G-protein modulation of N-type calcium channel gating current in human embryonic kidney cells (HEK 293).
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.
Jones
L P
LP
Department of Biomedical Engineering, Johns Hopkins University School of Medicine, Baltimore, MD 21205, USA.
Patil
P G
PG
Snutch
T P
TP
Yue
D T
DT
D015220:Calcium Channels / Q000187:drug effects / Q000502:physiology*; D002460:Cell Line; D018076:DNA, Complementary / Q000096:biosynthesis; D004594:Electrophysiology; D019204:GTP-Binding Proteins / Q000502:physiology*; D016244:Guanosine 5'-O-(3-Thiotriphosphate) / Q000494:pharmacology; D006153:Guanosine Diphosphate / Q000031:analogs & derivatives / Q000494:pharmacology; D006801:Humans; D015640:Ion Channel Gating / Q000187:drug effects / Q000502:physiology*; D007668:Kidney / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D011994:Recombinant Proteins / Q000378:metabolism; D015398:Signal Transduction / Q000187:drug effects / Q000502:physiology; D013873:Thionucleotides / Q000494:pharmacology
D015220:Calcium Channels; D018076:DNA, Complementary; D011994:Recombinant Proteins; D013873:Thionucleotides; D006153:Guanosine Diphosphate; D016244:Guanosine 5'-O-(3-Thiotriphosphate); C023427:guanosine 5'-O-(2-thiodiphosphate); D019204:GTP-Binding Proteins