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.

Agency
National Institute of Health (NIH)
Institute
National Heart, Lung, and Blood Institute (NHLBI)
Type
Research Project (R01)
Project #
5R01HL056441-04
Application #
2771525
Study Section
Pharmacology A Study Section (PHRA)
Project Start
1995-09-01
Project End
2000-08-31
Budget Start
1998-09-01
Budget End
1999-08-31
Support Year
4
Fiscal Year
1998
Total Cost
Indirect Cost
Name
University of Wisconsin Madison
Department
Internal Medicine/Medicine
Type
Schools of Medicine
DUNS #
161202122
City
Madison
State
WI
Country
United States
Zip Code
53715
Nagatomo, Toshihisa; January, Craig T; Ye, Bin et al. (2002) Rate-dependent QT shortening mechanism for the LQT3 deltaKPQ mutant. Cardiovasc Res 54:624-9
Nagatomo, T; January, C T; Makielski, J C (2000) Preferential block of late sodium current in the LQT3 DeltaKPQ mutant by the class I(C) antiarrhythmic flecainide. Mol Pharmacol 57:101-7
Zhang, S; Zhou, Z; Gong, Q et al. (1999) Mechanism of block and identification of the verapamil binding domain to HERG potassium channels. Circ Res 84:989-98
Makielski, J C; Limberis, J; Fan, Z et al. (1999) Intrinsic lidocaine affinity for Na channels expressed in Xenopus oocytes depends on alpha (hH1 vs. rSkM1) and beta 1 subunits. Cardiovasc Res 42:503-9
Nagatomo, T; Fan, Z; Ye, B et al. (1998) Temperature dependence of early and late currents in human cardiac wild-type and long Q-T DeltaKPQ Na+ channels. Am J Physiol 275:H2016-24
Fan, Z; George Jr, A L; Kyle, J W et al. (1996) Two human paramyotonia congenita mutations have opposite effects on lidocaine block of Na+ channels expressed in a mammalian cell line. J Physiol 496 ( Pt 1):275-86
Undrovinas, A I; Makielski, J C (1996) Blockade of lysophosphatidylcholine-modified cardiac Na channels by a lidocaine derivative QX-222. Am J Physiol 271:H790-7
Shander, G S; Undrovinas, A I; Makielski, J C (1996) Rapid onset of lysophosphatidylcholine-induced modification of whole cell cardiac sodium current kinetics. J Mol Cell Cardiol 28:743-53
Makielski, J C (1996) The heart sodium channel phenotype for inactivation and lidocaine block. Jpn Heart J 37:733-9
Makielski, J C; Limberis, J T; Chang, S Y et al. (1996) Coexpression of beta 1 with cardiac sodium channel alpha subunits in oocytes decreases lidocaine block. Mol Pharmacol 49:30-9