Separable Gating Mechanisms in a Mammalian Pacemaker Channel

Despite permeability to both K+and Na+, hyperpolarization-activated cyclic nucleotide-gated (HCN) pacemaker channels contain the K+channel signature sequence, GYG, within the selectivity filter of the pore. Here, we show that this region is involved in regulating gating in a mouse isoform of the pacemaker channel (mHCN2). A mutation in the GYG sequence of the selectivity filter (G404S) had different effects on the two components of the wild-type current; it eliminated the slowly activating current (If) but, surprisingly, did not affect the instantaneous current (Iinst). Confocal imaging and immunocytochemistry showed G404S protein on the periphery of the cells, consistent with the presence of channels on the plasma membrane. Experiments with the wild-type channel showed that the rate of If deactivation and If amplitude had a parallel dependence on the ratio of K+/Na+ driving forces. In addition, the amplitude of fully activated If, unlike Iinst, was not well predicted by equal and independent flow of K+ and Na+. The data are consistent with two separable gating mechanisms associated with pacemaker channels: one (If) that is sensitive to voltage, to a mutation in the selectivity filter, and to driving forces for permeating cations and another (Iinst) that is insensitive to these influences. Despite permeability to both K+and Na+, hyperpolarization-activated cyclic nucleotide-gated (HCN) pacemaker channels contain the K+channel signature sequence, GYG, within the selectivity filter of the pore. Here, we show that this region is involved in regulating gating in a mouse isoform of the pacemaker channel (mHCN2). A mutation in the GYG sequence of the selectivity filter (G404S) had different effects on the two components of the wild-type current; it eliminated the slowly activating current (If) but, surprisingly, did not affect the instantaneous current (Iinst). Confocal imaging and immunocytochemistry showed G404S protein on the periphery of the cells, consistent with the presence of channels on the plasma membrane. Experiments with the wild-type channel showed that the rate of If deactivation and If amplitude had a parallel dependence on the ratio of K+/Na+ driving forces. In addition, the amplitude of fully activated If, unlike Iinst, was not well predicted by equal and independent flow of K+ and Na+. The data are consistent with two separable gating mechanisms associated with pacemaker channels: one (If) that is sensitive to voltage, to a mutation in the selectivity filter, and to driving forces for permeating cations and another (Iinst) that is insensitive to these influences. hyperpolarization-activated cyclic nucleotide-gated channel Ih, or Iq, hyperpolarization-activated current instantaneous current in CHO cells expressing HCN2 HCN2 selectivity filter mutant instantaneous current in CHO cells expressing the G404S HCN2 mutant green fluorescent protein instantaneous current in CHO cells expressing GFP Chinese hamster ovary cells phosphate-buffered saline Pacemaker channels (also known asHyperpolarization-activated CyclicNucleotide-gated or HCN1 channels) are expressed by many excitable cells of the central nervous system and heart where they produce a slowly activating current known as If, Ih, or Iq (1Shi W. Wymore R. Yu H. Wu J. Wymore R.T. Pan Z. Robinson R.B. Dixon J.E. McKinnon D. Cohen I.S. Circ. Res. 1999; 85: e1-e6Crossref PubMed Google Scholar, 2Moosmang S. Biel M. Hofmann F. Ludwig A. J. Biol. Chem. 1999; 380: 975-980Crossref Scopus (207) Google Scholar, 3Monteggia L.M. Eisch A.J. Tang M.D. Kaczmarek L.K. Nestler E.J. Brain Res. Mol. Brain Res. 2000; 81: 129-139Crossref PubMed Scopus (186) Google Scholar, 4Santoro B. Liu D.T. Yao H. Bartsch D. Kandel E.R. Siegelbaum S.A. Tibbs G.R. Cell. 1998; 93: 717-729Abstract Full Text Full Text PDF PubMed Scopus (585) Google Scholar, 5Santoro B. Chen S. Luthi A. Pavlidis P. Shumyatsky G.P. Tibbs G.R. Siegelbaum S.A. J. Neurosci. 2000; 20: 5264-5275Crossref PubMed Google Scholar, 6Ludwig A. Zong X. Jeglitsch M. Hofmann F. Biel M. Nature. 1998; 393: 587-591Crossref PubMed Scopus (790) Google Scholar, 7Stevens D.R. Seifert R. Bufe B. Muller F. Kremmer E. Gauss R. Meyerhof W. Kaupp U.B. Lindemann B. Nature. 2001; 413: 631-635Crossref PubMed Scopus (193) Google Scholar). If is involved in regulating membrane potential and spontaneous activity in a variety of excitable cells (8Pape H.C. Annu. Rev. Physiol. 1996; 58: 299-327Crossref PubMed Scopus (984) Google Scholar, 9DiFrancesco D. Annu. Rev. Physiol. 1993; 55: 455-472Crossref PubMed Scopus (678) Google Scholar). If has a clear role in the sino-atrial node, where its activation upon hyperpolarization, deactivation upon depolarization, and permeability to both Na+ and K+, are important for regulating the rate of diastolic depolarization (9DiFrancesco D. Annu. Rev. Physiol. 1993; 55: 455-472Crossref PubMed Scopus (678) Google Scholar). If is also important in thalamocortical neurons where its deactivation produces a slowly decaying after-depolarization that determines the length of refractory periods separating episodes of synchronized oscillations (8Pape H.C. Annu. Rev. Physiol. 1996; 58: 299-327Crossref PubMed Scopus (984) Google Scholar, 10Bal T. McCormick D. J. Neurophys. 1997; 77: 3145-3156Crossref PubMed Scopus (170) Google Scholar). We have recently shown that HCN2 channels produce an instantaneous and Cs+-insensitive current component (Iinst) in addition to the hyperpolarization-activated and Cs+-sensitive If component (11Proenza C. Angoli D. Agranovich E. Macri V. Accili E.A. J. Biol. Chem. 2002; 277: 5101-5109Abstract Full Text Full Text PDF PubMed Scopus (91) Google Scholar). Iinst was not affected by a mutation in the S4 transmembrane segment (S306Q) whereas If was greatly reduced. Thus, our data support a role for the S4 segment in voltage-dependent gating (If), as suggested previously for HCN channels (12Chen J. Mitcheson J.S. Lin M. Sanguinetti M.C. J. Biol. Chem. 2000; 275: 36465-36471Abstract Full Text Full Text PDF PubMed Scopus (69) Google Scholar, 13Chen J. Mitcheson J.S. Tristani-Firouzi M. Lin M. Sanguinetti M.C. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 11277-11282Crossref PubMed Scopus (133) Google Scholar) but not in voltage-independent gating (Iinst). Despite permeability to both K+ and Na+, the HCN pore contains a conserved GYG sequence that is found in many potassium-selective channels (14Lipkind G.M. Hanck D.A. Fozzard H.A. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 9215-9219Crossref PubMed Scopus (41) Google Scholar, 15Heginbotham L., Lu, Z. Abramson T. MacKinnon R. Biophys. J. 1994; 66: 1061-1067Abstract Full Text PDF PubMed Scopus (690) Google Scholar). Studies examining the effects of mutations in the selectivity filter of K+ channels have shown that conformational changes in this region contribute directly to channel gating (16Lu T., Wu, L. Xiao J. Yang J. J. Gen. Physiol. 2001; 118: 509-522Crossref PubMed Scopus (34) Google Scholar, 17So I. Ashmole I. Davies N.W. Sutcliffe M.J. Stanfield P.R. J. Physiol. 2001; 531: 37-50Crossref PubMed Scopus (27) Google Scholar). Permeant and blocking ions such as Rb+, K+, and Cs+ also affect the closing of K+ channels, implicating the selectivity filter in voltage-dependent gating (18Lu T. Ting A.Y. Mainland J. Jan L.Y. Schultz P.G. Yang J. Nat. Neurosci. 2001; 4: 239-246Crossref PubMed Scopus (116) Google Scholar, 19Wang Z. Zhang X. Fedida D. J. Physiol. 1999; 515: 331-339Crossref PubMed Scopus (19) Google Scholar, 20Baukrowitz T. Yellen G. Neuron. 1995; 15: 951-960Abstract Full Text PDF PubMed Scopus (326) Google Scholar, 21Armstrong C.M. Bezanilla F. Rojas E. J. Gen. Physiol. 1973; 62: 375-391Crossref PubMed Scopus (365) Google Scholar). Finally, the KcsA K+ channel selectivity filter and activation gate have recently been shown to have different conformations in conditions of low K+ and high K+, which could explain the effects of permeant ions on gating (22Zhou Y. Morais-Cabral J.H. Kaufman A. MacKinnon R. Nature. 2001; 414: 43-48Crossref PubMed Scopus (1739) Google Scholar). Based on the similarity of the pore structure in HCN and K+ channels, it seems likely that the selectivity filter and the voltage-dependent gate of HCN channels are linked structurally such that changes in one influence the function of the other. However, this has not been directly demonstrated. In this study, we carried out experiments to determine whether the selectivity filter is coupled to gating in HCN channels using mouse HCN2 subunits expressed in Chinese hamster ovary (CHO) cells. We found that If was sensitive to a mutation in the selectivity filter and to different concentrations of permeating cations, whereas Iinst was insensitive to these influences. The results may be explained by changes in pore conformation upon hyperpolarization, and/or by the presence of a second pore that is: 1) found within the same channel, 2) formed by a second population of the same channel subunits, or 3) associated with HCN channels in the form of up-regulated endogenous channels. The G404S mutant was constructed by overlapping PCR mutagenesis from a mouse HCN2 template as previously described (11Proenza C. Angoli D. Agranovich E. Macri V. Accili E.A. J. Biol. Chem. 2002; 277: 5101-5109Abstract Full Text Full Text PDF PubMed Scopus (91) Google Scholar). The amplified mutagenic product and wild-type mHCN2, in the mammalian expression vector pcDNA3 (6Ludwig A. Zong X. Jeglitsch M. Hofmann F. Biel M. Nature. 1998; 393: 587-591Crossref PubMed Scopus (790) Google Scholar), were subsequently digested using NheI and BlpI. The fragment digested out of wild-type mHCN2 was replaced by the complementary fragment carrying the mutation. The mutation was confirmed by restriction analysis and automated sequencing (Biotechnology Laboratory, University of British Columbia, Vancouver Canada). CHO-K1 cells were obtained from ATCC (Manassas, VA), maintained in Hams F-12 media supplemented with antibiotics and 10% fetal bovine serum (Invitrogen) and incubated at 37 °C with 5% CO2. Cells were plated onto glass coverslips and 1 day after splitting were transiently co-transfected with mammalian expression vectors encoding wild-type and/or mutant mHCN2 channels (4 μg per 35-mm dish) along with a green fluorescent protein (GFP) reporter plasmid (0.3 μg per dish) using the FuGENE 6 transfection reagent (Roche Molecular Biochemicals). Cells expressing GFP were chosen for whole-cell recordings 24–48 h after transfection or co-transfection with mHCN2. The pipette solution contained (in mm): 130, KCl; 10, NaCl; 0.5, MgCl2; 1, EGTA; 5, HEPES; pH adjusted to 7.4 with KOH. The extracellular solution contained (in mm) 140, XCl (X = sum of K+ and Na+); 1.8, CaCl2; 0.5, MgCl2; 5, HEPES; pH adjusted to 7.4 with NaOH. For solution changes, a 200-μl was and for at 1 to were using an and at were not and was not were from glass and were is were at and were using and The current in and were by by the which was by the from the of the current by a at the of whole-cell to of activation and deactivation were using a to If deactivation that was not well described by a as described previously B. Chen S. Luthi A. Pavlidis P. Shumyatsky G.P. Tibbs G.R. Siegelbaum S.A. J. Neurosci. 2000; 20: 5264-5275Crossref PubMed Google C. A. E. M. C. A. D. J. Gen. Physiol. 2001; PubMed Scopus Google Scholar) and was not in our of Iinst for the presence of endogenous instantaneous we found that the amplitude of Iinst was and the instantaneous current in cells with GFP or with the membrane protein to the of the transmembrane from the that for the to be and on the extracellular of the CHO plasma at and (11Proenza C. Angoli D. Agranovich E. Macri V. Accili E.A. J. Biol. Chem. 2002; 277: 5101-5109Abstract Full Text Full Text PDF PubMed Scopus (91) Google Scholar). were to the of instantaneous current to in and 24–48 h cells on coverslips were with and in in for The cells were with using and with 10% serum one with the cells were incubated with a to HCN2 at a of in with for h at The was and cells were with Cells were incubated with a with at a of in with for 1 h at in the The was cells were in and the coverslips with cells were on using Cells were using and with an with and and an For were in using a and an of to the of the selectivity filter in gating was to a mutation the GYG sequence of mHCN2 (G404S) and determine its effects on expressed 1 that the region of the pore the selectivity filter has different of K+ channels and cyclic nucleotide-gated channels. We the at to a 1, was chosen the mutation in the channel which is to HCN channels, eliminated slowly activating current but not to the plasma membrane Z. J. Biol. Chem. 1998; Full Text Full Text PDF PubMed Scopus Google Scholar). Cells expressing G404S If, in a high K+ mm) extracellular solution We If and Iinst in cells expressing wild-type HCN2 and G404S in to for in Cells expressing G404S If did cells expressing wild-type HCN2 The instantaneous current associated with G404S and wild-type HCN2 were the instantaneous current in cells expressing GFP but were not different from A and The of and were the potential of but not different from that the expression of wild-type HCN2 or G404S a with a potential to that of the endogenous channels found in CHO cells expressing The for and for the wild-type HCN2 and G404S channels. was and by with of an of that the of and an of that = = = and after 1 in and and after a Finally, we found that was by (11Proenza C. Angoli D. Agranovich E. Macri V. Accili E.A. J. Biol. Chem. 2002; 277: 5101-5109Abstract Full Text Full Text PDF PubMed Scopus (91) Google Scholar) but unlike If, which is by D. J. Physiol. PubMed Scopus Google Scholar). For of the presence of G404S on the plasma we in CHO cells of wild-type or mutant channels and with an and a shown by of cells expressing wild-type HCN2 or was a of along the periphery of the cells the presence of HCN2 protein on the plasma membrane of were in the of the cells, which channel protein in such as the to or from the plasma or was in cells in the of or in cells We have shown previously that the wild-type channel and the mutant a of whereas a mutant the and did not and a different of that did not on the periphery of CHO cells (11Proenza C. Angoli D. Agranovich E. Macri V. Accili E.A. J. Biol. Chem. 2002; 277: 5101-5109Abstract Full Text Full Text PDF PubMed Scopus (91) Google Scholar). The imaging data and the in instantaneous current support the presence of G404S on the plasma membrane and the of hyperpolarization-activated gating (If) by the G404S mutation. the role of the selectivity filter in voltage-dependent gating of HCN channels, we the effects of permeating cations on If current from cells expressing HCN2 are shown in A. We of deactivation and of fully activated If at the same potential but at different of extracellular K+/Na+ both cations HCN channels A. A. C. C. J. M. D. 2000; PubMed Scopus Google Scholar). We a of a to by a to were chosen for the channels are to fully activated at and fully at in CHO cells (11Proenza C. Angoli D. Agranovich E. Macri V. Accili E.A. J. Biol. Chem. 2002; 277: 5101-5109Abstract Full Text Full Text PDF PubMed Scopus (91) Google Scholar). fully activated If amplitude and the rate of were at the of extracellular K+ and Na+ concentrations Finally, is within the of in many excitable cells expressing If (8Pape H.C. Annu. Rev. Physiol. 1996; 58: 299-327Crossref PubMed Scopus (984) Google Scholar, 9DiFrancesco D. Annu. Rev. Physiol. 1993; 55: 455-472Crossref PubMed Scopus (678) Google Scholar). the K+/Na+ ratio in the amplitude of If at and of the current at activation for at and deactivation at were both well with a function from which were the K+/Na+ ratio did not the rate of If activation at but the rate of If deactivation at and also a that has been to changes in conformation channel closing in both and HCN channels C. A. E. M. C. A. D. J. Gen. Physiol. 2001; PubMed Scopus Google Scholar, D. J. Physiol. PubMed Scopus Google Scholar). is in the K+/Na+ ratio as with the K+/Na+ ratio and be in the K+/Na+ the deactivation rate and in that permeant cations affect If deactivation We If and Iinst on the changes in the K+ and Na+ driving and these with current using the current in 1, 1 where and are the predicted current for If, the Na+ and the K+ and are the driving forces for Na+ and and and are the of for Na+ and We the pore and = 1 a that was obtained using the and the If at a ratio of Na+ the A the fully activated If as a function of the ratio of K+ and Na+ driving forces at of data at K+ and to determine the which the changes The in If and where a of ratio of driving forces was A Na+ and Na+ where a of ratio of driving forces was The in with the in of K+ flow a ratio of equal to we found that the If on at where the K+ driving was of the If to a ratio of Thus, the K+ driving in the to be of the in the to a second for was that was equal to these a to the that different of could for the the If and current the fully activated If at was as a function of the ratio of driving forces with a function showed that the in the If Na+ and Na+, as it did at The = or = the at at of the If an of that If was eliminated the that driving forces were for both Na+ and has also been in of If (8Pape H.C. Annu. Rev. Physiol. 1996; 58: 299-327Crossref PubMed Scopus (984) Google Scholar, 1995; PubMed Scopus Google Scholar, F. Y. M. A. J. Physiol. 1994; PubMed Scopus Google Scholar, D. J. Physiol. PubMed Scopus Google Scholar). The concentrations at the were K+ and which to K+and Na+ at concentrations of K+ and Na+ are in a found in that the of is a for regulating If in different determine whether Iinst could be predicted by independent flow of Na+ and K+ the we the Iinst for of endogenous as described the ratio of driving forces. was with current where = at and as was carried out for A for was using the for Iinst where the ratio of driving forces = Na+ the for The for Iinst were to the current were the of fully activated If from and data that produces conformational changes that the flow of K+ Na+ the HCN pore in the fully activated to predicted for the If these data could be explained by a where the two and flow two different both to Na+ and where pore has a gating We If deactivation at to determine whether this was by permeating cations in the same of driving forces that If Here, we the of data at for both If amplitude and We found that the in rate of If deactivation the in fully activated at K+/Na+ driving forces. of in the of driving forces found in cells and where K+ in the for the fully activated If at deactivation were with two on of the K+ The K+ Na+ and Na+ where a of = ratio of driving forces these A Na+ and Na+, where a of = ratio of driving forces the both of were to the the for the component were and and for the component were and for the fully activated If and deactivation The parallel changes in both of support the that permeating cations affect If deactivation as well as If also that the conformational changes by slowly unlike was not greatly affected by changes in K+/Na+ driving forces. Thus, after the changes in pore conformation to changes in the rate of If in the of K+ Na+, and in the fully activated If show that in the HCN2 G404S a in K+ or of Cs+ or If blocking with our the of a Cs+-sensitive current (If) and an instantaneous Cs+-insensitive current (Iinst) (11Proenza C. Angoli D. Agranovich E. Macri V. Accili E.A. J. Biol. Chem. 2002; 277: 5101-5109Abstract Full Text Full Text PDF PubMed Scopus (91) Google Scholar), these data that two gating which be and are associated with HCN channels. it for two with different and to the same The of If by Cs+ and G404S may be to the of in channel of by the permeant and by mutations of the to in HCN2 (16Lu T., Wu, L. Xiao J. Yang J. J. Gen. Physiol. 2001; 118: 509-522Crossref PubMed Scopus (34) Google Scholar, T. Ting A.Y. Mainland J. Jan L.Y. Schultz P.G. Yang J. Nat. 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