Ionic blockage of the delayed potassium conductance of skeletal muscle fibres

The interactions of 4-aminopyridine with the delayed potassium conductance of skeletal muscle have been investigated. It is ! fo~nd that 4-aminopyridine (4-AP) is 10 times more potent than TEA ions in blocking the potassium conductance, and in addition 4-AP appears to block in a different way. (2) 4-AP is a weak base with a pKa of 9.17. The distribution of such a weak base across the cell membrane and means for determining which class of the base is responsible for the biological activity are discussed. (3) When 4-AP is present on both sides of the membrane, the peak potassium conductance is reduced and there is an initial faster component to the timecourse of inactivati.on. When 4-AP is present on the outside of the fibre only the peak conductance is reduced (Kdiss =l.lmH). (4) It is proposed that the cationic form of 4-AP is the active form and that it acts at two sites. One is accessible from the extracellular solution and is responsible for the reduction in peak current; and the other accessible from the intracellular solution and responsible for the initial faster component of inactivation. (5) After an initial delay the inactivation induced by 4-AP inside the fibre, 4-AP:, is found to d.ecay exponentially at a rate which ~ increases as the internal potential is made more positive. (iv) (6) The fraction of channels that become inactivated by 4-AP+. in the' 1 course of a depolarizing step is found to increase as the internal potential is made more positive. In the voltage range -4OmV to Omv this fraction undergoes an e-fold increase every l2mV; beyond +lOmV this fraction reaches a limit, the value of which depends only on the concentration of 4-AP. (7) The blocking actions of 4-AP outside the fibre, 4-AP + , can be o separated into two components. One component is not influenced by the membrane electric field and the level of block depends only on the concentration of 4-AP. The second component is influenced by the membrane electric field and undergoes an e-fold change every 38mV, increasing as the internal potential is made negative. (8) Upon early 'repolarization of the membrane to -8OmV this voltage dependent component of 4-AP+ block proceeds exponentially with a o time constant of 8 msec. (9) The blockage of the delayed channel by 4-AP: and 4-AP+ is described 1 0 in mathematical form; and the results discussed in terms of possible molecular structures and events within the channel. (10) The repolarizingphase of the action potential is slowed in the presence of lmM 4-AP (pH 7.2). The resting potential, the r~te of rise and overshoot of the action potential are little affected. (11) The inwardly rectifying component of the potassium conductance is reduced by 4-AP. This block is intensified in alkaline solution which' suggests that the cationic form inside the cell is the active form. (12) The delayed potassium channel has also been studied ~n solutions " of different pH. At pH 5.0, gK is reduced by 16.3% and the threshold shifted by 20.2 mV in the depolarizing direction. At pH 9.8, gK is red"uced by 13% and the threshold shifted by l5mV in the depolarizing direction. (13) The delayed potassium current is abolished after treatment with 5mM 4-AP (pH 9.8). In these experiments, the transient current which remains is associated with the charging of the membrane capacity. (14) Asymmetries in the membrane capacity are observed, similar to those described by Schneider and Chandler (1973). These asymmetries are thought to be intra membrane currents associated \-lith exitation contraction ~ coupling. Formaldehyde, which interrupts exitation contraction coupling, reduces the asymmetry in the membrane capacity