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Correspondence to Gareth R. Tibbs: GRT1{at}columbia.edu
 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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–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.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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–34 mV (Santoro and Tibbs, 1999
; Accili et al., 2002; Robinson and Siegelbaum, 2002). Gating of these channels is slow with respect to many physiological processes, a finding that has influenced the way in which the channels are conceived to contribute to cellular physiology. Thus, while opening and closing of channels formed predominantly from HCN2 and 4 are thought to provide a drive for slow rhythmic events (such as the cardiac action potential and thalamocortical delta and spindle oscillations; McCormick and Bal, 1997; Accili et al., 2002; Biel et al., 2002; Robinson and Siegelbaum, 2002), the contribution of IH to fast nonrhythmic processes (as exemplified by the role of HCN1 and 2 in normalization of the somatic time course of proximal and distal synaptic inputs) is generally considered with respect to the resting input resistance and slow changes therein (Hausser et al., 2000; Reyes, 2001; Robinson and Siegelbaum, 2002; Magee and Johnston, 2005; Ying et al., 2007). By analyzing the behavior of expressed HCN2 channels we show that pacemaker channels undergo rapid changes in conductance that seem poised to allow these slow channels to help shape the fastest cellular processes.
Architecturally, HCN channels are members of the voltage-gated K (KV) channel superfamily. Thus, they have six transmembrane helices (S1–S6) and cytoplasmic amino and carboxy termini. S1–S4 form the voltage sensor while S5, S6, and the intervening loop form the ion conducting pore (Santoro and Tibbs, 1999; Robinson and Siegelbaum, 2002). Functionally, hyperpolarization increases the intracellular accessibility of S4 in both HCN and KV channels (Bell et al., 2003; Vemana et al., 2003), albeit this is linked to opening of the helical bundle at the cytoplasmic end of S6 in HCN channels but closing in other KV channels (Shin et al., 2001; Rothberg et al., 2002, 2003).
HCN2 activation gating is slow due, in part, to the presence of a slow, voltage-independent opening transition (see the representative activation records herein and Shin et al., 2004; Bruening-Wright et al., 2007; Chen et al., 2007) but deactivation is faster than would be expected based on return along such a reaction path (Bruening-Wright et al., 2007; Chen et al., 2007). Further complexity in channel gating is revealed by the finding that the kinetics of deactivation of both native (DiFrancesco, 1984; Maruoka et al., 1994) and expressed (Mannikko et al., 2005; Elinder et al., 2006; Bruening-Wright and Larsson, 2007) channels change as a function of the strength and duration of the hyperpolarizing step used to open the channels. Mechanistically, the origin of this hysteresis was originally ascribed to the existence of multiple closed and open states linearly coupled by voltage-dependent reactions (Maruoka et al., 1994). However, the ability of cAMP binding to the C-terminal gating ring to increase the fully activated open probability, (Shin et al., 2004; unpublished data), of anesthetics to lower this (Lyashchenko et al., 2007), and the observation that the rate of opening of HCN2 saturates at very negative voltages (Chen et al., 2007) argues against this. More recently, HCN channel gating has been interpreted within models wherein the voltage sensors are decoupled from opening such that they can deactivate before the pore closes (Bruening-Wright et al., 2007; Chen et al., 2007) and they can undergo a modal shift in their gating energetics such that activation and/or opening makes gating easier (Mannikko et al., 2005; Elinder et al., 2006; Bruening-Wright and Larsson, 2007).
Architectural conservation between HCN and other K channels extends to the selectivity filter (where HCN channels have a canonical K-selective sequence of CIGYG) but not to the pore helix, S5, or S6, and differences in these regions presumably account for the physiologically critical weak K selectivity (PK:PNa 4:1). Despite these differences, the behavior of native IH and expressed HCN channels suggest their pores, like those of other K channels, contain multiple ion binding sites and are plastic. First, increasing K or lowering Na externally increases the permeability of Na relative to K in native IH (Hestrin, 1987; Frace et al., 1992; Ho et al., 1993; Solomon and Nerbonne, 1993) and expressed HCN channels (Moroni et al., 2000; unpublished data). Second, in the limiting case when K is absent, the ability of IH (Edman and Grampp, 1989; Frace et al., 1992; Wollmuth and Hille, 1992; Solomon and Nerbonne, 1993) and HCN channels (data presented herein) to carry an inward Na current appears to be lost. Third, kinetic and equilibrium parameters describing voltage-dependent gating of both native IH (Hart, 1983; McCormick and Pape, 1990; Wollmuth and Hille, 1992; Maruoka et al., 1994; Wollmuth, 1995) and expressed HCN channels (Azene et al., 2003; Mannikko et al., 2005; Elinder et al., 2006; Lyashchenko et al., 2007; see also data reported herein) are generally, though not universally (Hestrin, 1987), reported to be sensitive to changes in K and Na concentrations. While the effects on ion selectivity could be attributed to anomalous mole fraction effects of permeant ions, the other behaviors indicate plastic coupling of the pore to the energetics of opening or to intrinsic or extrinsic conductance blocking mechanisms. Indeed, it has been suggested that the K dependence of current flow and/or of gating could represent rearrangements of the pore analogous to P/C-type inactivation in KV channels (Kurata and Fedida, 2006), but the relationships between the architecture and operation of the pore in HCN channels remains poorly understood.
Here we show that the current carried by open HCN channels is shaped by at least two distinct ion sensitive mechanisms: (1) a submillisecond block by intracellular Mg that has a similar voltage dependence to that observed in Shaker K channels and (2) a K depletion–mediated collapse of conduction that is asymmetrical with respect to flux and that occurs with kinetics close to the maximal K ion transit rate through the HCN channel pore. The former mechanism indicates that the architecture of the inner face of the selectivity filter of HCN and Shaker channels may be similar while the latter observation helps define the origin of the small, K-dependent conductance of pacemaker channels. The relationship between these processes and ion-sensitive modifications of activation and deactivation gating reactions remains to be explored.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Animal procedures
Xenopus oocytes were harvested according to a Columbia University approved protocol (PI#366G CU#2928) (Lyashchenko et al., 2007).
Electrophysiology
Two-electrode voltage clamp (TEVC) and excised inside-out patch clamp (IOPC) recordings were made at 22–25°C using a Warner Instruments OC-725C amplifier and an Axon Instruments Axopatch 200B amplifier (in the resistive mode), respectively. In both configurations, the amplifier gain was kept below the point where the fastest transients would be internally clipped and signals were digitized (at 20 kHz in TEVC and 20, 100, or 200 kHz in IOPC) using an ITC-18 interface (Instrutech Corporation) controlled by Pulse software (HEKA Elektronik). In IOPC, but not TEVC, analogue compensation of linear ionic and capacitive currents was applied. In initial experiments the Axopatch 200B stimulus filter was set to 20 µs but in wide bandwidth experiments this was routinely set to 2 µs. If IOPC currents exceeded 500 pA at the most extreme voltages, we applied analogue series resistance compensation (90% correction, 20 µs lag, resistance set to that of the electrode before seal formation). In TEVC, the wide bandwidth output from the clamp was filtered at 1 kHz using a Frequency devices 902 8-pole Bessel low pass filter while in IOPC the 100 kHz four-pole amplifier output was sampled directly or following additional filtering through a Warner Instruments LPF-8 eight-pole low pass Bessel filter. Sampling and filter corner frequencies are indicated in each figure legend.
Microelectrodes (V-sensing, 1–4 M
; I-passing, 0.1–0.4 M) and patch electrodes (1–2 M) were fabricated from 1B120-F4 (World Precision Instruments) and Kimax-51 (Kimble Glass) borosilicate glass, respectively. Patch electrodes were coated with Sylgard (Dow Corning Corporation). Ag-AgCl ground wires were connected to the bath by 3 M KCl-2% agar salt bridges placed close to, but downstream of, the oocyte. Patch electrodes were moved upstream of the cell after excision.
In all experiments, the extracellular solution was (in mM) 112 MCl, 1 MgCl2, 1 CaCl2, 10 HEPES-free acid, pH 7.4. In IOPC the intracellular solution was (in mM) 112 MCl, 1 EGTA-free acid, 10 HEPES-free acid, pH 7.4 with either MgCl2 (at 1 or 2 mM) or 1 EDTA-free acid, as indicated. The alkali metal (M) salts and the hydroxide used to pH the solutions were chosen to achieve the ion concentrations indicated on each figure. Unless otherwise indicated, 30 µM cAMP was included in intracellular IOPC solutions to saturate cyclic nucleotide enhancement of activation gating as this permitted use of higher voltages and shorter times to fully activate the channels.
To prevent contamination of the patch pipette solution, cells were bathed in the pipette solution during seal formation and the bath was switched to the appropriate intracellular solution before patch excision. As HCN channel activation shifts hyperpolarized upon patch excision, we waited at least 4 min before acquiring IOPC data to allow gating to stabilize to the cell free level.
Paradigms and Analysis
In TEVC, cells were held at –30 mV while the holding potential in IOPC was –40 mV. The voltage paradigms used to collect data are depicted on each figure or described in the appropriate legend. In pilot experiments, we confirmed that the activation voltages were adequate to saturate gating under the indicated conditions (not depicted).
The kinetics of Mg block were analyzed within the context of the model shown in Scheme 1. Within this framework, activation of the voltage sensors is represented by the equilibrium between closed resting (CR) and closed activated (CA) states and is defined by the equilibrium constant, K(V) (all equilibrium constants are reported in the forward direction).
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| (SCHEME 1) |
Opening of the channel is represented by the reaction from CA to O and is defined by the equilibrium constant L. In keeping with the results from several groups, we assume that the opening and closing transitions underlying L are voltage independent (Shin et al., 2004; Bruening-Wright and Larsson, 2007; Bruening-Wright et al., 2007; Chen et al., 2007; Lyashchenko et al., 2007). For simplicity, we further assume that Mg ions (B) associate with a single class of binding sites and do so only when the channel is open such that this association is defined by the equilibrium constant KA, that the Mg bound state, OB, is nonconducting, and that channels do not significantly redistribute between closed and open states in the intervals where Mg block is examined at depolarized potentials. The validity of these assumptions is considered further in the Discussion. Within this framework, we can ignore changes in the open probability (as defined as the sum of being either open or open blocked) and treat the reaction between O and OB as a simple two state process. Accordingly, the kinetics of block and unblock can be described according to Eqs. 1–3.
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 | (2) |
 | (3) |
BLOCK is the single exponential time constant of the decay of the transient component of the predeactivation tail current, and [B] is the free concentration of Mg (0.924 and 1.859 mM) in our standard solutions wherein we add 1 or 2 mM MgCl2 and 1 mM EGTA (determined using WebmaxC http://www.stanford.edu/%7Ecpatton/webmaxc/webmaxcE.htm). IT and IP represent the amplitudes of the transient and plateau components of the predeactivation phase of the tail currents with IT determined from the zero time extrapolation of the block exponential (see Fig. 4 B and Fig. 5 B) or from the current in the absence of internal Mg (I-Mg) minus IP determined in the presence of internal Mg (see Fig. 5 B).
The relationships of kON and kOFF to the applied membrane voltage were fit with Eqs. 4 and 5, respectively, while the fractional block at equilibrium was fit with Eq. 6.
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 | (5) |
 | (6) |
ON and OFF are the effective electrical distance Mg travels across the field (V) to the transition state in the on and off reactions assuming that all the effect of the field arises from a discrete effect on the Mg ion, = ON + OFF, while R, T, and F have their usual meaning.
To facilitate comparison between the IV relationships under different conditions, we superimposed the normalized inward (IM INWARD in Eq. 7), outward (IM OUTWARD in Eq. 8) and net fluxes (sum of inward and outward components) predicted by the GHK current equation. To do so, we let PM equal the relative permeability of ion M with respect to K and then scaled the results such that the net flux was equal to the flux observed at the extreme positive (Fig. 8 E) or negative (all other IV plots) potential of the particular paradigm. ZM is the valence of ion M, while [M]O and [M]I are the external and internal concentrations of M, respectively. In TEVC, we assumed that the internal concentrations of Na and K were 14 and 122 mM, respectively (Zeuthen et al., 2002).
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 | (8) |
Data analysis was performed in PulseFit (HEKA Elektronik) and IgorPro (Wavemetrics Corporation). Data are presented as mean ± SEM or range except for quotients, which are reported with respect to their 95% confidence interval.
Reagents
ZD7288 (Tocris bioscience) solutions were prepared fresh each day by dilution of a 100 mM stock solution (deionized water) that was stored at –20°C for no longer than 1 mo. Spermine and spermidine (obtained as Ultra grade, >99.5% purity, hydrochloride salts from Fluka) solutions were prepared from 30 mM stock solutions (deionized water) that were prepared fresh each day. Other electrophysiology reagents were of the highest purity from Sigma-Aldrich.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Fig. 1 H provides a further demonstration of the complexity of the conductance behavior. Here, the relative amplitude of the inward and outward currents carried in the presence of elevated external K are plotted as a function of the currents carried when physiological levels of Na and K are presented externally. In each case the currents are determined 150 mV positive and negative to the observed reversal potential. These results show that while the inward current shows the strong (6.1-fold) enhancement of the inward flux in the presence of elevated K, the outward flux (which should, in both cases, be dominated by the high internal K concentration) is markedly (0.6 times) smaller when external K is elevated.
Fig. 2 (A, C, and E) shows superimposed representative HCN2 channel currents (left) and expanded views of the tail currents (right) collected from IOPC recordings wherein patches were first stepped to –145 mV for 1.5 s and then stepped to a series of tail potentials between –100 and + 80 mV in 20-mV increments (as indicated next to select tail current traces). In these recordings, currents were acquired in the presence of quasi-physiological gradients of Na and K (Fig. 2 A), symmetrical high K (Fig. 2 C), or in the presence of an inverted Na and K gradient (Fig. 2 E). Fig. 2 (B, D, and F) plots the IV relationships for the three representative recordings with current amplitudes determined during the plateau phase of the tails.
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Further paralleling the findings shown in Fig. 1, elevation of external K results in a slowing of the activation time course, enhancement of the inward current (though, in these experiments, this is only qualitatively demonstrated by comparison across patches), depolarization of the reversal potential (to EREV = -2.7 mV ± 0.4, n = 13), and, most significantly, an emergence of a clear inward rectification of the open channel current.
To explore the role of Na and K in altering channel function in general and influencing alterations in conductance in particular, we inverted the gradients of these ions. If the only effects of Na and K on HCN2 channels arise as a result of a mole fraction–determined occupancy within a static, modestly K-selective selectivity filter, inverting the Na and K ion gradients should result in an enhancement of the inward current amplitude, a slowing of activation gating, and generation of a linear (based on the behavior observed with quasi-physiological concentrations of Na and K, see Fig. 2 B) or modestly inwardly rectifying (according to the GHK equation, gray lines in Fig. 2 F) IV relationship that reverses around +44 mV. Inspection of the current records and the plateau IV plot (Fig. 2, E and F, respectively) reveals that the HCN2 conductance does not hew to the above predictions. Thus, while gating and the current amplitude at negative potentials appear to follow the external K concentration, the channel becomes strongly inwardly rectifying such that it carries no detectable current positive to the anticipated reversal potential. Together, these findings indicate that open channel rectification is influenced by both permeant ions and other mechanisms.
Inward Rectification of the HCN2 Channel Current in Symmetric Elevated K Is Accompanied by a Rapidly Decaying Outward Transient
During the course of the experiments with symmetrical K, we observed, at depolarized potentials, a transient component of the initial phase of the tail current that did not appear to arise from uncompensated capacity transients (this component is not well resolved in recordings such as those in Fig. 2 so was omitted there for clarity). We hypothesized that this transient may arise from a rapid deactivation, inactivation, or block of the HCN2 channels and such a process could account for the emergence of inward rectification.
To address these possibilities, we recorded HCN2 currents at a wide bandwidth and incorporated an interleaved leak subtraction protocol to eliminate any linear capacitive or ionic components that were not excluded from the recordings by analogue compensation. Fig. 3, A and C, shows the voltage paradigm while Fig. 3, B and D, shows a representative recording. In this paradigm, the patch was stepped to –135 mV for 2 s (Fig. 3, A and B, bottom "active" paradigm and record, respectively) or 5 ms (Fig. 3, A and B, top "leak" paradigm and record) and then stepped to + 80 mV for 1 or 3 ms before returning to –135 mV. The longer preconditioning step in the "active" paradigm was sufficient to fully activate the channels while the shorter step in the "leak" paradigm was sufficient to allow the uncompensated capacity transients to decay without evoking channel opening.
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The Transient Outward Current in Symmetric Elevated K Is Carried by the Expressed HCN2 Channels but Does Not Arise from Fast Deactivation or Restricted Diffusional Access of Permeant Ions to the Pore
As a first test of the origin of the outward transient, we asked if it was sensitive to HCN channel blockade and if its suppression correlated with the efficacy of the blocker on the plateau tail current and the inward HCN2 ionic current. Fig. 4 A1 shows averaged, but not otherwise post hoc processed, HCN2 currents (recorded with voltage paradigms as described in Fig. 3 A, bottom) before, during, and after inclusion of the HCN-selective inhibitor, ZD7288, in the bath.
It is apparent that both the inward current at –135 mV and the tail currents on return to the holding potential were reversibly inhibited by ZD7288. Importantly, the stability of the –135-mV current trajectory either side of the brief sojourn (3 ms) at +80 mV reveals that there was no significant change in channel block during the depolarizing interval. Fig. 4 A2 (left) shows, on an expanded time scale, the leak-subtracted currents recorded before, during, and after the step to + 80 mV in the absence, presence, and following washout of ZD7288. These records show that in addition to suppressing the steady-state current at –135 mV and the inward tail currents at –40 mV (Fig. 4 A1), ZD7288 also reduced the amplitudes of both the fast transient and the plateau phases of the outward +80-mV tail current. Fig. 4 A2 (right) shows that a scaled version of the ZD7288 unblocked transient (normalized with respect to the current amplitudes at –135 mV preceding each +80-mV step) superimposed with the control current (and with a scaled version of the ZD7288-washout current). Similarly, Fig. 4 A3 shows the time course of the ZD7288-sensitive component of the outward current (obtained by subtraction of the ZD7288-unblocked current from the control current) and a scaled version plotted with respect to the outward transient obtained in the absence of ZD7288 (Fig. 4 A3, left and right, respectively). Together, these results show that the ZD7288-blocked and unblocked components of the outward transient had identical time courses, indicating that a homogenous population of channels carries the transient.
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These findings confirm that the transient is carried by the expressed HCN2 channels and does not represent a gating current or ionic current arising from recruitment of an endogenous channel.
The data obtained in the experiments reported in Figs. 3 and Figs.4 also serve to eliminate fast deactivation or ion accumulation and/or depletion as mechanisms underlying or contributing to the outward transient based rectification of the HCN2 current.
With respect to gating, several lines of evidence suggest HCN channels display hysteresis in gating such that (a) deactivation is faster than would be anticipated if this were to proceed by reversal of the slow, voltage-independent opening transition of activated channels, a finding that has been interpreted to mean that open channels can deactivate permitting them to close rapidly (Chen et al., 2007); (b) movement of the voltage sensors is thought to be dependent on the holding potential such that open channels activate more readily than closed channels (Mannikko et al., 2005; Elinder et al., 2006; Bruening-Wright and Larsson, 2007). However, in the former canon, deactivated open channels would remain conducting at +80 mV while any channels that closed would be obliged to reopen along the original, slow path. As the channels reestablish the full conductance at –135 mV without the emergence of slow reactivation (see, for example, Fig. 3, B and D), this route can be discounted. Similarly, a key effect of the modal shift in activation gating is that tail currents develop a sigmoidicity upon prolonged or strong activation, that is, closing slows down, an effect that is opposite to the observed fast loss of conduction on depolarization.
Does ion accumulation and/or depletion in the unstirred layers outside of the channel contribute to the observed current decline? This hypothesis predicts that the time constant of decay should be faster when the current flow is larger. To explore this, we plotted the time constants of current decay at +80 mV against the amplitude of the current determined at –135 mV immediately before the depolarizing step. Inspection of Fig. 4 F shows that there was no marked dependency of the time constant of decay at +80 mV in seven patches wherein the control current amplitude varied between 98 and 1120 pA (black regression line). To eliminate the possibility that differences in patch geometry were occluding observation of a negative correlation between the time constant and the current density, we examined the time constants obtained in the absence and presence of ZD7288 within three separate recordings (symbols linked by red regression lines represent data from each separate patch). In two out of three such experiments inclusion of intracellular ZD7288 suppressed the maximal current by 90% but had no marked effect on the time constant. Although, in a third recording, the time constant accelerated upon wash on of the inhibitor, this response is the inverse of that predicted by the ion accumulation/depletion model.
Together, the above results indicate the mechanism for rectification of the K conductance is an intrinsic property of the channel but is distinct from voltage-dependent closing of the S6 activation gate. Accordingly, we hypothesized that the inward rectification could arise as a consequence of intrinsic conductance asymmetry, an ion-dependent collapse of conduction (perhaps akin to P/C-type inactivation) or a voltage-dependent block by an internal cation. Below, we present evidence that suggests each of these processes may contribute to the behavior of open HCN2 channels as a function of permeant ion concentrations.
Inward Rectification of the HCN2 Channel K Current Arises as a Result of a Fast, Voltage-dependent Block by Internal Mg
To explore the possibility that current rectification arises from a voltage-dependent block by internal polyvalent cations, we asked what effect the presence or absence of internal divalent ions or polyamines would have on the current carried by symmetrical elevated K. To do so, we used a hybrid of the paradigms described in Figs. 1 and Figs.3. Thus, we recorded interleaved "active" and "leak" IV families (wherein the patch was stepped to voltages between ±200 mV for 1–3 ms with steps applied at 10 or 4 Hz in 50-mV increments) that were distinguished by the duration of the conditioning steps to the activation voltage. In the "active" paradigm the patch was held at the activation voltage for 2 s and returned to this potential between test steps, while in the "leak" paradigm the patch was held at –40 mV and stepped to the activation voltage for only 5 ms before (and after) each test step.
Fig. 5 A shows the averaged but unsubtracted records obtained in response to the "active" protocol when the internal solution contained 1 mM EDTA (top) or 1 mM Mg (bottom) in addition to 1 mM EGTA in both cases. Fig. 5 B shows the same records following subtraction of the cognate averaged leak records (in each case the current responses have been aligned according to the onset of the test step for clarity). In the presence of internal Mg, the outward currents decay with a monoexponential time course (solid red lines) that accelerates upon depolarization (note the crossover of the currents recorded at the more depolarized potentials with respect to those obtained at the less positive voltages). In the absence of internal divalent ions this current decay is abolished. The close correspondence of the zero time intercept of the exponential fit of the currents obtained in the presence of Mg to the amplitudes observed in the absence of internal Mg supports the conclusion that the decay of the current under these conditions is due to Mg block.
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To determine the equilibrium voltage dependence of the internal Mg block, we plotted the ratio of the current available in the presence versus the absence of internal Mg and fit this relationship with Eq. 6 (Fig. 5 D). Under conditions of symmetrical K, a fit of Eq. 6 yielded a value of the equilibrium constant in the absence of an applied field (KA0) of 431 M–1 and an effective electrical distance of 0.36, suggesting that Mg crosses a significant fraction of the electric field to reach its binding site (but see also the Discussion with respect to KIR channels).
We next examined the origin of the voltage dependence by determining kON and kOFF at depolarized potentials. We performed this analysis in two ways. First, we evaluated the rate constants and the association constant (KA) from the values of BLOCK (Fig. 5, B and F1) and the unblocked fraction of the current (Fig. 5 D) by solution of Eqs. 1–3
(see Materials and methods). We performed this analysis with values of IT determined from either the extrapolated amplitude of the exponential fits or from scaled versions of the Mg-free IV relationships. These two approaches yielded essentially identical estimates of each parameter (compare open upright and inverted triangles in Figs. 5, F2–4). Second, we determined the rate constants from linear regression to plots of 1/BLOCK versus the free Mg concentration (Fig. 5 E), where kON at each voltage is the slope of the appropriate line and kOFF its zero concentration intercept. These estimates of kON and kOFF (and the derived value of KA) are plotted as upright filled triangles in Fig. 5 (F2–4).
Several things are apparent. First, all approaches yielded similar measures of kON, kOFF, and KA. Second, the variation of kON with voltage is well described by Eq. 4 (solid blue line in Fig. 5 F3, R2 = 0.9997), yielding values for kON0 and ON of 7.97 * 105 M–1s–1 and 0.19, respectively. Third, kOFF is only poorly described by Eq. 5 (solid blue line in Fig. 5 F4, R2 = 0.246), suggesting the derived values of kOFF0 and OFF (936 s–1 and 0.03, respectively) are unlikely to be accurate.
We considered two possibilities for the deviation in behavior of kOFF from a simple exponential relationship. First, that this arises from an experimental error in estimation of the blocking parameters. Second, that this reflects complexity in the blocking mechanism.
The most likely sources of experimental error are (a) contamination of BLOCK as a consequence of the finite clamp response time and (b) errors in the determination of PUNBLOCK. Although BLOCK is fast, 66 µs at +200 mV, it is only at this potential that it approaches the clamp response kinetics (10–90% rise time is on the order of 10–20 µs, see Fig. 8 and discussion thereof), suggesting the clamp kinetics should not have a marked impact at lower voltages. However, not only is kON well behaved across all voltages, the deviations in kOFF are marked at lower potentials. Accordingly, we consider the clamp kinetics to be a relatively minor factor. Similarly, while modest errors in PUNBLOCK (which is a small value at very depolarized potentials, see Fig. 5 D) could translate into a significant error in the estimation of kOFF based on Eq. 2, this will not impact estimation of kOFF based on regression to the inverse of the time constants at different free Mg concentrations (which relies only on BLOCK). As kOFF determined by these two approaches shows close correspondence, we conclude that errors in PUNBLOCK are unlikely to fully account for the deviant behavior of kOFF.
In light of the above arguments, we conclude that the concave relationship of kOFF with voltage represents true deviation of the Mg block reaction from that shown in Scheme 1. Accordingly, we have used our measures of kON and of KA0 and (as obtained from the fit to the equilibrium block) to reevaluate kOFF0 and OFF (estimated to be 1848 s–1 and 0.17 based on the 0 mV solution of Eq. 3 and – ON, respectively) and then explored the predicted behavior of kOFF to consider the reasonableness of these new parameter estimates. Two observations suggest these estimates may be more reasonable. First, the predicted values of kOFF correspond very closely to the values observed at +50 to +100 mV with deviations becoming marked only at higher field strengths (compare the blue dashed line and open symbols in Fig. 5 F4). Second, the value of BLOCK at negative potentials predicted from these estimates of the kOFF parameters (83 µs at –140 mV) is an order of magnitude faster than that estimated if the voltage dependence follows the shallow relationship determined by the regression analysis (when BLOCK at –140 mV is estimated to be 718 µs, see the inset in Fig. 5 F1 for a full comparison of the predicted voltage dependence of BLOCK for the two kOFF parameter sets). The faster unblock accords more closely with the very fast reversal of block observed at such potentials (see, for example, Fig. 3 D). The origin of the nonexponential behavior of kOFF and the implications of kOFF kinetics on the physiological impact of Mg block are considered further in the Discussion.
Does Mg block account quantitatively for the rectification observed in TEVC? A comparison of the normalized IV relationships obtained in IOPC and TEVC (Fig. 6 A) reveals that while inclusion of Mg in the internal solution in IOPC can largely recapitulate the rectification observed in the whole cell currents obtained in TEVC there are two notable discrepancies between the datasets (see inset in Fig. 6 A for an expanded view of the IV relations at depolarized potentials).
First, an increase in the IOPC current at extreme positive potentials is not observed in the whole cell current. Second, the extent of rectification observed in TEVC is only quantitatively reproduced in IOPC when the free Mg concentration is increased to 1.9 mM while the bulk free concentration in an oocyte is estimated to be closer to 0.3 mM (Gabriel and Gunzel, 2007). These observations suggest that either block by other polyvalent cations contributes to the shape of the whole cell current or the action of Mg is modified in the context of an intact cell. Given that polyamines act as important blockers of other classes of Mg sensitive channels, we asked whether the open HCN2 current was sensitive to spermine or spermidine when these ions were presented in the absence of Mg. Although both ions exerted a weak slow block at very depolarized potentials (see left panel of Fig. 6 B for an example of the effect of 10 µM spermidine on the HCN2 current at +200 mV) neither ion had any noticeable effect on the current amplitude at lower voltages. Thus, while block by polyamines may account for the divergence in the TEVC and IOPC IV relationships above 150 mV, these molecules are not responsible for the suppression of the whole cell current at lower potentials. We consider possible explanations for the quantitative discrepancy between the effectiveness of Mg in IOPC and suppression of the TEVC current further in the Discussion.
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Fig. 7 A shows averaged "active" and "leak" records collected using the voltage paradigm described in Fig. 3 but with our quasi-physiological ion gradients. As observed when elevated concentrations of K were present on both the internal and external face, elimination of uncompensated linear leakage currents (by subtraction of the "leak" from the "active" records) isolates the outward HCN2 current and reveals that this has a clear predeactivation transient component (Fig. 7 B, black trace). Importantly, this record superimposes on a scaled version of a predeactivation tail obtained in the presence of elevated K (Fig. 7 B, gray trace) while the relative amplitudes of IT and IP (Fig. 7 C, top) and the time constant of the predeactivation decay (Fig. 7 C, bottom) under the two ion conditions are indistinguishable. These findings demonstrate that Mg block contributes to shaping the HCN2 current under physiological ion gradients. Interestingly, the close correspondence of the IOPC-determined block parameters (Fig. 7 C) suggests that the dissonance in the strength of rectification observed in the data shown in Fig. 1 has a different origin to Na and K sensitivity of the Mg block but we have not explored this further.
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To explore these questions, we adopted the same protocol as shown in Fig. 5 with the exception that K was present only on the external (Fig. 8, A–C) or internal (Fig. 8, D–F) face with Na as the only alkali metal ion on the opposite side and EDTA replacing Mg in the intracellular solution. Inspection of the "on" and "off" phases of the steps to ±200 mV (Fig. 8, A and D, wherein records have been averaged, subtracted, and temporally aligned with respect to the onset of each step) and the current voltage plots obtained there from (Figs. 8, B and E) shows that, even in the absence of internal Mg, Na does not carry a detectable current when K was only present on the trans face irrespective of the orientation of the ion gradients. These results reveal that the loss of Na flux does not arise because Na cannot displace the block by internal Mg but, rather, K association with a control site within the pore of the channel permits the passage of Na.
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10 µs to evolve. The later observation suggests that the rising phase of the black traces is predominantly accounted for by the clamp's kinetics. Does this mean that the slower rate of rise of the blue traces informs us that the rate of restoration of the channel function as K reenters the pore is slow? We suspect not. It seems probable that this simply reflects the longer time constant of the system when the channels are initially nonconducting (and the patch resistance high). Nonetheless, these records show that current recovery cannot be slower than 10–20 µs. That is the rate of reentry of K and/or any conformational changes required to reestablish flux is not markedly different from the optimal ion transit time observed when the pore is multiply occupied by K (0.5 to 3 µs based on the single channel conductance determined at negative potentials). |
DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Biophysical Properties of Internal Cation Block in HCN Channels and Comparison with KIR and KV Channels
Where is the Mg site located and what do the results tell us about the shape of the field in an open HCN channel pore? To explore this question, it is instructive to consider the behavior of other channels that are sensitive to cation block.
In KIR channels, polyvalent cations interact with at least two sites, one formed from S6 residues lining the inner vestibule and a second formed from residues in the cytoplasmic extension of S6. However, these sites are thought to lie outside the field per se such that the voltage dependence of block (which resides predominantly in the off rate) arises from coupling of blocker occupancy to the inward K flux (Bichet et al., 2003
