A rate theory model for Mg2+ block of ATP‐dependent potassium channels of rat skeletal muscle.

Abstract

1. We have studied the block by intracellular Mg21 (0-08-4 mM) of ATP-dependent potassium channels (KATP channels) from rat skeletal muscle using inside-out excised sarcolemmal patches. The block is voltage dependent, is relieved by extracellular potassium and has rapid kinetics, allowing the use of amplitude distribution analysis to estimate on and off rates. 2. To gain insight into the pore properties necessary to produce such a block, we have used an energy barrier model based on Eyring rate theory. (Findlay, 1987;Ashcroft & Kakei, 1989;Vivaudou, Arnoult & Villaz, 1991), but, secondly, Mg2+ produces a rapid voltagedependent block of the channel which causes mild inward rectification of the unitary current-voltage relation (i-V) at physiological intracellular Mg2+ concentrations (Findlay, 1987;Horie, Irisawa & Noma, 1987;Woll, Lonnendonker & Neumcke, 1989). Voltage-dependent channel block by cations is usually assumed to result from the blocking ion having to enter the channel, and so experience some of the transmembrane voltage field, in order to reach its binding site (Woodhull, 1973). On this basis Mg2+ appears to pass about 0-2-0-4 of the electrical distance into the channel to exert its fast block of KATP channels (Horie et al. 1987;Ciani & Ribalet, 1988). Mg2+ block of KATP channels can be relieved by increasing [K+]O (Horie et al. 1987).In the present paper, we report a detailed study of the voltage-dependent block by Mg2+ of KATP channels from rat skeletal muscle. We find that both voltage and extracellular K+ affect the off rate for Mg2+, and we have used rate theory analysis to develop a model that describes K+ permeation and the kinetics of Mg2+ block in this channel. METHODS PreparationAdult Wistar rats were killed by stunning followed by cervical dislocation, and the flexor digitorum brevis muscle was dissected from the hindlimb. To dissociate single fibres, the muscle was incubated in 0 3 % collagenase (Type 1; Sigma) in Ringer solution (containing (mM): NaCl, 146-3; KCI, 4-75; CaCl2, 1; CaHPO4, 0 95; MgCl2, 0 5; Hepes, 9.5, adjusted to pH 7-4 with NaOH) for 30 min at 4°C and then for 90 min at 37 'C. Fibres were subsequently isolated in collagenase-free Ringer solution by triturating the muscle with a fire-polished Pasteur pipette. Sarcolemmal vesicles were formed by placing the isolated fibres in a 155 mm K+ solution, and inside-out patches were excised from these into flowing intracellular solution containing 40 mM gluconate

Block of cloned BK Ca channels ( rSlo ) expressed in HEK 293 cells by N- methyl d - glucamine

“…NMDG + , when present, was treated as a second permeating cation. The values and positions of the barriers and wells for both K + and NMDG + were fitted as described in Materials and methods and elsewhere [4], and are shown in graphical form in the inset of Fig. 2B.…”

“…The model also includes repulsion factors to account for the effect of electrostatic repulsion between ions when both sites are occupied. We have previously published a detailed description of this procedure [4].…”

Permeation and block of rat glur6 glutamate receptor channels by internal and external polyamines

“…A matrix, Q, of rate constants qij, where the row index i defines the initial state and the column j defines the final state for a given transition, was used to calculate the equilibrium occupancies of all n states as described previously (Begenisich & Cahalan, 1980;Colquhoun & Hawkes, 1987;Davies, McKillen, Stanfield & Standen, 1996). At any given membrane potential Vm, values for qij were calculated as:…”

“…Barrier-well values (outside to inside) for Na¤ (10·81, −2·07, 10·08, −2·08 and 7·87RT) were chosen to give a conductance of 21 pS based on estimates for the main conductance state of GluR6(Q) (Swanson, Feldmeyer, Kaneda & Cull-Candy, 1996); values for spermine were 14·27, −3·50, 0, −13·96 and 11·80RT; electrical distances (ä) between adjacent barriers and wells were 0·08, 0·152, 0·17, 0·082, 0·405 and 0·111; the barriers were asymmetrical to increase the steepness of the voltage dependence of onset and relief from block by spermine. The model also included electrostatic repulsion between ions when both sites were occupied as described by Davies et al (1996); the repulsion factors were rNa-Na = 3·0, rNa-Spm = 7·0, rSpm-Spm = 20. Fits of a Woodhull infinite barrier model for block by 30 ìÒ internal spermine over the range −100 to +20 mV gave zè = 1·7 and Kd(0) = 2·4 ìÒ, similar to experimentally recorded values.…”

Two mechanisms for inward rectification of current flow through the purinoceptor P2X2 class of ATP‐gated channels

“…Muscle nAChR channels show a very small degree of inward rectification because channel gating is weakly voltage dependent (Magleby & Stevens, 1972), and neuronal nAChR channels show substantial inward rectification due to a combination of open channel block by internal Mg 2+ and voltage‐dependent gating (Ifune & Steinbach, 1990; Sands & Barish, 1992). Intracellular Mg 2+ is also capable of producing an open channel block of several different inwardly rectifying K + channels in excised patches (Matsuda, Saigusa & Irisawa, 1987; Vandenberg, 1987; Davies, McKillen, Stanfield & Standen, 1996). However, Mg 2+ block cannot account quantitatively for the degree of rectification found in some inwardly rectifying K + channels.…”