through a calcium-activated potassium channel.

1988 ◽  
Vol 92 (5) ◽  
pp. 549-567 ◽  
Author(s):  
J Neyton ◽  
C Miller
Keyword(s):  
Skeletal Muscle ◽  
Lipid Bilayers ◽  
Dissociation Rate ◽  
Planar Lipid Bilayers ◽  
Rat Skeletal Muscle ◽  
K Channels ◽  
Calcium Activated Potassium Channel ◽  
Micromolar Range ◽  
High Conductance ◽  
External K

Single high-conductance Ca2+-activated K+ channels from rat skeletal muscle were inserted into planar lipid bilayers, and discrete blocking by the Ba2+ ion was studied. Specifically, the ability of external K+ to reduce the Ba2+ dissociation rate was investigated. In the presence of 150 mM internal K+, 1-5 microM internal Ba2+, and 150 mM external Na+, Ba2+ dissociation is rapid (5 s-1) in external solutions that are kept rigorously K+ free. The addition of external K+ in the low millimolar range reduces the Ba2+ off-rate 20-fold. Other permeant ions, such as Tl+, Rb+, and NH4+ show a similar effect. The half-inhibition constants rise in the order: Tl+ (0.08 mM) less than Rb+ (0.1 mM) less than K+ (0.3 mM) less than Cs+ (0.5 mM) less than NH4+ (3 mM). When external Na+ is replaced by 150 mM N-methyl glucamine, the Ba2+ off-rate is even higher, 20 s-1. External K+ and other permeant ions reduce this rate by approximately 100-fold in the micromolar range of concentrations. Na+ also reduces the Ba2+ off-rate, but at much higher concentrations. The half-inhibition concentrations rise in the order: Rb+ (4 microM) less than K+ (19 microM) much less than Na+ (27 mM) less than Li+ (greater than 50 mM). The results require that the conduction pore of this channel contains at least three sites that may all be occupied simultaneously by conducting ions.

Modulation of skeletal muscle Ca2(+)-release channel activity by sphingosine

1997 ◽  
Vol 272 (5) ◽  
pp. C1465-C1474 ◽  
Author(s):  
D. H. Needleman ◽  
B. Aghdasi ◽  
A. B. Seryshev ◽  
G. J. Schroepfer ◽  
S. L. Hamilton
Keyword(s):  
Skeletal Muscle ◽  
Lipid Bilayers ◽  
Binding Protein ◽  
Planar Lipid Bilayers ◽  
Dependent Manner ◽  
Carboxy Terminus ◽  
Release Channel ◽  
Proteolytic Fragment ◽  
High Affinity ◽  
High Affinity Site

The effect of D-erythro-C18-sphingosine (sphingosine) and related compounds on the Ca(2+)-release channel (ryanodine binding protein) was examined on rabbit skeletal muscle membranes, on the purified ryanodine binding protein, and on the channel reconstituted into planar lipid bilayers. Sphingosine inhibited [3H]ryanodine binding to sarcoplasmic reticulum (SR) membranes in a dose-dependent manner similar to published results (R. A. Sabbadini, R. Betto, A. Teresi, G. Fachechi-Cassano, and G. Salviati. J. Biol. Chem. 267: 15475-15484, 1992). The sphingolipid also inhibited [3H]ryanodine binding to the purified ryanodine binding protein. Our results demonstrate that the inhibition of [3H]ryanodine binding by sphingosine is due to an increased rate of dissociation of bound [3H]ryanodine from SR membranes and a decreased rate of association of [3H]ryanodine to the high-affinity site. Unlike other modulators of the Ca(2+)-release channel, sphingosine can remove bound [3H]ryanodine from the high-affinity site within minutes. Sphingosine increased the rate of dissociation of [3H]ryanodine bound to a solubilized proteolytic fragment derived from the carboxy terminus of the ryanodine binding protein (cleavage at Arg4475). Sphingosine also inhibited the activity of the Ca(2+)-release channel incorporated into planar lipid bilayers. Taken together, the data provide evidence for a direct effect of sphingosine on the Ca(2+)-release channel. Sphingosine is a noncompetitive inhibitor at the high-affinity ryanodine binding site, and it interacts with a site between Arg4475 and the carboxy terminus of the Ca(2+)-release channel.

scholarly journals Coupling of voltage-dependent gating and Ba++ block in the high-conductance, Ca++-activated K+ channel.

1987 ◽  
Vol 90 (3) ◽  
pp. 427-449 ◽  
Author(s):  
C Miller ◽  
R Latorre ◽  
I Reisin
Keyword(s):  
Lipid Bilayers ◽  
Dissociation Rate ◽  
K Channel ◽  
Single Channels ◽  
Closed Channel ◽  
Association Rate ◽  
Conduction Pathway ◽  
Voltage Dependent ◽  
Kinetics Of ◽  
High Conductance

Voltage-dependent Ca++-activated K+ channels from rat skeletal muscle were reconstituted into planar lipid bilayers, and the kinetics of block of single channels by Ba++ were studied. The Ba++ association rate varies linearly with the probability of the channel being open, while the dissociation rate follows a rectangular hyperbolic relationship with open-state probability. Ba ions can be occluded within the channel by closing the channel with a strongly hyperpolarizing voltage applied during a Ba++-blocked interval. Occluded Ba ions cannot dissociate from the blocking site until after the channel opens. The ability of the closed channel to occlude Ba++ is used as an assay to study the channel's gating equilibrium in the blocked state. The blocked channel opens and closes in a voltage-dependent process similar to that of the unblocked channel. The presence of a Ba ion destabilizes the closed state of the blocked channel, however, by 1.5 kcal/mol. The results confirm that Ba ions block this channel by binding in the K+-conduction pathway. They further show that the blocking site is inaccessible to Ba++ from both the cytoplasmic and external solutions when the channel is closed.

scholarly journals Unitary Ca2+ Current through Mammalian Cardiac and Amphibian Skeletal Muscle Ryanodine Receptor Channels under Near-physiological Ionic Conditions

2003 ◽  
Vol 122 (4) ◽  
pp. 407-417 ◽  
Author(s):  
Claudia Kettlun ◽  
Adom González ◽  
Eduardo Ríos ◽  
Michael Fill
Keyword(s):  
Skeletal Muscle ◽  
Charge Carrier ◽  
Lipid Bilayers ◽  
Ryanodine Receptor ◽  
Skeletal Muscles ◽  
Planar Lipid Bilayers ◽  
High Concentrations

Ryanodine receptor (RyR) channels from mammalian cardiac and amphibian skeletal muscle were incorporated into planar lipid bilayers. Unitary Ca2+ currents in the SR lumen-to-cytosol direction were recorded at 0 mV in the presence of caffeine (to minimize gating fluctuations). Currents measured with 20 mM lumenal Ca2+ as exclusive charge carrier were 4.00 and 4.07 pA, respectively, and not significantly different. Currents recorded at 1–30 mM lumenal Ca2+ concentrations were attenuated by physiological [K+] (150 mM) and [Mg2+] (1 mM), in the same proportion (∼55%) in mammalian and amphibian channels. Two amplitudes, differing by ∼35%, were found in amphibian channel studies, probably corresponding to α and β RyR isoforms. In physiological [Mg2+], [K+], and lumenal [Ca2+] (1 mM), the Ca2+ current was just less than 0.5 pA. Comparison of this value with the Ca2+ flux underlying Ca2+ sparks suggests that sparks in mammalian cardiac and amphibian skeletal muscles are generated by opening of multiple RyR channels. Further, symmetric high concentrations of Mg2+ substantially reduced the current carried by 10 mM Ca2+ (∼40% at 10 mM Mg2+), suggesting that high Mg2+ may make sparks smaller by both inhibiting RyR gating and reducing unitary current.

scholarly journals Time-irreversible Subconductance Gating Associated with Ba2+ Block of Large Conductance Ca2+-activated K+ Channels

1998 ◽  
Vol 111 (2) ◽  
pp. 343-362 ◽  
Author(s):  
Ricardo A. Bello ◽  
Karl L. Magleby
Keyword(s):  
Skeletal Muscle ◽  
Membrane Potential ◽  
External Solution ◽  
Single Step ◽  
Rat Skeletal Muscle ◽  
Patch Clamp Technique ◽  
K Channels ◽  
Open Time ◽  
Channel Open Time ◽  
A Site

Ba2+ block of large conductance Ca2+-activated K+ channels was studied in patches of membrane excised from cultures of rat skeletal muscle using the patch clamp technique. Under conditions in which a blocking Ba2+ ion would dissociate to the external solution (150 mM N-methyl-d-glucamine+o, 500 mM K+i, 10 μM Ba2+i, +30 mV, and 100 μM Ca2+i to fully activate the channel), Ba2+ blocks with a mean duration of ∼2 s occurred, on average, once every ∼100 ms of channel open time. Of these Ba2+ blocks, 78% terminated with a single step in the current to the fully open level and 22% terminated with a transition to a subconductance level at ∼0.26 of the fully open level (preopening) before stepping to the fully open level. Only one apparent preclosing was observed in ∼10,000 Ba2+ blocks. Thus, the preopenings represent Ba2+-induced time-irreversible subconductance gating. The fraction of Ba2+ blocks terminating with a preopening and the duration of preopenings (exponentially distributed, mean = 0.75 ms) appeared independent of changes in [Ba2+]i or membrane potential. The fractional conductance of the preopenings increased from 0.24 at +10 mV to 0.39 at +90 mV. In contrast, the average subconductance level during normal gating in the absence of Ba2+ was independent of membrane potential, suggesting different mechanisms for preopenings and normal subconductance levels. Preopenings were also observed with 10 mM Ba2+o and no added Ba2+i. Adding K+, Rb+, or Na+ to the external solution decreased the fraction of Ba2+ blocks with preopenings, with K+ and Rb+ being more effective than Na+. These results are consistent with models in which the blocking Ba2+ ion either induces a preopening gate, and then dissociates to the external solution, or moves to a site located on the external side of the Ba2+ blocking site and acts directly as the preopening gate.

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