The extracellular domain of the beta1 subunit is both necessary and sufficient for beta1-like modulation of sodium channel gating.
The type IIA voltage-gated sodium Na(+) channel from rat brain is composed of a large, pore-forming alpha subunit and the auxiliary subunits beta1 and beta2. When expressed in Xenopus oocytes, the beta1 subunit modulates the gating properties of the type IIA alpha subunit, resulting in acceleration of both inactivation and recovery from inactivation and in a negative shift in the voltage dependence of fast inactivation. The beta1 subunit is composed of an extracellular domain with a single immunoglobulin-like fold, a single transmembrane segment, and a small intracellular domain. A series of chimeras with exchanges of domains between the Na(+) channel beta1 and beta2 subunits and between beta1 and the structurally related protein myelin P0 were constructed and analyzed by two-microelectrode voltage clamp in Xenopus oocytes. Only chimeras containing the beta1 extracellular domain were capable of beta1-like modulation of Na(+) channel gating. Neither the transmembrane segment nor the intracellular domain was required for modulation, although mutation of Glu(158) within the transmembrane domain altered the voltage dependence of steady-state inactivation. A truncated beta1 subunit was engineered in which the beta1 extracellular domain was fused to a recognition sequence for attachment of a glycosylphosphatidylinositol membrane anchor. The beta1(ec)-glycosylphosphatidylinositol protein fully reproduced modulation of Na(+) channel inactivation and recovery from inactivation by wild-type beta1. Our findings demonstrate that extracellular domain of the beta1 subunit is both necessary and sufficient for the modulation of Na(+) channel gating.
McCormick
K A
KA
Department of Pharmacology, University of Washington, Seattle, Washington 98195, USA.
Srinivasan
J
J
White
K
K
Scheuer
T
T
Catterall
W A
WA
D000595:Amino Acid Sequence; D000818:Animals; D001921:Brain / Q000378:metabolism; D002462:Cell Membrane / Q000378:metabolism; D017261:Glycosylphosphatidylinositols / Q000235:genetics; D015640:Ion Channel Gating / Q000235:genetics*; D008565:Membrane Proteins / Q000737:chemistry / Q000235:genetics; D008845:Microinjections; D008969:Molecular Sequence Data; D009154:Mutation; D018993:Myelin P0 Protein / Q000235:genetics; D009865:Oocytes; D018408:Patch-Clamp Techniques; D012333:RNA, Messenger / Q000235:genetics; D051381:Rats; D011993:Recombinant Fusion Proteins / Q000235:genetics; D015222:Sodium Channels / Q000737:chemistry / Q000235:genetics*; D014981:Xenopus
D017261:Glycosylphosphatidylinositols; D008565:Membrane Proteins; D018993:Myelin P0 Protein; D012333:RNA, Messenger; D011993:Recombinant Fusion Proteins; D015222:Sodium Channels