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Movements near the gate of a hyperpolarization-activated cation channel
Brad S Rothberg1, Ki Soon Shin, Gary Yellen
1Department of Neurobiology, Harvard Medical School, 220 Longwood Avenue, Boston, MA 02115.
Insights
Cadmium ions (Cd2+) reveal new insights into hyperpolarization-activated cyclic nucleotide-gated (HCN) channel gating. Metal interactions with substituted cysteines in S6 segments modulate channel opening and closing dynamics.
Area of Science:
- Molecular and Cellular Biology
- Biophysics
- Ion Channel Physiology
Background:
- Hyperpolarization-activated cyclic nucleotide-gated (HCN) channels are crucial for pacemaking in cardiac and neuronal cells.
- HCN channels, similar to voltage-gated potassium (Kv) channels, possess an intracellular activation gate within the S6 transmembrane segments.
- Understanding the precise mechanisms of HCN channel gating is essential for comprehending cellular excitability.
Purpose of the Study:
- To investigate the role of metal ion interactions with substituted cysteines in the S6 region of HCN channels.
- To elucidate how these interactions affect the voltage-dependent gating of HCN channels.
- To characterize novel metal-dependent gating effects, including stabilization of open and closed states.
Main Methods:
- Site-directed mutagenesis to introduce cysteine substitutions at specific positions (L466, Q468) in the HCN channel S6 segments.
- Electrophysiological recordings to assess channel function and gating kinetics.
- Application of cadmium ions (Cd2+) at nanomolar concentrations to probe metal-binding sites and their effects on channel gating.
Main Results:
- Cd2+ ions stabilize the open state of HCN channels with L466C substitutions, dependent on native histidines at position 462.
- A Cd2+-dependent "lock-open" effect at L466 involves a bridge between His462 and Cys466 within the same subunit.
- Cysteine substitution at Q468 results in both Cd2+-dependent "lock-open" and "lock-closed" effects, with the latter involving stabilization of the closed state by up to four Cd2+ ions.
Conclusions:
- Metal ion interactions with S6 cysteines provide powerful tools to dissect HCN channel gating mechanisms.
- Cd2+ binding at different S6 positions differentially stabilizes either the open or closed channel states.
- These findings offer novel insights into the conformational changes underlying HCN channel voltage-dependent activation.
Abstract:
Hyperpolarization-activated cation (HCN) channels regulate pacemaking activity in cardiac cells and neurons. Like the related depolarization-activated K+ channels (Kv channels), HCN channels use an intracellular activation gate to regulate access to an inner cavity, lined by the S6 transmembrane regions, which leads to the selectivity filter near the extracellular surface. Here we describe two types of metal interactions with substituted cysteines in the S6, which alter the voltage-controlled movements of the gate. At one position (L466), substitution of cysteine in all four subunits allows Cd2+ ions at nanomolar concentration to stabilize the open state (a "lock-open" effect). This effect depends on native histidines at a nearby position (H462); the lock-open effect can be abolished by changing the histidines to tyrosines, or enhanced by changing them to cysteines. Unlike a similar effect in Kv channels, this effect depends on a Cd2+ bridge between 462 and 466 in the same subunit. Cysteine substitution at another position (Q468) produces two effects of Cd2+: both a lock-open effect and a dramatic slowing of channel activation-a "lock-closed" effect. The two effects can be separated, because the lock-open effect depends on the histidine at position 462. The novel lock-closed effect results from stabilization of the closed state by the binding of up to four Cd2+ ions. During the opening conformational change, the S6 apparently moves from one position in which the 468C cysteines can bind four Cd2+ ions, possibly as a cluster of cysteines and cadmium ions near the central axis of the pore, to another position (or flexible range of positions) where either 466C or 468C can bind Cd2+ in association with the histidine at 462.
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