Ion binding in the Open HCN Pacemaker Channel Pore: Fast Mechanisms to Shape “Slow” Channels

IH pacemaker channels carry a mixed monovalent cation current that, under physiological ion gradients, reverses at ∼−34 mV, reflecting a 4:1 selectivity for K over Na. However, IH channels display anomalous behavior with respect to permeant ions such that (a) open channels do not exhibit the outward rectification anticipated assuming independence; (b) gating and selectivity are sensitive to the identity and concentrations of externally presented permeant ions; (c) the channels' ability to carry an inward Na current requires the presence of external K even though K is a minor charge carrier at negative voltages. Here we show that open HCN channels (the hyperpolarization-activated, cyclic nucleotide sensitive pore forming subunits of IH) undergo a fast, voltage-dependent block by intracellular Mg in a manner that suggests the ion binds close to, or within, the selectivity filter. Eliminating internal divalent ion block reveals that (a) the K dependence of conduction is mediated via K occupancy of site(s) within the pore and that asymmetrical occupancy and/or coupling of these sites to flux further shapes ion flow, and (b) the kinetics of equilibration between K-vacant and K-occupied states of the pore (10–20 μs or faster) is close to the ion transit time when the pore is occupied by K alone (∼0.5–3 μs), a finding that indicates that either ion:ion repulsion involving Na is adequate to support flux (albeit at a rate below our detection threshold) and/or the pore undergoes rapid, permeant ion-sensitive equilibration between nonconducting and conducting configurations. Biophysically, further exploration of the Mg site and of interactions of Na and K within the pore will tell us much about the architecture and operation of this unusual pore. Physiologically, these results suggest ways in which “slow” pacemaker channels may contribute dynamically to the shaping of fast processes such as Na-K or Ca action potentials

Subunit Stoichiometry of Cyclic Nucleotide-Gated Channels and Effects of Subunit Order on Channel Function

AbstractCyclic nucleotide-gated (CNG) ion channels are multimeric structures containing at least two subunits. However, the total number of subunits per functional channel is unknown. To determine the subunit stoichiometry of CNG ion channels, we have coexpressed the 30 pS conductance bovine retinal channel (RET) with an 85 pS conductance chimeric retinal channel containing the catfish olfactory channel P region (RO133). When RO133 and RET monomers are coexpressed, channels with four distinct intermediate conductances are observed. Dimer constructs reveal that two of these conductance levels arise from channels with the same subunit composition (2 RO133:2 RET) but distinct subunit order (like subunits adjacent to each other versus like subunits across from each other). Thus, the data demonstrate that cyclic nucleotide-gated ion channels are tetrameric like the related voltage-gated potassium ion channels; the order of subunits affects the conductance of the channel; and the channel has 4-fold symmetry in which four asymmetric subunits assemble head to tail around a central axis

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

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

[Mg²⁺]_in does not modify closing kinetics and closing does not intrude into the block time domain.

<p><b>A,B.</b> Leak sweep subtracted tail currents in the absence or presence of 2²⁺ and absence of cAMP normalized to the peak amplitude of each recording then averaged (<b>A</b>: 10 and 16 separate recordings) or same records after scaling of the 0 Mg²⁺ record (<b>B</b>). The SEM of these averaged records is included as a pixilated halo around the records in <b>A</b> and <b>D</b>. <b>C.</b> Deactivation envelopes determined in the absence (open circles) and presence (filled circles) of 2 mM Mg²⁺ (3–8 determinations per point). The continuous line represents a mean +100 mV tail current (14 separate recordings each normalized to the peak amplitude before averaging). At no time were the envelope amplitudes in the absence and presence of Mg²⁺ significantly different (Student's t-tests). <b>D.</b> The initial 2 ms of +100 mV tail currents collected in the absence of internal Mg²⁺ and the absence or presence of cAMP (normalized to the peak amplitude during the 2 ms window then averaged).</p

A modular model describes the cAMP enhancement of HCN2 activation and acceleration of [Mg²⁺]_in block.

<p><b>A.</b> Observed (obs) and model generated (model) values of the V_1/2 and P_MAX of channel activation and P_CL (the probability that the linker is in the resting configuration which we assume is reported as the slow component of block) each in the absence and presence of cAMP. The observed apparent affinities (K_1/2) were either determined by fits of the Hill equation to model-generated concentration response curves or, for the observed K_1/2 of cAMP modulation of gating, taken from published values <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0101236#pone.0101236-Chen2" target="_blank">[72]</a>. <b>B.</b> Predicted behavior of τ_FAST (thick line) and τ_SLOW (thin line) as a function of the membrane potential. Curves were generated using <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0101236#pone.0101236.e003" target="_blank">equation 3</a> with k₂ set to zero (see text for details).</p

cAMP acceleration of [Mg²⁺]_in block is mediated <i>via</i> ligand occupancy of the cyclic nucleotide-gating ring.

<p><b>A.</b> Average of 8 consecutive active sweeps acquired from a patch expressing HCN2-R591E channels in response to the sequential IV voltage paradigm. Intracellular Mg²⁺ was 1 mM. <b>B.</b> Expanded view of activation (Left panel) and deactivation (at the holding potential of −40 mV; Right Panel) of HCN2-R591E obtained in the absence (Pre), presence (Plus) and following washout (Post) of 30 µM cAMP. Records are from same patch as A and are each averages of 8 sweeps acquired in response to the active paradigm before subtraction of the averaged interlaced leak records. <b>C.</b> Expanded views of the leak subtracted currents recorded at +50 and +200 mV (as indicated) in the absence, presence and following washout of 30 µM cAMP (traces and legend as in <b>B</b>). Red lines are fits of a single exponential function. Residuals are shown vertically offset for clarity. <b>D,E.</b> Time constant of block by 1 mM intracellular Mg²⁺ of HCN2-R591E (<b>D</b>) and HCN2 (<b>E</b>) in the absence or presence of 30 or 300 µM cAMP. For HCN2 but not the cAMP-disabled construct, HCN2-R591E, block kinetics in the presence of cAMP were significantly different from block in the absence of cAMP while the speed of block of HCN2-R591E in the absence or presence of cAMP was not different from that of block of HCN2 in the absence of the nucleotide (one-way ANOVA at each voltage with 11–20 determinations per point).</p

Optimized values of rate constants and gating modifier variables used in the simulations shown in Figure 8.

<p>Gating parameters were estimated using time course fitting of HCN2 currents while those describing Mg²⁺ block kinetics were derived from block in the presence of cAMP as shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0101236#pone-0101236-g004" target="_blank">figures 4</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0101236#pone-0101236-g005" target="_blank">5</a> (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0101236#s2" target="_blank">methods</a> for details). Where plus or minus cAMP parameter windows are left blank, the values are constrained to be equivalent to that shown in the other condition for that model. Superscripted #'s refer to the appropriate equations in the methods that were used to determine the value of the indicated parameter.</p

cAMP abolishes a slow blocking population of channels.

<p><b>A.</b> k₁ determined from the slopes of the regression lines in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0101236#pone-0101236-g004" target="_blank">Figure 4C–E</a> plotted against the depolarizing step potential in the presence or absence of cAMP. Dashed lines represent fits of <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0101236#pone.0101236.e001" target="_blank">equation 1</a> (see text for details). <b>B,C.</b> Compound rate constants determined in the presence (<b>B</b>) and absence (<b>C</b>) of cAMP. Black symbols: k′′′ (equal to k₂[Mg²⁺]_out+k₋₁+k₋₂ at 1 mM [Mg²⁺]_out) as obtained from the y-intercepts in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0101236#pone-0101236-g004" target="_blank">Figure 4C–E</a>. Teal and blue symbols: k′′ (equal to k₋₁+k₋₂ obtained according to <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0101236#pone.0101236.e015" target="_blank">equation 6</a>) at 2 mM and 3 mM [Mg²⁺]_in, respectively. The red symbol at −135 mV is set to 10⁵ s⁻¹ in keeping with the observation that recovery of current is faster than the time constant of the clamp at that voltage <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0101236#pone.0101236-Lyashchenko1" target="_blank">[64]</a>. The long and short dashed lines (<b>B</b>) represent the optimized behavior of k₋₁ and k₋₂, respectively obtained from fits to the black and red circles. This fit reported s⁻¹, δ-₁  = 0.306, s⁻¹ and δ₋₂  = 0.303. <b>D.</b> Black and grey symbols show the fractional unblocked current. The ratios at 0 mV are omitted as this potential is close to the reversal potential and, therefore, poorly defined. Teal and blue lines: the probability channels are unblocked (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0101236#pone.0101236.e004" target="_blank">equation 4</a>) using the scheme II parameters determined in <b>A–C</b>. The black line is a fit of <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0101236#pone.0101236.e004" target="_blank">equation 4</a> wherein both k₂ and k₋₂ are zero; it represents the predicted exponential behavior if Mg²⁺ block were to accord to Scheme I. <b>E,F.</b> Plots of the fractional unblocked current and the relative amplitude of the fast component of block (zero time extrapolation of the fast component with respect to the sum of zero time amplitudes of the fast and slow components, A_f and A_s respectively – right hand aspect of <b>F</b>). Open red symbols (<b>F</b>) represent the estimates obtained in the presence of 2 mM Mg²⁺ and absence of cAMP when a block window of 10 ms was employed in place of the normal 2 ms window. The dashed line (<b>F</b>) is the mean of the fractional fast amplitude determined in the presence of 0.3, 1, 2 and 3 mM Mg²⁺ at 200 mV.</p

cAMP control of [Mg²⁺]_in block and channel opening are kinetically decoupled processes.

<p><b>A,B.</b> Simulated HCN2 currents at −155 mV (Left) and +100 mV (Right) in the absence (Gray) and presence (Black) of cAMP and the absence of intracellular Mg²⁺. The current records were simulated using the rate constants shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0101236#pone-0101236-t001" target="_blank">Table 1</a> wherein cAMP did (B) or did not (A) alter activation transitions. <b>C,D.</b> Probability of occupancy of sum of open and open blocked states with the indicated number of activated voltage sensors (upper panels) and open unblocked probability (lower panels) when cAMP alters only the opening isomerization (<b>C</b>) or both activation and opening reactions (<b>D</b>). In all panels, the probabilities were normalized to the initial maximal open probability under the specified conditions to simplify comparison of simulations generated in the presence and absence of cAMP. Note that the plus and minus cAMP traces in the lower panels superimpose.</p