Intracellular Mg²⁺ is a voltage-dependent pore blocker of HCN channels

Abstract

Hyperpolarization-activated cyclic nucleotide-gated (HCN) channels are activated by membrane hyperpolarization that creates time-dependent, inward rectifying currents, gated by the movement of the intrinsic voltage sensor S4. However, inward rectification of the HCN currents is not only observed in the time-dependent HCN currents, but also in the instantaneous HCN tail currents. Inward rectification can also be seen in mutant HCN channels that have mainly time-independent currents (5). In the present study, we show that intracellular Mg 2ϩ functions as a voltagedependent blocker of HCN channels, acting to reduce the outward currents. The affinity of HCN channels for Mg 2ϩ is in the physiological range, with Mg 2ϩ binding with an IC50 of 0.53 mM in HCN2 channels and 0.82 mM in HCN1 channels at ϩ50 mV. The effective electrical distance for the Mg 2ϩ binding site was found to be 0.19 for HCN1 channels, suggesting that the binding site is in the pore. Removing a cysteine in the selectivity filter of HCN1 channels reduced the affinity for Mg 2ϩ , suggesting that this residue forms part of the binding site deep within the pore. Our results suggest that Mg 2ϩ acts as a voltage-dependent pore blocker and, therefore, reduces outward currents through HCN channels. The pore-blocking action of Mg 2ϩ may play an important physiological role, especially for the slowly gating HCN2 and HCN4 channels. Mg 2ϩ could potentially block outward hyperpolarizing HCN currents at the plateau of action potentials, thus preventing a premature termination of the action potential. inward rectifying; hyperpolarization-activated current; instantaneous currents; divalent block; cysteine; hyperpolarization-activated cyclic nucleotide RHYTHMIC ACTIVITY OF PACEMAKER cells in the heart and thalamic neurons in the brain are dependent on the inward I h current through hyperpolarization-activated cyclic nucleotide-gated (HCN) channels (29,36). Following an action potential in pacemaker cells, I h contributes to the currents that slowly depolarize the membrane potential to threshold, thereby initiating another action potential (7). Four mammalian HCN channels have been cloned: HCN1-HCN4 (11,21,35). The deletion of HCN channels, or the presence of naturally occurring mutations in HCN channels, has been shown to have significant physiological consequences (20,25,37,40). HCN2 knockout mice, for example, showed spontaneous absence seizures and cardiac sinus dysrhythmia (20).HCN channels are members of the superfamily of voltagegated ion channels, possessing features such as a tetrameric structure, with each subunit containing six transmembrane domains (S1-S6) (29,36). In addition, HCN channels have a pore domain that shows conservation with voltage-gated potassium (Kv) channels, including a GYG signature motif in the selectivity filter, even though HCN channels are only modestly more selective (3:1) for K ϩ over Na ϩ (11, 21). Similar to Kv channels, HCN channels have an intracellular gate at the base of S6 that prevents access of ions to the pore (32, 33)....

Functionalized ZnO nanorod-based selective magnesium ion sensor for intracellular measurements

“…Once the microelectrodes were inside the cell, that is isolated from the extracellular buffer solution, an electrochemical potential difference signal was detected identifying the activity of Mg 2+ . The measured intracellular Mg 2+ concentration for human adipocytes was 0.4 to 0.5 mM and for frog oocyte it was 0.8 to 0.9 mM, closely consistent with the intracellular concentration reported in the literature (Nadler and Scott, 1994;Gabriel and Gunzel, 2007;Vemana et al, 2008;Lyashchenko and Tibbs, 2008).…”

cAMP Control of HCN2 Channel Mg2+ Block Reveals Loose Coupling between the Cyclic Nucleotide-Gating Ring and the Pore

“…It has previously been shown that intracellular Mg 2+ acts as a voltage dependent blocker of open HCN channels [64], [65]. The voltage dependence of the block and its sensitivity to mutation of residues forming the inner face of the selectivity filter is consistent with the Mg 2+ binding site lying close to or within the selectivity filter itself.…”

Stress Impairs Prefrontal Cortical Function via D1 Dopamine Receptor Interactions With Hyperpolarization-Activated Cyclic Nucleotide-Gated Channels

“…It should be noted that I h does not necessarily require hyperpolarization to open, as HCN channels have a tonically active leak current component (67–71) that is blocked by ZD7288 (67, 72). Furthermore, HCN channels and D1Rs are found near a constellation of cAMP signaling proteins at dendritic spines, whereas HCN channels on dendrites have few cAMP signaling proteins nearby (26).…”