Local anesthetic (LA) block of sodium current (INa) exhibits different phasic and non-phasic components depending in part on the charge of the LA molecule. Charged LA's cause little tonic block, but do cause slowly developing phasic block that does not depend upon the duration of depolarization (transient-state block). Uncharged LA cause both tonic block and rapidly developing phasic block that depends upon the duration of depolarization (maintained-state block). The overall goal of this project is to define structures on the primary sequence of the voltage- dependent Na+ channel accounting for different types of LA block, and also, secondarily, the structures affecting inactivation and recovery from inactivation. Pilot data show isoform differences in LA block and also inactivation recovery kinetics. We will first refine a protocol for the components of LA block using prototype charged (QX-222), uncharged (benzocaine), and mixed charge (lidocaine) LA (Aim 1). We will apply the protocol for LA block on Na+ channels isoforms in native tissues and Na+ channels expressed in oocytes and transfected mammalian cells. This will define the functional isoform differences or phenotype for the components of LA block (Aim 2). LA block of channels with mutations making them inactivation-defective (Aim 3) will test the relationship between inactivation gating and block. Domains responsible for LA block will be identified using isoform chimeras (Aim 4); specific amino acid residues responsible for LA block will be identified using site-directed mutagenesis (Aim 5). The working hypothesis guiding the experiments is that polar or charged amino acid residues on the S45 transmembrane segments account for charged LA block, and that hydrophobic residues on S6 transmembrane segments account for uncharged LA block. Studies of inactivation and recovery from inactivation (necessary control studies for the previous aims) for the different isoforms, chimeras, and point- mutations will provide insight into structure-function of these processes (Aim 6). Achieving these aims will help define the inner pore of the Na channel and also provide insight into the mechanism of drug interactions with ion channels. Understanding the structures and mechanisms involved with tissue-specific drug-channel interactions may lead to better therapeutic strategies by, for example, improving rational drug design and assisting in molecular approaches to clinical arrhythmias.
