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      Single-Channel Kinetics, Inactivation, and Spatial Distribution of Inositol Trisphosphate (IP 3) Receptors in Xenopus Oocyte Nucleus

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          Abstract

          Single-channel properties of the Xenopus inositol trisphosphate receptor (IP 3R) ion channel were examined by patch clamp electrophysiology of the outer nuclear membrane of isolated oocyte nuclei. With 140 mM K + as the charge carrier (cytoplasmic [IP 3] = 10 μM, free [Ca 2+] = 200 nM), the IP 3R exhibited four and possibly five conductance states. The conductance of the most-frequently observed state M was 113 pS around 0 mV and ∼300 pS at 60 mV. The channel was frequently observed with high open probability (mean P o = 0.4 at 20 mV). Dwell time distribution analysis revealed at least two kinetic states of M with time constants τ < 5 ms and ∼20 ms; and at least three closed states with τ ∼1 ms, ∼10 ms, and >1 s. Higher cytoplasmic potential increased the relative frequency and τ of the longest closed state. A novel “flicker” kinetic mode was observed, in which the channel alternated rapidly between two new conductance states: F 1 and F 2. The relative occupation probability of the flicker states exhibited voltage dependence described by a Boltzmann distribution corresponding to 1.33 electron charges moving across the entire electric field during F 1 to F 2 transitions. Channel run-down or inactivation (τ ∼ 30 s) was consistently observed in the continuous presence of IP 3 and the absence of change in [Ca 2+]. Some (∼10%) channel disappearances could be reversed by an increase in voltage before irreversible inactivation. A model for voltage-dependent channel gating is proposed in which one mechanism controls channel opening in both the normal and flicker modes, whereas a separate independent mechanism generates flicker activity and voltage- reversible inactivation. Mapping of functional channels indicates that the IP 3R tends to aggregate into microscopic (<1 μm) as well as macroscopic (∼10 μm) clusters. Ca 2+-independent inactivation of IP 3R and channel clustering may contribute to complex [Ca 2+] signals in cells.

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          Most cited references73

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          Improved patch-clamp techniques for high-resolution current recording from cells and cell-free membrane patches.

          1. The extracellular patch clamp method, which first allowed the detection of single channel currents in biological membranes, has been further refined to enable higher current resolution, direct membrane patch potential control, and physical isolation of membrane patches. 2. A description of a convenient method for the fabrication of patch recording pipettes is given together with procedures followed to achieve giga-seals i.e. pipette-membrane seals with resistances of 10(9) - 10(11) omega. 3. The basic patch clamp recording circuit, and designs for improved frequency response are described along with the present limitations in recording the currents from single channels. 4. Procedures for preparation and recording from three representative cell types are given. Some properties of single acetylcholine-activated channels in muscle membrane are described to illustrate the improved current and time resolution achieved with giga-seals. 5. A description is given of the various ways that patches of membrane can be physically isolated from cells. This isolation enables the recording of single channel currents with well-defined solutions on both sides of the membrane. Two types of isolated cell-free patch configurations can be formed: an inside-out patch with its cytoplasmic membrane face exposed to the bath solution, and an outside-out patch with its extracellular membrane face exposed to the bath solution. 6. The application of the method for the recording of ionic currents and internal dialysis of small cells is considered. Single channel resolution can be achieved when recording from whole cells, if the cell diameter is small (less than 20 micrometer). 7. The wide range of cell types amenable to giga-seal formation is discussed.
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            Local anaesthetics transiently block currents through single acetylcholine-receptor channels.

            1. Single channel currents through acetylcholine receptor channels (ACh channels) were recorded at chronically denervated frog muscle extrajunctional membranes in the absence and presence of the lidocaine derivatives QX-222 and QX-314. 2. The current wave forms due to the opening and closing of single ACh channels (activated by suberyldicholine) normally are square pulses. These single pulses appear to be chopped into bursts of much shorter pulses, when the drug QX-222 is present in addition to the agonist. 3. The mean duration of the bursts is comparable to or longer than the normal channel open time, and increases with increasing drug concentration. 4. The duration of the short pulses within a burst decreases with increasing drug concentration. 5. It is concluded that drug molecules reversibly block open end-plate channels and that the flickering within a burst represents this fast, repeatedly occurring reaction. 6. The voltage dependence of the reaction rates involved, suggested that the site of the blocking reaction is in the centre of the membrane, probably inside the ionic channel.
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              Inositol (1,4,5)-trisphosphate (InsP3)-gated Ca channels from cerebellum: conduction properties for divalent cations and regulation by intraluminal calcium

              The conduction properties of inositol (1,4,5)-trisphosphate (InsP3)- gated calcium (Ca) channels (InsP3R) from canine cerebellum for divalent cations and the regulation of the channels by intraluminal Ca were studied using channels reconstituted into planar lipid bilayers. Analysis of single-channel recordings performed with different divalent cations present at 55 mM on the trans (intraluminal) side of the membrane revealed that the current amplitude at 0 mV and the single- channel slope conductance fell in the sequence: Ba (2.2 pA, 85 pS) > Sr (2.0 pA, 77 pS) > Ca (1.4 pA, 53 pS) > Mg (1.1 pA, 42 pS). The mean open time of the InsP3R recorded with Ca (2.9 ms) was significantly shorter than with other divalent cations (approximately 5.5 ms). The "anomalous mole fraction effect" was not observed in mixtures of divalent cations (Mg and Ba), suggesting that these channels are single- ion pores. Measurements of InsP3R activity at different intraluminal Ca levels demonstrated that Ca in the submillimolar range did not potentiate channel activity, and that very high levels of intraluminal Ca (> or = 10 mM) decreased channel open probability 5-10-fold. When InsP3R were measured with Ba as a current carrier in the presence of 110 mM cis potassium, a PBa/PK of 6.3 was estimated from the extrapolated value for the reversal potential. When the unitary current through the InsP3R at 0 mV was measured as a function of the permeant ion (Ba) concentration, the half-maximal current occurred at 10 mM trans Ba. The following conclusions are drawn from these data: (a) the conduction properties of InsP3R are similar to the properties of the ryanodine receptor, another intracellular Ca channel, and differ dramatically from the properties of voltage-gated Ca channels of the plasma membrane. (b) The estimated size of the Ca current through the InsP3R under physiological conditions is 0.5 pA, approximately four times less than the Ca current through the ryanodine receptor. (c) The potentiation of InsP3R by intraluminal Ca in the submillimolar range remains controversial. (d) A quantitative model that explains the inhibitory effects of high trans Ca on InsP3R activity was developed and the kinetic parameters of InsP3R gating were determined.
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                Author and article information

                Journal
                J Gen Physiol
                The Journal of General Physiology
                The Rockefeller University Press
                0022-1295
                1540-7748
                1 May 1997
                : 109
                : 5
                : 571-587
                Affiliations
                From the Department of Physiology, University of Pennsylvania, Philadelphia, Pennsylvania 19104-6100
                Author notes

                Address correspondence to Dr. Don-On Daniel Mak, Department of Physiology, University of Pennsylvania, Stellar-Chance Laboratories, Rm. 314, Philadelphia, PA 19104-6100. Fax: 215-573-8590; E-mail: dmak@ 123456mail.med.upenn.edu

                Article
                2217068
                9154905
                bab8571a-a314-4a8e-a022-1940b4df1e59
                Copyright @ 1997
                History
                : 14 November 1996
                : 24 February 1997
                Categories
                Article

                Anatomy & Physiology
                ca2+ signaling,inositol phosphates,calcium release channel,patch clamp,signal transduction

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