Kinetics of Ca2+-activated K+ channels from rabbit muscle incorporated into planar bilayers. Evidence for a Ca2+ and Ba2+ blockade.

219

269

SUMMARY1. Kinetic states and modes of a large-conductance Ca2+-activated K+ channel in excised patches of membrane from cultured rat skeletal muscle were studied with the patch clamp technique. Up to 106 open and shut intervals were analysed from each of seven different excised membrane patches containing a single channel.2. Plots of the mean durations of consecutive groups of ten to fifty open and shut intervals were made to assess kinetic stability of the channel. Occasional abrupt decreases in the mean open interval duration from normal to different distinct levels, which were maintained for hundreds to thousands of consecutive intervals, indicated entry of the channel into different modes. 5. During normal activity the distributions of interval durations were typically described by the sum of three to four exponential components for the open intervals and six to eight exponential components for the shut intervals, and this was the case for data obtained over a wide range of open channel probability resulting from different CaF+. These observations suggest that the channel can enter at least three to four open and six to eight shut states during normal activity.6. The numbers of detected states for data sets of different sample sizes drawn from normal activity agreed with theoretical predictions, and were essentially independent of the segment of normal activity from which the data sets were drawn. These observations are consistent with relative stability of channel kinetics during normal activity. Detection of each additional open or shut state after the first was found to require a 3-to 10-fold increase in the number of analysed events.

Section: B Mcmanus and K L Maglebysupporting

confidence: 90%

Kinetic states and modes of single large‐conductance calcium‐activated potassium channels in cultured rat skeletal muscle.

McManus

Magleby

1988

219

269

“…Ba2+ has been reported to block large-conductance Ca2+-activated K+ channels (Vergara & Latorre, 1983). In all four terminals tested, 200 /2M Ba2+ applied extracellularly markedly inhibited the neurohypophysial Ca2+-activated K+ current.…”

Section: Pharmacology Of the Outward K+ Currentmentioning

confidence: 79%

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

Wang

Thorn

Lemos

1992

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...

“…We used the high Ba¥ sensitivity of IK(IR) to evaluate its contribution to the resting membrane potential (Vm) in physiological saline as millimolar concentrations of external Ba¥ are generally needed to inhibit most voltage-sensitive K¤ channels (Vergara & Latorre, 1983;Hurst, Latorre, Toro & Stefani, 1995). According to eqn (2), the extrapolated Kd(V) for inhibition by Ba¥ at potentials between −60 and −50 mV is 16 ìÒ or less.…”

Section: Characterization Of the Inwardly Rectifying Current In Rat Cmentioning

confidence: 99%

“…One possible target of Ba¥ is the large-conductance, voltage-sensitive Ca¥-activated K¤ channel. Although this channel is relatively insensitive to external Ba¥, it is inhibited by micromolar concentrations of internal Ba¥ (Vergara & Latorre, 1983). In physiological saline, the Ca¥-activated K¤ currents are activated by Ca¥ influx through voltage-dependent Ca¥ channels and may possibly be blocked intracellularly by Ba¥ that enters the cell through the Ca¥ channels.…”

Section: Characterization Of the Inwardly Rectifying Current In Rat Cmentioning

confidence: 99%

Corticotropin releasing hormone inhibits an inwardly rectifying potassium current in rat corticotropes

Kuryshev

Haak

Childs

et al. 1997

The perforated‐patch‐clamp technique was used to identify an inwardly rectifying K+ current (IK(IR)) in cultured rat anterior pituitary cells highly enriched in corticotropes. IK(IR) was rapidly activating and highly selective for K+. The K+ conductance was approximately proportional to the square root of the extracellular K+ concentration. I K(IR) was blocked in a voltage‐dependent manner by external Ba2+ and Cs+, slightly attenuated by 5 mM 4‐aminopyridine (15% inhibition) and insensitive to 10 mM tetra‐ethylammonium, 2 mM Ca2+, 1 mM Cd2+ and 50 μM La3+. In physiological saline, 100 μM Ba2+, which inhibits 86% of IK(IR) at the cell resting potential, depolarized cells by 6.1 ± 0.7 mV from a mean resting potential of −59.6 ± 0.8 mV. Corticotropin releasing hormone (CRH), which activates adenylyl cyclase and stimulates adrenocorticotropic hormone (ACTH) secretion from corticotropes, inhibited IK(IR) by 25% and depolarized the cells by 10.2 ± 1.0 mV. Dibutyryl cAMP ((Bu)2cAMP) mimicked these effects. The membrane depolarization evoked by Ba2+ or CRH increased the cell firing frequency. Comparison of cells exhibiting a membrane potential of approximately −50 mV revealed that spike frequency in the presence of CRH (109 ± 7 spikes (5 min)−1) was greater than in control (60 ± 5 spikes (5 min)−1) or Ba2+‐treated (77 ± 15 spikes (5 min)−1) corticotropes. The data suggest that IK(IR) contributes to maintenance of the resting membrane potential of rat corticotropes. Inhibition of IK(IR) plays a role in, but does not account for all of, the membrane depolarization and enhancement of firing frequency evoked by CRH.