Local Anesthetic Block of Mutant Rat Skeletal Muscle Na+ Channels Lacking Fast Inactivation: Evidence for Activation Gate Trapping

Abstract

Local anesthetics (LAs) inhibit voltage-gated Na+ channels in a complex time-, voltage-, and state-dependent fashion that may involve a “guarded intrapore receptor” where closed channel gates (m - activation, and h - inactivation) preclude LA escape via the inner channel mouth leading to trapping and long-lived, drug blocked states. Recent evidence discounts the involvement of the h gate in this mechanism, but m gate trapping may contribute importantly to use-dependent block. Critical steps in m gate trapping are the initial binding of a local anesthetic molecule to the intrapore receptor followed by m gate closure upon deactivation. Consequently, the mechanism predicts that the fraction of long-lived blocked channels is directly related to the probability of intrapore receptor occupancy immediately preceding m gate closure.To test this prediction we explored the relationship between open channel block and the long-lived drug blocked states in a mutant Na+ channel lacking fact inactivation. Disabling fast inactivation in this manner provided for the study of open channel blockade and use-dependent block in the absence of a functional h gate. We studied macro- and microscopic currents of a mutant rat skeletal muscle Na+ channel (μ1, Nav 1.4) lacking fast inactivation through the triple point mutation (IFM/QQQ) in the III-IV interdomain (QQQ) expressed in Xenopus oocytes. Lidocaine (LIDO) caused use-dependent block of QQQ involving a long-lived drug blocked state (recovery τ=0.12s, -100mV). LIDO produced time dependent reduction of non-inactivating macroscopic currents and discrete and rapid block of single channel currents both of which report occupancy of the intrapore receptor. The fraction of long-lived states was tightly linked with the degree of receptor occupancy. The findings provide strong support for the m gate trapping of LIDO.