Abstract
We searched for sites on the α-subunit of the fast Na+channel responsible for the difference in GTX (grayanotoxin) sensitivity of the skeletal- and cardiac-muscle Na+current. cDNA clones, encoding the skeletal or cardiac isoforms of the α-subunit, were inserted into a mammalian expression vector and transiently transfected into human embryonic kidney cells. The expressed channels were measured using whole-cell patch-clamp techniques and examined for GTX sensitivity. As a measure of GTX sensitivity, we used relative chord conductance (ratio of maximum chord conductance of noninactivating GTX-modified Na+ currents to that of unmodified peak currents). Wild-type channels from skeletal muscle (μ1) were more sensitive to GTX modification than wild-type cardiac channels (rH1) by a factor of 1.6. To facilitate exploration of α-subunit sites determining GTX sensitivity, we used SHHH, a chimera of skeletal muscle (S) domain D1 and heart muscle (H) domains D2D3D4 with supernormal sensitivity to GTX I (1.5-fold of wild-type μ1). Successive replacement of Ser-251 (D1S4–S5 intracellular loop) and Ile-433 (D1S6 transmembrane segment), with corresponding rH1 residues Ala and Val, reduced, in a stepwise manner, the GTX sensitivity of the chimera and related mutants to that of wild-type rHl. We concluded that, in addition to Ile-433, known as the GTX-binding site, Ser-251 represents a novel site for GTX modification.
Footnotes
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This work was supported by Grants 11470011 (to K.Y.) and 11770023 (to E.K.) from the Ministry of Education and Culture of Japan and by the research Grant 11C-1 (to I.S.) for cardiovascular diseases from the Ministry of Health and Welfare.
- Abbreviations:
- GTX
- grayanotoxin
- D
- domain
- S
- segment
- HEK
- human embryonic kidney
- Po
- channel open probability
- I-V
- current-voltage
- INa
- Na+ current
- μ1
- α-subunit of rat skeletal muscle Na+ channel
- rH1
- α-subunit of rat heart Na+ channel
- Received April 23, 2001.
- Accepted June 13, 2001.
- The American Society for Pharmacology and Experimental Therapeutics
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