The proposal is to examine mechanisms of local anesthetic action on sodium channels with particular reference to the antiarrhythmic properties of local anesthetics in cardiac muscle sodium channels following an ischemic episode. The central hypothesis of the proposed studies is that local anesthetics stabilize inactivated states of the sodium channel by altering the coupling between channel activation and inactivation, functioning as allosteric effectors of channel gating. This hypothesis differs from the """"""""modulated receptor"""""""" hypothesis which has been influential in the past two decades; the latter proposes that the inactivated state has a higher affinity for local anesthetics than the resting state. The strategy is to employ mutations that modify fast and slow inactivation to examine the molecular determinants of local anesthetic properties, using standard electrophysiologic voltage clamp techniques to study channels expressed either in Xenopus oocytes or in a mammalian kidney cell line. There are 3 specific aims: Using 4 types of mutations which disrupt fast inactivation in rat skeletal muscle by different mechanisms, studies will be carried out to determine how each type alters time- and voltage-dependent local anesthetic effects. The mutations are in the hydrophobic triplet (IFM) on the III-IV interdomain linker that may be the inactivation latch; residues on the cytoplasmic end of domain III S4-S5 and IV-S6 that may be the inactivation receptor; hinge residues of the linker lateral to IFM; and external residues in domain IV that influence activation-inactivation. Amino acids of different size, charge and hydrophobicity will be substituted at the 402 position to determine how their properties influence coupling between slow inactivation and other gating processes, and how such modifications alter local anesthetic action. Two point mutations, W402C, tryptophan to cysteine in domain I and T698M in domain II, will be used in studies to determine how coupling interactions among gating processes influence differences between local anesthetic action on skeletal muscle and cardiac sodium channels. These mutations both modify slow inactivation in skeletal muscle channels, and alter fast gating processes via different mechanisms.
Showing the most recent 10 out of 27 publications
