Differential effects of RGK proteins on L-type channel function in adult mouse skeletal muscle

Biophys J. 2014 May 6;106(9):1950-7. doi: 10.1016/j.bpj.2014.03.033.

Abstract

Work in heterologous systems has revealed that members of the Rad, Rem, Rem2, Gem/Kir (RGK) family of small GTP-binding proteins profoundly inhibit L-type Ca(2+) channels via three mechanisms: 1), reduction of membrane expression; 2), immobilization of the voltage-sensors; and 3), reduction of Po without impaired voltage-sensor movement. However, the question of which mode is the critical one for inhibition of L-type channels in their native environments persists. To address this conundrum in skeletal muscle, we overexpressed Rad and Rem in flexor digitorum brevis (FDB) fibers via in vivo electroporation and examined the abilities of these two RGK isoforms to modulate the L-type Ca(2+) channel (CaV1.1). We found that Rad and Rem both potently inhibit L-type current in FDB fibers. However, intramembrane charge movement was only reduced in fibers transfected with Rad; charge movement for Rem-expressing fibers was virtually identical to charge movement observed in naïve fibers. This result indicated that Rem supports inhibition solely through a mechanism that allows for translocation of CaV1.1's voltage-sensors, whereas Rad utilizes at least one mode that limits voltage-sensor movement. Because Rad and Rem differ significantly only in their amino-termini, we constructed Rad-Rem chimeras to probe the structural basis for the distinct specificities of Rad- and Rem-mediated inhibition. Using this approach, a chimera composed of the amino-terminus of Rem and the core/carboxyl-terminus of Rad inhibited L-type current without reducing charge movement. Conversely, a chimera having the amino-terminus of Rad fused to the core/carboxyl-terminus of Rem inhibited L-type current with a concurrent reduction in charge movement. Thus, we have identified the amino-termini of Rad and Rem as the structural elements dictating the specific modes of inhibition of CaV1.1.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Amino Acid Sequence
  • Animals
  • Calcium Channels, L-Type / metabolism*
  • Cell Membrane / metabolism
  • Electrophysiological Phenomena
  • Male
  • Mice
  • Molecular Sequence Data
  • Monomeric GTP-Binding Proteins / chemistry
  • Monomeric GTP-Binding Proteins / metabolism*
  • Muscle Fibers, Skeletal / cytology
  • Muscle Fibers, Skeletal / metabolism*
  • Muscle Fibers, Skeletal / physiology
  • ras Proteins / chemistry
  • ras Proteins / metabolism*

Substances

  • CACNA1S protein, mouse
  • Calcium Channels, L-Type
  • Rem protein, mouse
  • Rrad protein, mouse
  • Monomeric GTP-Binding Proteins
  • ras Proteins