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G‐protein modulation of N‐type calcium channel gating current in human embryonic kidney cells (HEK 293).
Author(s) -
Jones L P,
Patil P G,
Snutch T P,
Yue D T
Publication year - 1997
Publication title -
the journal of physiology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.802
H-Index - 240
eISSN - 1469-7793
pISSN - 0022-3751
DOI - 10.1113/jphysiol.1997.sp021886
Subject(s) - hek 293 cells , embryonic stem cell , microbiology and biotechnology , human kidney , modulation (music) , kidney , chemistry , gating , calcium , biophysics , biology , endocrinology , medicine , physics , biochemistry , receptor , gene , acoustics
1. Voltage‐dependent inhibition of N‐type calcium currents by G‐proteins contributes importantly to presynaptic inhibition. To examine the effect of G‐proteins on key intermediary transitions leading to channel opening, we measured both gating and ionic currents arising from recombinant N‐type channels (alpha 1B, beta 1b and alpha 2) expressed in transiently transfected human embryonic kidney cells (HEK 293). Recombinant expression of a homogeneous population of channels provided a favourable system for rigorous examination of the mechanisms underlying G‐protein modulation. 2. During intracellular dialysis with GTP gamma S to activate G‐proteins, ionic currents demonstrated classic features of voltage‐dependent inhibition, i.e. strong depolarizing prepulses increased ionic currents and produced hyperpolarizing shifts in the voltage‐dependent activation of ionic current. No such effects were observed with GDP beta S present to minimize G‐protein activity. 3. Gating currents were clearly resolved after ionic current blockade with 0.1 mM free La3+, enabling this first report of gating charge translocation arising exclusively from N‐type channels. G‐proteins decreased the amplitude of gating currents and produced depolarizing shifts in the voltage‐dependent activation of gating charge movement. However, the greatest effect was to induce a approximately 20 mV separation between the voltage‐dependent activation of gating charge movement and ionic current. Strong depolarizing prepulses largely reversed these effects. These modulatory features provide telling clues about the kinetic steps affected by G‐proteins because gating currents arise from the movement of voltage sensors that trigger channel activation. 4. The mechanistic implications of concomitant G‐protein‐mediated changes in gating and ionic currents are discussed. We argue that G‐proteins act to inhibit both voltage‐sensor movement and the transduction of voltage‐sensor activation into channel opening.