Home | Help | Feedback | Subscriptions | Archive | Search | Table of Contents

This Article Full Text Full Text (PDF, 304K) PPT slides of all figures Alert me when this article is cited Citation Map Services Email this article Similar articles in this journal Similar articles in PubMed Alert me to new content in the JGP Download to citation manager Citing Articles Citing Articles via CrossRef Citing Articles via Google Scholar Google Scholar Articles by Yamagishi, T. Articles by Tomaselli, G. F. Search for Related Content PubMed PubMed Citation Articles by Yamagishi, T. Articles by Tomaselli, G. F. Social Bookmarking                            What's this?

Original Article

Functional Evidence for Helices in the Pore

Toshio Yamagishi^a, Ronald A. Li^a, Kate Hsu^a, Eduardo Marbán^a, and Gordon F. Tomaselli^a

^a Institute of Molecular and Cellular Cardiology, Department of Medicine, The Johns Hopkins University, Baltimore, MD 21205
Department of Medicine, Division of Cardiology, The Johns Hopkins University School of Medicine, 844 Ross Building, Baltimore, MD 21205.(410) 955-7953

gtomasel{at}jhmi.edu

The permeation pathway of the Na channel is formed by asymmetric loops (P segments) contributed by each of the four domains of the protein. In contrast to the analogous region of K channels, previously we (Yamagishi, T., M. Janecki, E. Marban, and G. Tomaselli. 1997. Biophys. J. 73:195–204) have shown that the P segments do not span the selectivity region, that is, they are accessible only from the extracellular surface. The portion of the P-segment NH₂-terminal to the selectivity region is referred to as SS1. To explore further the topology and functional role of the SS1 region, 40 amino acids NH₂-terminal to the selectivity ring (10 in each of the P segments) of the rat skeletal muscle Na channel were substituted by cysteine and expressed in tsA-201 cells. Selected mutants in each domain could be blocked with high affinity by externally applied Cd²+ and were resistant to tetrodotoxin as compared with the wild-type channel. None of the externally applied sulfhydryl-specific methanethiosulfonate reagents modified the current through any of the mutant channels. Both R395C and R750C altered ionic selectivity, producing significant increases in K⁺ and NH₄⁺ currents. The pattern of side chain accessibility is consistent with a pore helix like that observed in the crystal structure of the bacterial K channel, KcsA. Structure prediction of the Na channel using the program PHDhtm suggests an helix in the SS1 region of each domain channel. We conclude that each of the P segments undergoes a hairpin turn in the permeation pathway, such that amino acids on both sides of the putative selectivity filter line the outer mouth of the pore. Evolutionary conservation of the pore helix motif from bacterial K channels to mammalian Na channels identifies this structure as a critical feature in the architecture of ion selective pores.

Key Words: pore helix • cysteine mutagenesis • tetrodotoxin • methanethiosulfonates • cadmium

CiteULike    Complore    Connotea    Del.icio.us    Digg    Facebook    Reddit    Technorati    Twitter    What's this?

Home | Help | Feedback | Subscriptions | Archive | Search | Table of Contents