CAMP control of HCN2 channel Mg2+ block reveals loose coupling between the cyclic nucleotide-gating ring and the pore.

Alex K Lyashchenko,Peter A Goldstein,Gareth R Tibbs,Kacy J Redd

doi:10.1371/journal.pone.0101236

Alex K Lyashchenko, Peter A Goldstein + Show 2 more

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https://doi.org/10.1371/journal.pone.0101236

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Journal: PloS one	Publication Date: Jul 1, 2014
Citations: 93	License type: CC BY 4.0

Affiliation: Columbia University, Cornell University

Abstract

Hyperpolarization-activated cyclic nucleotide-regulated HCN channels underlie the Na+-K+ permeable IH pacemaker current. As with other voltage-gated members of the 6-transmembrane KV channel superfamily, opening of HCN channels involves dilation of a helical bundle formed by the intracellular ends of S6 albeit this is promoted by inward, not outward, displacement of S4. Direct agonist binding to a ring of cyclic nucleotide-binding sites, one of which lies immediately distal to each S6 helix, imparts cAMP sensitivity to HCN channel opening. At depolarized potentials, HCN channels are further modulated by intracellular Mg2+ which blocks the open channel pore and blunts the inhibitory effect of outward K+ flux. Here, we show that cAMP binding to the gating ring enhances not only channel opening but also the kinetics of Mg2+ block. A combination of experimental and simulation studies demonstrates that agonist acceleration of block is mediated via acceleration of the blocking reaction itself rather than as a secondary consequence of the cAMP enhancement of channel opening. These results suggest that the activation status of the gating ring and the open state of the pore are not coupled in an obligate manner (as required by the often invoked Monod-Wyman-Changeux allosteric model) but couple more loosely (as envisioned in a modular model of protein activation). Importantly, the emergence of second messenger sensitivity of open channel rectification suggests that loose coupling may have an unexpected consequence: it may endow these erstwhile “slow” channels with an ability to exert voltage and ligand-modulated control over cellular excitability on the fastest of physiologically relevant time scales.

Highlights

HCN channels represent the structural and functional fusion of two major branches of the potassium channel superfamily depolarization-activated, K+-selective, KV channels and the weakly voltage-sensitive, mono- and divalent cation permeable, cyclic nucleotide-gated CNG channels.Gating in both HCN and depolarization-activated Kv channels involves stabilization of a dilated arrangement of their S6 helical bundles
Inclusion of 30 mM cAMP appears to reversibly accelerate the block. This impression is reinforced by comparison of the records after scaling each record to the maximal current amplitude observed in the presence of the nucleotide (Fig. 2C)
To quantify the effect of cAMP on the Mg2+ block kinetics, we fit the initial phase of the tail currents with a single exponential function

Summary

Introduction

Gating in both HCN and depolarization-activated Kv channels involves stabilization of a dilated arrangement of their S6 helical bundles In both channel classes this rearrangement is energetically coupled to motion of the four S1–S4 voltage-sensing domains and the concomitant reorientation of S4 positive charges with respect to the transmembrane field - albeit with an inverted coupling between the orientation of the sensors and opening of the gate [1,2,3,4,5,6,7,8,9,10,11,12,13,14]. Such conservation suggests that propagated changes that alter the pore of CNG channels may be conserved and serve to alter the permeability properties of HCN channels, a hypothesis that has recently received support at least with respect to blocker binding sites in the inner vestibule of HCN2 [40]

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