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Related Experiment Videos

Separable gating mechanisms in a Mammalian pacemaker channel.

Vincenzo Macri1, Catherine Proenza, Eugene Agranovich

  • 1Ion Channel Laboratory, Simon Fraser University, Burnaby, British Columbia V5A 1S6, Canada.

The Journal of Biological Chemistry
|July 18, 2002
PubMed
Summary

The GYG motif in hyperpolarization-activated cyclic nucleotide-gated (HCN) pacemaker channels regulates channel gating. A G404S mutation eliminated the slowly activating current (I(f)) but not the instantaneous current (I(inst)).

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Area of Science:

  • Biophysics
  • Molecular Biology
  • Cardiovascular Physiology

Background:

  • Hyperpolarization-activated cyclic nucleotide-gated (HCN) channels are crucial for cardiac pacemaking.
  • These channels conduct both K(+) and Na(+) ions, featuring a K(+)-like GYG motif in their selectivity filter.
  • The precise role of the GYG motif in HCN channel gating remains incompletely understood.

Purpose of the Study:

  • To investigate the role of the GYG signature sequence in the selectivity filter of mouse HCN2 (mHCN2) channels in regulating channel gating.
  • To determine the differential effects of a GYG mutation on the slowly activating (I(f)) and instantaneous (I(inst)) current components of mHCN2 channels.

Main Methods:

  • Site-directed mutagenesis to introduce a G404S mutation in the mHCN2 GYG sequence.

Related Experiment Videos

  • Electrophysiological recordings (whole-cell patch-clamp) to measure ionic currents.
  • Confocal imaging and immunocytochemistry to assess protein localization.

    • Main Results:

    • The G404S mutation selectively eliminated the slowly activating current (I(f)) while leaving the instantaneous current (I(inst)) unaffected.
    • G404S protein localized to the cell periphery, indicating proper plasma membrane insertion.
    • The deactivation rate and amplitude of I(f) were dependent on the K(+)/Na(+) driving force ratio, unlike I(inst).
    • I(f) amplitude was not accurately predicted by independent K(+) and Na(+) flow, suggesting complex gating.

    Conclusions:

    • The GYG motif in the mHCN2 selectivity filter plays a critical role in the voltage-dependent gating of the slowly activating current (I(f)).
    • Pacemaker channels exhibit at least two distinct gating mechanisms: one sensitive to voltage and ion driving forces (I(f)) and another insensitive (I(inst)).
    • These findings provide insights into the molecular basis of pacemaker channel function and ion permeation selectivity.