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

Miller, Christopher; Latorre, Ramón; Reisin, Ignacio L.

doi:10.1085/jgp.90.3.427

Cited by 117 publications

(134 citation statements)

References 24 publications

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“…Data were collected on video tape and analyzed by a pattern-recognition program using a laboratory computer (Indec, Sunnyvale, CA). The details of the computer analysis were identical to those used to analyze block of this channel by Ba 2+ (Miller, 1987;Miller et al, 1987). Apparent rate constants of CTX association and dissociation were measured from the mean blocked and unblocked intervals in a given channel record, rb and ru, respectively:…”

Section: Discussionmentioning

confidence: 99%

Charybdotoxin block of single Ca2+-activated K+ channels. Effects of channel gating, voltage, and ionic strength.

Anderson

MacKinnon

Smith

et al. 1988

The Journal of General Physiology

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247

155

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Charybdotoxin (CTX), a small, basic protein from scorpion venom, strongly inhibits the conduction of K ions through high-conductance, Ca2+-activated K + channels. The interaction of CTX with Ca2+-activated K + channels from rat skeletal muscle plasma membranes was studied by inserting single channels into uncharged planar phospholipid bilayers. CTX blocks K § conduction by binding to the external side of the channel, with an apparent dissociation constant of ---10 nM at physiological ionic strength. The dwelltime distributions of both blocked and unblocked states are single-exponential. The toxin association rate varies linearly with the CTX concentration, and the dissociation rate is independent of it. CTX is competent to block both open and closed channels; the association rate is sevenfold faster for the open channel, while the dissociation rate is the same for both channel conformations. Membrane depolarization enhances the CTX dissociation rate e-fold/28 mV; if the channel's open probability is maintained constant as voltage varies, then the toxin association rate is voltage independent. Increasing the external solution ionic strength from 20 to 300 mM (with K § Na +, or arginine +) reduces the association rate by two orders of magnitude, with little effect on the dissociation rate. We conclude that CTX binding to the Ca2+-activated K § channel is a bimolecular process, and that the CTX interaction senses both voltage and the channel's conformational state. We further propose that a region of fixed negative charge exists near the channel's CTX-binding site. INTRODUGTIONThe high-conductance, Ca~+-activated K + channel is present in the plasma membranes of many types of cells, both excitable and inexcitable (Latorre, 1086). Its

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Section: Discussionmentioning

confidence: 99%

Charybdotoxin block of single Ca2+-activated K+ channels. Effects of channel gating, voltage, and ionic strength.

Anderson

MacKinnon

Smith

et al. 1988

The Journal of General Physiology

Self Cite

247

155

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“…For simplicity, we assume that there is a single ion binding site in the pore (but see Discussion). A gate could flank the extracellular side of the binding site (model A), the intracellular side (model B), or both sides (model C) as suggested by Miller, Latorre, and Reisin (1987) for Cae+-activated K ÷ channels of rat skeletal muscle. The Ni 2+ blocking kinetics seem most consistent with model A, in which internal ions have access to the ion binding Stimulated by the work of Miller et al (1987), we attempted to trap Ni 2+ in the closed channel as a further test of the models in Fig.…”

Section: Where Is the Gate In Relation To The Binding Site(s)for Ions ?mentioning

confidence: 99%

Interactions between divalent cations and the gating machinery of cyclic GMP-activated channels in salamander retinal rods.

Karpen

Brown

Stryer

et al. 1993

The Journal of General Physiology

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The effects of divalent cations on the gating of the cGMP-activated channel, and the effects of gating on the movement of divalent cations in and out of the channel's pore were studied by recording macroscopic currents in excised membrane patches from salamander retinal rods. The fractional block of cGMPactivated Na + currents by internal and external Mg 2+ as well as internal Ca ~+ was nearly independent ofcGMP concentration. This indicates that Mg 2÷ and Ca ~÷ bind with similar affinity to open and closed states of the channel. In contrast, the efficiency of block by internal Cd 2÷ or Zn 2÷ increased in proportion to the fraction of open channels, indicating that these ions preferentially occupy open channels. The kinetics of block by internal Ni 2+, which competes with Mg z÷ but blocks more slowly, were found to be unaffected by the fraction of channels open. External Ni z÷, however, blocked and unblocked much more rapidly when channels were mostly open. This suggests that within the pore a gate is located between the binding site(s) for ions and the extraceUular mouth of the channel. Micromolar concentrations of the transition metal divalent cations Ni 2÷, Cd 2+, Zn ~+, and Mn 2÷ applied to the cytoplasmic surface of a patch potentiated the response to subsaturating concentrations of cGMP without affecting the maximum current induced by saturating cGMP. The concentration of cGMP that opened half the channels was often lowered by a factor of three or more. Potentiation persisted after the experimental chamber was washed with divalent-free solution and fresh cGMP was applied, indicating that it does not result from an interaction between divalent cations and cGMP in solution; 1 mM EDTA or isotonic MgCl~ reversed potentiation. Voltage-jump experiments suggest that potentiation results from an increase in the rate of cGMP binding. Lowering the ionic strength of the bathing solution enhanced potentiation, suggesting that it involves electrostatic interactions. The strong electrostatic effect on cGMP binding and absence of effect on ion permeation through open channels implies that the cGMP binding sites on the channel are well separated from the permeation pathway.Address reprint requests to Dr.

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“…5C). This could be accounted for by two possible mechanisms: (1) an exceptional sensitivity of the neurohypophysial Ca2+-activated K+ channels to external Ba2+; or (2) enough Ba2+ was accumulated inside the terminal to block the channels after leakage across the membrane (Miller et al 1987) or entering through the voltage-gated Ca2+ channels (see Wang et al 1992 Channel block by extracellular TEA is well described for the maxi-K+ channel (Blatz & Magleby, 1986) and is comparable to its effects on the neurohypophysial channel and current. The sensitivity of the neurohypophysial Ca2+-activated K+ current to low concentrations of TEA implies that this macroscopic current may be due to the large-conductance K'a channel (Latorre, 1986;Rudy, 1988).…”

Section: Pharmacology Of the K~ca Channelmentioning

confidence: 99%

A novel large‐conductance Ca(2+)‐activated potassium channel and current in nerve terminals of the rat neurohypophysis.

Wang

Thorn

Lemos

1992

The Journal of Physiology

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SUMMARY1. Nerve terminals of the rat posterior pituitary were acutely dissociated and identified using a combination of morphological and immunohistochemical techniques. Terminal membrane currents were studied using the 'whole-cell' patch clamp technique and channels were studied using inside-out and outside-out patches.2. In physiological solutions, but with 7 mm 4-aminopyridine (4-AP), depolarizing voltage clamp steps from different holding potentials (-90 or -50 mV) elicited a fast, inward current followed by a slow, sustained, outward current. This outward current did not appear to show any steady-state inactivation.3. The threshold for activation of the outward current was -30 mV and the current-voltage relation was 'bell-shaped'. The amplitude increased with increasingly depolarized potential steps. The outward current reversal potential was measured using tail current analysis and was consistent with that of a potassium current.4. The sustained potassium current was determined to be dependent on the concentration of intracellular calcium. Extracellular Cd2+ (80 /LM), a calcium channel blocker, also reversibly abolished the outward current.5. The current was delayed in onset and was sustained over the length of a 150 msduration depolarizing pulse. The outward current reached a peak plateau and then decayed slowly. The decay was fitted by a single exponential with a time constant of 9 0 + 2-2 s. The decay constants did not show a dependence on voltage but rather on intracellular Caa2+. The time course of recovery from this decay was complex with full recovery taking > 190 s. 6. 4-AP (7 mM), dendrotoxin (100 nM), apamin (40-80 nM), and charybdotoxin (10-100 nM) had no effect on the sustained outward current. In contrast Ba21 G. WANG, P. THORN AND J. R. LEMOS most closely to a Ca2+-activated K+ current (IK(ca)) and not to a delayed rectifier or IA-like current. It also has properties different from that of the Ca2+-dependent outward current described in the magnocellular neuronal cell bodies of the hypothalamus.8. A large conductance channel is often observed in isolated rat neurohypophysial nerve terminals. The channel had a unit conductance of 231 pS in symmetrical 150 mM K+.9. With different potassium gradients across the patch of 150:5 and 5:130 (mM), the conductance decreased to 140 and 121 pS, respectively, and the shift in apparent reversal potentials (+ 42 and -25 mV) were consistent with that of a potassiumselective channel.10. The open probability (P.) of the channel was shown to be dependent on the concentration of calcium (Ca2+) at the intracellular side of the patch. The channel was activated at Ca2+ concentrations greater than 0-5 /M, and reached apparent full activation at around 5 /M with a half-maximal effective concentration (EC50) for Ca2+ = 1-53 JM.11. Most patches showed a voltage-sensitive increase in open probability (14-3 mV/e-fold change) with increasing depolarizations of the patch. Other patches showed little voltage-dependent properties. The activation curve for the probability of...

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

Cited by 117 publications

References 24 publications

Charybdotoxin block of single Ca2+-activated K+ channels. Effects of channel gating, voltage, and ionic strength.

Charybdotoxin block of single Ca2+-activated K+ channels. Effects of channel gating, voltage, and ionic strength.

Interactions between divalent cations and the gating machinery of cyclic GMP-activated channels in salamander retinal rods.

A novel large‐conductance Ca(2+)‐activated potassium channel and current in nerve terminals of the rat neurohypophysis.

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