Lyashchenko AK and Tibbs GR (2008), Ion binding in the open HCN pacemaker channel p...

XB-ART-37966

J Gen Physiol 2008 Mar 01;1313:227-43. doi: 10.1085/jgp.200709868.

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Ion binding in the open HCN pacemaker channel pore: fast mechanisms to shape "slow" channels.

Lyashchenko AK , Tibbs GR .

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I(H) pacemaker channels carry a mixed monovalent cation current that, under physiological ion gradients, reverses at approximately -34 mV, reflecting a 4:1 selectivity for K over Na. However, I(H) 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 I(H)) 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 micros or faster) is close to the ion transit time when the pore is occupied by K alone ( approximately 0.5-3 micros), 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.

???displayArticle.pubmedLink??? 18270171
???displayArticle.pmcLink??? PMC2248720
???displayArticle.link??? J Gen Physiol

Species referenced: Xenopus
Genes referenced: camp hcn2

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	Figure 1. Permeant ion-dependent rectification in the IV relationship of fully activated HCN2 channels. (A and D) HCN2 TEVC currents (from a single Xenopus oocyte) obtained in response to 50-ms steps to potentials ranging from −200 to +200 mV following channel activation at −115 mV for 3 s or after a 5-ms step to −115 mV (left and right parts of sweeps in top panels and dark and light gray sweeps in bottom panels, respectively) and the difference currents (blue lines) at test potentials of +200 mV (bottom left) and −200 mV (bottom right). In this and subsequent figures, text insets indicate the Na and K concentrations in the intracellular (IN) and extracellular (OUT) solutions (see Materials and methods for estimations of internal concentrations in TEVC) while the red dashed line shows the zero current level. Sweeps were filtered at 1 kHz and sampled at 20 kHz. (B and E) Expanded views of the difference current families (from recordings shown in A and D, respectively) with −200 and +200 mV sweeps highlighted in blue. (C and F) Difference current amplitudes (determined by averaging records in B and E between 2.5 to 5 ms after the onset of the voltage step) plotted versus observed voltages. EREV and current amplitudes with respect thereunto were determined from fits of an eighth order polynomial (thin black line). In this and subsequent IV plots, superimposed gray lines show component (dashed) and net current (solid) predictions of the GHK current equation assuming a 4:1 K:Na selectivity. (G) Expanded view around zero current of the IV plots shown in C and F. (H) Ratio of the currents recorded in elevated external K (IK) with respect to those obtained in physiological concentrations of Na and K (IPHYS) 150 mV negative (left) or positive (right) to EREV. Data are mean ± SEM from five cells recorded as shown in A–G.
	Figure 2. Ion-dependent asymmetry in the IV relationship of fully activated HCN2 channels is maintained in excised inside-out patches. (A, C, and E) HCN2 currents obtained in response to activation at −145 mV for 1.5 s (left) and expanded views of tail currents upon depolarization to potentials between −100 and + 80 mV (right) when K was the only alkali metal presented to the channels (C) or Na and K were presented with quasi-physiological (A) and inverted (E) gradients. Each record is a single sweep filtered at 5 kHz and sampled at 20 kHz with no post hoc digital correction for uncompensated linear ionic or capacitive components. (B, D, and F) Plateau tail currents (determined by averaging current amplitudes between 7 and 9 ms after the test step) plotted versus the corresponding tail potential. Data are from records shown in A, C, and E, respectively. Data are representative of 4 (A and B), 6 (C and D), and 4 (E and F) similar recordings.
	Figure 3. Rectification of the hyperpolarization-activated current is associated with a submillisecond outward transient that does not arise from HCN2 channel deactivation. (A and C) Schematic representation of the complete voltage paradigms (A) and expanded views of the interval during the middle of the record (C, as indicated by the red ovals in A). In the top “leak” paradigm, the patch was stepped to −135 mV for 5 ms (a duration sufficient to allow uncompensated capacity transients to decay but insufficient to permit channels to open) and then stepped to +80 mV for 1 or 3 ms before being returned to −135 mV for 2 s. In the bottom “active” paradigm, channels were first opened at −135 mV for 2 s before the 1- or 3-ms sojourn at +80 mV and the return to −135 mV. Each sweep was filtered at 20 kHz and sampled at 100 kHz with “leak” and “active” sweeps recorded interlaced. (B) Averages of seven “leak” sweeps (top) and their cognate interleaved “active” sweeps (bottom) recorded with K as the only alkali metal. (D) Expanded views of the total current (including uncompensated capacity and ionic components) observed during the step to (top left) and return from (top right) +80 mV in the presence (dark gray lines) and absence (light gray lines) of activated HCN2 channels and of the “active” minus “leak” subtracted records before, during, and after the step to +80 mV for 1 ms (thin black line, bottom panel only) or 3 ms (thick black lines). Data are representative of five similar recordings of which three were collected at the widest bandwidth (filtered at 100 kHz and sampled at 200 kHz).
	Figure 4. The transient outward current is carried by the HCN2 channels. (A1) HCN2 currents obtained before (black), during (blue), and after (gray) application of 300 μM ZD7288 (ionic conditions and voltage paradigms as in Fig. 3). Each trace is the average of eight sweeps evoked in response to the 3-ms “active” protocol before subtraction of the interlaced “leak” sweeps (records not shown for simplicity). Currents were filtered and sampled at 20 and 100 kHz, respectively. (A2) Expanded views of the +80 mV sojourn following subtraction of the appropriate “leak” records before (raw) and after (scaled) scaling each trace to the amplitude of the preZD7288 −135 mV current (I –135). (A3) ZD7288-sensitive component of the current (obtained as the difference between the leak-subtracted pre and plus ZD7288 records in “A2, raw”) is shown before (subtracted) and after (subtracted and scaled) scaling the blocked component (blue traces) to the preZD7288 I –135 amplitude. (B) Outward transient in the absence of ZD7288 (black line) with superimposed fit of a single-exponential function (red line). Residuals from the fit (green line) are offset from zero for clarity. Arrows show the amplitudes of the plateau (Ip) and transient (It) components of the outward current. (C) Fractional ZD7288 inhibition of I –135, It, and Ip, where IC is the control amplitude and IZ the 300 μM ZD7288-resistant amplitude, respectively. Data are mean ± SEM from three recordings acquired and analyzed as described in A and B. (D and E) Linear regressions (superimposed lines) show the amplitudes of It (R2 = 0.955) and Ip (R2 = 0.986) are highly correlated with that of I –135 (D) and with each other (E; R2 = 0.945). Data are from seven independent patches. (F) Time constant of current decay at +80 mV as a function of the I –135 amplitude. The black line is a linear regression (R2 = 0.070) across the control data from seven independent recordings. Red lines connect data acquired in three independent recordings before, during, and after cut down of the current by inclusion of ZD7288 (data points obtained before and after washout of ZD7288 in the same patch are ringed by a red ellipse).
	Figure 5. Inward rectification of the open HCN2 channel current arises from a voltage-dependent block by intracellular Mg. (A and B) HCN2 currents activated at 2140 mV in the presence of 300 μM cAMP with either 1 mM EDTA plus 1 mM EGTA or 1 mM Mg plus 1 mM EGTA in the intracellular solution (records are from the same patch). In all panels K was the only alkali metal on either side of the membrane ([K]OUT 117 mM; [K]IN 119 mM or 121 mM when EDTA replaced MgCl2). After allowing activation to equilibrate (2 s), the patch was stepped to potentials between −200 and +200 mV in 50-mV intervals with test steps applied at 10 Hz (records were filtered at 100 kHz and sampled at 200 kHz). To ensure steps were long enough to determine the time constant of block but short enough to prevent deactivation, the durations of the steps were increased from 1 ms at +200 mV to 3 ms at 0 mV in 0.5-ms increments and then held at 3 ms for all negative potentials. Collection of such “active” sweeps was interlaced with “leak” sweeps wherein the patch was stepped to −140 mV for 5 ms before and after each test step (not shown for simplicity). For each condition, four averaged “leak” records were subtracted from the average of the four cognate “active” records (shown in A) and the subtracted currents aligned to the beginning of each test step (B). Activation at −155 mV with test steps applied at a lower frequency (4 Hz) gave identical results so data from these two paradigms were combined. Records collected at depolarized potentials in the presence of Mg are well fit (red lines) with a single exponential function (residuals from the fits shown as green lines offset from zero for clarity). Arrows show the correspondence between the zero time extrapolation of the fitted exponentials and the amplitudes of currents acquired in the absence of Mg. (C) Plateau tail currents (determined at the end of test steps as shown in B) in the presence (IP Mg) and absence (IP EDTA) of 1 mM Mg (mean ± SEM from nine separate recordings for each condition) plotted versus the corresponding test potential. (D) Equilibrium voltage dependence of the block by 1 mM Mg was determined by fitting Eq. 6 (smooth line) to the ratio of IP Mg/IP EDTA (same data as shown in C). The 95% confidence interval of this quotient was smaller than the symbol size at all voltages except −50 mV. The ratio is undefined at the reversal potential of 0 mV. (E) Plot of the inverse time constant of block at the indicated potentials as a function of the free Mg concentration. Data for low and high concentrations of Mg are mean ± SEM of eight and four recordings acquired and analyzed as described in A–D. (F) Kinetic parameters of Mg block plotted as a function of the test potential. Parameters were determined from τblock (such as shown for 1 mM Mg in B and F1) and the steady-state current inhibition (D) according to Eqs. 1–3 based on the zero time extrapolation of the block exponential (upright open triangle) or the instantaneous current predicted from scaled amplitudes determined in the absence of internal Mg (inverted open triangle) and from 1/τBLOCK versus free Mg concentration (E) (upright filled triangles). Solid blue lines are regressions to the data represented by the inverted open triangles. The dashed blue line represents the predicted value of kOFF based on δOFF = 0.17 (from δ-δON where δ and δON are 0.36 and 0.19, respectively) and kOFF0 = 1848 s−1 (from the 0 mV version of Eq. 3 where kon0 = 7.97 × 105 M−1s−1 and KA0 = 431 M−1). The inset in F1 shows relationship of τBLOCK to membrane voltage at 0.924 mM free Mg when kOFF0 and δOFF are 1848 s−1 and 0.17 (thick line) or 936 s−1 and 0.03 (thin line). The ordinates are logarithmic in the main panels but linear in the inset.
	Figure 6. Spermine and spermidine are inefficient blockers of the HCN2 current. (A) Plateau tail currents obtained from the TEVC and IOPC experiments shown in Figs. 1 and Figs.5 were normalized with respect to the amplitudes of the currents observed at −200 mV and plotted with respect to the observed (TEVC) and the command potential (IOPC). Data are plotted as mean ± SEM. The errors around the voltage in TEVC were always smaller than the symbols. The inset shows an expanded view of the IV relations at depolarized potentials. (B) Representative recording obtained upon stepping to +200 mV in the absence and presence of 10 μM spermidine (SPD) and normalized open channel IV relationships in the absence (n = 6) and presence of 10 μM SPD (n = 3) or spermine (SPM, n = 5) determined using the protocol described in Fig. 5 and plotted as mean ± SEM versus the command potential. Test steps were applied at 4 Hz from an activation potential of −155 mV in the absence of added divalent ions and the presence of 1 mM EGTA, 1 mM EDTA, and 30 μM cAMP with K as the only alkali metal on either side of the membrane ([K]OUT 117 mM; [K]IN 121 mM).
	Figure 7. Mg blocks the outward HCN2 current in the presence of physiological gradients of Na and K. (A and B) Averaged sweeps (A) and subtracted predeactivation HCN2 channel +80-mV tails (B) (three and two “leak” and “active” records collected as described in Fig. 3 except for the use of quasi-physiological gradients of Na and K, black lines). In B, the gray line is the K tail shown in Fig. 4 B scaled to the amplitude of Ip of the mixed ion tail. The red and green lines represent fits of a single exponential function and the resultant residuals for the two transients. (C) Plot of the extent (top) and time constant (bottom) of block at +80 mV in the presence of elevated K or physiological levels of Na and K in the extracellular solution. Data are mean ± SEM and range from five and two recordings, respectively.
	Figure 8. Na sensitivity of Mg block does not account for the collapse of conduction in a K-depleted pore. (A and D) Unblocked HCN2 currents (intracellular solution contained 1 mM EDTA and 1 mM EGTA and no added divalent cations) were collected and processed as described in Fig. 5, A and B, except the cAMP concentration was 30 μM and Na and K were presented biionically (with the direction of the gradients reversed in the two records as indicated). Red circles indicate a small transient present in the current collapse time course that is absent in the recovery time course. Data are representative of four and three similar recordings for A–C and D–F, respectively, with records in A–C from the same patch shown in Fig. 5, A and B. (B and E) Plateau tail current amplitudes (measured 1 ms after the beginning of each test step) plotted versus the corresponding step potential. (C and F) Expanded views of the collapse (black lines) and recovery (blue lines) of current observed when the potential was stepped between −140 mV and test potentials of +50 to +200 mV (thicker lines represent steps to more depolarized voltages). In each panel the currents have been normalized to change from 0 to 1 for both the step to, and the return from, the depolarized test potentials and each phase of the sweep shifted on the time axis so its beginning is aligned with respect to its onset.
	Figure 9. Asymmetry in the ability of low concentrations of K to support Na conduction gives rise to a Mg-independent form of inward rectification. (A) Unblocked HCN2 currents were collected and processed as described in Fig. 8 except the major charge carrier in both internal and external solutions was Na with K included at only 5 mM. Records are averages of 10 “active” sweeps before subtraction of the averaged cognate “leak” sweeps (not shown for simplicity). (B) Leak-subtracted records obtained in response to steps from −140 mV to potentials ranging from −200 to +200 mV with the later sweep highlighted in blue. (C) Plateau tail current amplitudes (measured 1 ms after the beginning of each test step) plotted versus the corresponding step potential.

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