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      Gating and Ionic Currents Reveal How the BK Ca Channel's Ca 2+ Sensitivity Is Enhanced by its β1 Subunit

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      The Journal of General Physiology
      The Rockefeller University Press

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          Abstract

          Large-conductance Ca 2+-activated K + channels (BK Ca channels) are regulated by the tissue-specific expression of auxiliary β subunits. β1 is predominately expressed in smooth muscle, where it greatly enhances the BK Ca channel's Ca 2+ sensitivity, an effect that is required for proper regulation of smooth muscle tone. Here, using gating current recordings, macroscopic ionic current recordings, and unitary ionic current recordings at very low open probabilities, we have investigated the mechanism that underlies this effect. Our results may be summarized as follows. The β1 subunit has little or no effect on the equilibrium constant of the conformational change by which the BK Ca channel opens, and it does not affect the gating charge on the channel's voltage sensors, but it does stabilize voltage sensor activation, both when the channel is open and when it is closed, such that voltage sensor activation occurs at more negative voltages with β1 present. Furthermore, β1 stabilizes the active voltage sensor more when the channel is closed than when it is open, and this reduces the factor D by which voltage sensor activation promotes opening by ∼24% (16.8→12.8). The effects of β1 on voltage sensing enhance the BK Ca channel's Ca 2+ sensitivity by decreasing at most voltages the work that Ca 2+ binding must do to open the channel. In addition, however, in order to fully account for the increase in efficacy and apparent Ca 2+ affinity brought about by β1 at negative voltages, our studies suggest that β1 also decreases the true Ca 2+ affinity of the closed channel, increasing its Ca 2+ dissociation constant from ∼3.7 μM to between 4.7 and 7.1 μM, depending on how many binding sites are affected.

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          Most cited references49

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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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            Physiological roles and properties of potassium channels in arterial smooth muscle.

            This review examines the properties and roles of the four types of K+ channels that have been identified in the cell membrane of arterial smooth muscle cells. 1) Voltage-dependent K+ (KV) channels increase their activity with membrane depolarization and are important regulators of smooth muscle membrane potential in response to depolarizing stimuli. 2) Ca(2+)-activated K+ (KCa) channels respond to changes in intracellular Ca2+ to regulate membrane potential and play an important role in the control of myogenic tone in small arteries. 3) Inward rectifier K+ (KIR) channels regulate membrane potential in smooth muscle cells from several types of resistance arteries and may be responsible for external K(+)-induced dilations. 4) ATP-sensitive K+ (KATP) channels respond to changes in cellular metabolism and are targets of a variety of vasodilating stimuli. The main conclusions of this review are: 1) regulation of arterial smooth muscle membrane potential through activation or inhibition of K+ channel activity provides an important mechanism to dilate or constrict arteries; 2) KV, KCa, KIR, and KATP channels serve unique functions in the regulation of arterial smooth muscle membrane potential; and 3) K+ channels integrate a variety of vasoactive signals to dilate or constrict arteries through regulation of the membrane potential in arterial smooth muscle.
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              Coupling between Voltage Sensor Activation, Ca2+ Binding and Channel Opening in Large Conductance (BK) Potassium Channels

              To determine how intracellular Ca2+ and membrane voltage regulate the gating of large conductance Ca2+-activated K+ (BK) channels, we examined the steady-state and kinetic properties of mSlo1 ionic and gating currents in the presence and absence of Ca2+ over a wide range of voltage. The activation of unliganded mSlo1 channels can be accounted for by allosteric coupling between voltage sensor activation and the closed (C) to open (O) conformational change (Horrigan, F.T., and R.W. Aldrich. 1999. J. Gen. Physiol. 114:305–336; Horrigan, F.T., J. Cui, and R.W. Aldrich. 1999. J. Gen. Physiol. 114:277–304). In 0 Ca2+, the steady-state gating charge-voltage (QSS-V) relationship is shallower and shifted to more negative voltages than the conductance-voltage (GK-V) relationship. Calcium alters the relationship between Q-V and G-V, shifting both to more negative voltages such that they almost superimpose in 70 μM Ca2+. This change reflects a differential effect of Ca2+ on voltage sensor activation and channel opening. Ca2+ has only a small effect on the fast component of ON gating current, indicating that Ca2+ binding has little effect on voltage sensor activation when channels are closed. In contrast, open probability measured at very negative voltages (less than −80 mV) increases more than 1,000-fold in 70 μM Ca2+, demonstrating that Ca2+ increases the C-O equilibrium constant under conditions where voltage sensors are not activated. Thus, Ca2+ binding and voltage sensor activation act almost independently, to enhance channel opening. This dual-allosteric mechanism can reproduce the steady-state behavior of mSlo1 over a wide range of conditions, with the assumption that activation of individual Ca2+ sensors or voltage sensors additively affect the energy of the C-O transition and that a weak interaction between Ca2+ sensors and voltage sensors occurs independent of channel opening. By contrast, macroscopic IK kinetics indicate that Ca2+ and voltage dependencies of C-O transition rates are complex, leading us to propose that the C-O conformational change may be described by a complex energy landscape.
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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
                October 2005
                : 126
                : 4
                : 393-412
                Affiliations
                Molecular Cardiology Research Institute, New England Medical Center, and Department of Neuroscience, Tufts University School of Medicine, Boston, MA 02111
                Author notes

                Correspondence to Daniel H. Cox: dan.cox@ 123456tufts.edu

                Article
                200509346
                10.1085/jgp.200509346
                2266624
                16186565
                cb990a4e-9541-4747-b69a-2fc33d616d01
                Copyright © 2005, The Rockefeller University Press
                History
                : 15 June 2005
                : 26 August 2005
                Categories
                Article

                Anatomy & Physiology
                Anatomy & Physiology

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