An External Determinant in the S5-P Linker of the Pacemaker (HCN) Channel Identified by Sulfhydryl Modification
2002; Elsevier BV; Volume: 277; Issue: 48 Linguagem: Inglês
10.1074/jbc.m204915200
ISSN1083-351X
Autores Tópico(s)Cardiac electrophysiology and arrhythmias
ResumoHyperpolarization-activated,cyclic nucleotide-gated (HCN) channels underlie spontaneous rhythmic activities in the heart and brain. Sulfhydryl modification of ion channels is a proven approach for studying their structure-function relationships; here we examined the effects of the hydrophilic sulfhydryl-modifying agents methanethiosulfonate ethylammonium (MTSEA+) and methanethiosulfonate ethylsulfonate (MTSES−) on wild-type (WT) and engineered HCN1 channels. External application of MTSEA+ to WT channels irreversibly reduced whole-cell currents (I MTSEA/I Control = 42 ± 2%), slowed activation and deactivation kinetics (∼7- and ∼3-fold at −140 and −20 mV, respectively), and produced hyperpolarizing shifts of steady-state activation (V12[MTSEA]=−125.8±9.0mVversusV12[control]=−76.4±1.6mV). Sequence inspection revealed the presence of five endogenous cysteines in the transmembrane domains of HCN1: three are putatively close to the extracellular milieu (Cys303, Cys318, and Cys347 in the S5, S5-P, and P segments, respectively), whereas the remaining two are likely to be cytoplasmic or buried. To identify the molecular constituent(s) responsible for the effects of MTSEA+, we mutated the three "external" cysteines individually to serine. C303S did not yield measurable currents. Whereas C347S channels remained sensitive to MTSEA+, C318S was not modified (I MTSEA/I Control = 101 ± 2%, V12(MTSEA)=−78.4±1.1 mV, and V12(Control)=−79.8±2.3 mV. Likewise, WT (but not C318S) channels were sensitive to MTSES−. Despite their opposite charges, MTSES− produced changes directionally similar to those effected by MTSEA+(I MTSES/I Control = 22 ± 1.6% and V12(MTSES)=−145.8±4.9 mV). We conclude that S5-P Cys318 of HCN1 is externally accessible and that the external pore vestibule and activation gating of HCN channels are allosterically coupled. Hyperpolarization-activated,cyclic nucleotide-gated (HCN) channels underlie spontaneous rhythmic activities in the heart and brain. Sulfhydryl modification of ion channels is a proven approach for studying their structure-function relationships; here we examined the effects of the hydrophilic sulfhydryl-modifying agents methanethiosulfonate ethylammonium (MTSEA+) and methanethiosulfonate ethylsulfonate (MTSES−) on wild-type (WT) and engineered HCN1 channels. External application of MTSEA+ to WT channels irreversibly reduced whole-cell currents (I MTSEA/I Control = 42 ± 2%), slowed activation and deactivation kinetics (∼7- and ∼3-fold at −140 and −20 mV, respectively), and produced hyperpolarizing shifts of steady-state activation (V12[MTSEA]=−125.8±9.0mVversusV12[control]=−76.4±1.6mV). Sequence inspection revealed the presence of five endogenous cysteines in the transmembrane domains of HCN1: three are putatively close to the extracellular milieu (Cys303, Cys318, and Cys347 in the S5, S5-P, and P segments, respectively), whereas the remaining two are likely to be cytoplasmic or buried. To identify the molecular constituent(s) responsible for the effects of MTSEA+, we mutated the three "external" cysteines individually to serine. C303S did not yield measurable currents. Whereas C347S channels remained sensitive to MTSEA+, C318S was not modified (I MTSEA/I Control = 101 ± 2%, V12(MTSEA)=−78.4±1.1 mV, and V12(Control)=−79.8±2.3 mV. Likewise, WT (but not C318S) channels were sensitive to MTSES−. Despite their opposite charges, MTSES− produced changes directionally similar to those effected by MTSEA+(I MTSES/I Control = 22 ± 1.6% and V12(MTSES)=−145.8±4.9 mV). We conclude that S5-P Cys318 of HCN1 is externally accessible and that the external pore vestibule and activation gating of HCN channels are allosterically coupled. The hyperpolarization-activated, cyclicnucleotide-gated (HCN1–4) or the so-called pacemaker channel gene family encode the pacemaking current I for I h that underlies the spontaneous periodic activities found in parts of the heart and brain (1Gauss R. Seifert R. Kaupp U.B. Nature. 1998; 393: 583-587Crossref PubMed Scopus (374) Google Scholar, 2Ludwig A. Zong X. Jeglitsch M. Hofmann F. Biel M. Nature. 1998; 393: 587-591Crossref PubMed Scopus (779) Google Scholar, 3Santoro 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 (577) Google Scholar). Like voltage-gated K+ channels, HCN channels are tetramers (4Xue T. Marbán E. Li R.A. Circ. Res. 2002; 90: 1267-1273Crossref PubMed Scopus (65) Google Scholar) made up of monomeric subunits consisting of six transmembrane segments (S1–6) with a pore-forming P-loop between S5 and S6 (1Gauss R. Seifert R. Kaupp U.B. Nature. 1998; 393: 583-587Crossref PubMed Scopus (374) Google Scholar, 2Ludwig A. Zong X. Jeglitsch M. Hofmann F. Biel M. Nature. 1998; 393: 587-591Crossref PubMed Scopus (779) Google Scholar, 3Santoro 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 (577) Google Scholar). Different HCN isoforms can co-assemble with each other to form heteromultimeric complexes, greatly increasing the diversity of the molecular identity of native I f (4Xue T. Marbán E. Li R.A. Circ. Res. 2002; 90: 1267-1273Crossref PubMed Scopus (65) Google Scholar, 5Chen S. Wang J. Siegelbaum S.A. J. Gen. Physiol. 2001; 117: 491-504Crossref PubMed Scopus (324) Google Scholar, 6Ulens C. Tytgat J. J. Biol. Chem. 2001; 276: 6069-6072Abstract Full Text Full Text PDF PubMed Scopus (174) Google Scholar). Despite the presumed structural similarity to K+ channels, direct evidence regarding the topology of HCN channels is relatively sparse.Inspection of the primary sequences of HCN channels (1Gauss R. Seifert R. Kaupp U.B. Nature. 1998; 393: 583-587Crossref PubMed Scopus (374) Google Scholar, 2Ludwig A. Zong X. Jeglitsch M. Hofmann F. Biel M. Nature. 1998; 393: 587-591Crossref PubMed Scopus (779) Google Scholar, 3Santoro 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 (577) Google Scholar) reveals the presence of a total of five endogenous cysteines in the transmembrane domains: three appear to be close to the extracellular side (Cys303, Cys318, and Cys347 in the S5, S5-P, and P segments, respectively; HCN1 numbering), whereas the remaining two are likely to be cytoplasmic or buried (S5 Cys298 and S6 Cys374) (Fig.1). It is known that the cysteine sulfhydryl, in its reduced form, may readily undergo chemical reactions (such as alkylation, acylation, and arylation) or even cross-link with a second nearby cysteine to form a disulfide bridge (7Benitah J.P. Tomaselli G.F. Marbán E. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 7392-7396Crossref PubMed Scopus (57) Google Scholar, 8Tsushima R.G. Li R.A. Backx P.H. J. Gen. Physiol. 1997; 110: 59-72Crossref PubMed Scopus (57) Google Scholar). These properties render free cysteinyl the most chemically reactive amino acid side chain (9Creighton T. Proteins: Structures and Molecular Properties. 5th Ed. W. H. Freeman & Co., New York1997Google Scholar). When exposed to the aqueous phase, cysteinyls can be selectively and covalently modified by hydrophilic sulfhydryl-reactive agents (10Karlin A. Akabas M.H. Methods Enzymol. 1998; 293: 123-145Crossref PubMed Scopus (541) Google Scholar). Indeed, engineering cysteines into proteins, followed by the assessment of their accessibility pattern using a variety of sulfhydryl-reactive probes with different physical and chemical properties (i.e. cysteine scanning mutagenesis), has been a proven approach to study the structure-function relationships of numerous ion channels, receptors, and proteins (7Benitah J.P. Tomaselli G.F. Marbán E. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 7392-7396Crossref PubMed Scopus (57) Google Scholar, 8Tsushima R.G. Li R.A. Backx P.H. J. Gen. Physiol. 1997; 110: 59-72Crossref PubMed Scopus (57) Google Scholar, 10Karlin A. Akabas M.H. Methods Enzymol. 1998; 293: 123-145Crossref PubMed Scopus (541) Google Scholar, 11Li R.A. Velez P. Chiamvimonvat N. Tomaselli G.F. Marbán E. J. Gen. Physiol. 2000; 115: 81-92Crossref PubMed Scopus (32) Google Scholar, 12Perez-Garcia M.T. Chiamvimonvat N. Marbán E. Tomaselli G.F. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 300-304Crossref PubMed Scopus (107) Google Scholar, 13Lu Q. Miller C. Science. 1995; 268: 304-307Crossref PubMed Scopus (172) Google Scholar, 14Akabas M.H. Stauffer D.A. Xu M. Karlin A. Science. 1992; 258: 307-310Crossref PubMed Scopus (595) Google Scholar, 15Akabas M.H. Karlin A. Biochemistry. 1995; 34: 12496-12500Crossref PubMed Scopus (159) Google Scholar, 16Akabas M.H. Kaufmann C. Archdeacon P. Karlin A. Neuron. 1994; 13: 919-927Abstract Full Text PDF PubMed Scopus (356) Google Scholar, 17Sun Z.P. Akabas M.H. Goulding E.H. Karlin A. Siegelbaum S.A. Neuron. 1996; 16: 141-149Abstract Full Text Full Text PDF PubMed Scopus (105) Google Scholar).The presence of endogenous cysteines in HCN channels that are putatively exposed to the extracellular milieu raises the possibility that wild-type (WT) 1The abbreviations used for: WT, wild-type; MTS, methanethiosulfonate; MTSEA, methanethiosulfonate ethylammonium; MTSES, methanethiosulfonate ethylsulfonate; DTT, dithiothreitol. 1The abbreviations used for: WT, wild-type; MTS, methanethiosulfonate; MTSEA, methanethiosulfonate ethylammonium; MTSES, methanethiosulfonate ethylsulfonate; DTT, dithiothreitol. channels are susceptible to modifications by sulfhydryl-reactive compounds, which in turn provide an excellent opportunity for localizing structurally and functionally important channel domains. In this report, we examined the effects of the hydrophilic covalent sulfhydryl modifiers methanethiosulfonate ethylammonium (MTSEA) and methanethiosulfonate ethylsulfonate (MTSES), which are positively and negatively charged, respectively, on the functions of HCN1 channels. We found that WT HCN1 channels were indeed modified by these agents. Using site-directed mutagenesis, we identified the native cysteine in the S5-P linker (i.e. Cys318) as the residue responsible for the post-translational changes caused by these agents. Novel insights into the structure-function relationships of HCN channels from these results are discussed. The hyperpolarization-activated, cyclicnucleotide-gated (HCN1–4) or the so-called pacemaker channel gene family encode the pacemaking current I for I h that underlies the spontaneous periodic activities found in parts of the heart and brain (1Gauss R. Seifert R. Kaupp U.B. Nature. 1998; 393: 583-587Crossref PubMed Scopus (374) Google Scholar, 2Ludwig A. Zong X. Jeglitsch M. Hofmann F. Biel M. Nature. 1998; 393: 587-591Crossref PubMed Scopus (779) Google Scholar, 3Santoro 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 (577) Google Scholar). Like voltage-gated K+ channels, HCN channels are tetramers (4Xue T. Marbán E. Li R.A. Circ. Res. 2002; 90: 1267-1273Crossref PubMed Scopus (65) Google Scholar) made up of monomeric subunits consisting of six transmembrane segments (S1–6) with a pore-forming P-loop between S5 and S6 (1Gauss R. Seifert R. Kaupp U.B. Nature. 1998; 393: 583-587Crossref PubMed Scopus (374) Google Scholar, 2Ludwig A. Zong X. Jeglitsch M. Hofmann F. Biel M. Nature. 1998; 393: 587-591Crossref PubMed Scopus (779) Google Scholar, 3Santoro 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 (577) Google Scholar). Different HCN isoforms can co-assemble with each other to form heteromultimeric complexes, greatly increasing the diversity of the molecular identity of native I f (4Xue T. Marbán E. Li R.A. Circ. Res. 2002; 90: 1267-1273Crossref PubMed Scopus (65) Google Scholar, 5Chen S. Wang J. Siegelbaum S.A. J. Gen. Physiol. 2001; 117: 491-504Crossref PubMed Scopus (324) Google Scholar, 6Ulens C. Tytgat J. J. Biol. Chem. 2001; 276: 6069-6072Abstract Full Text Full Text PDF PubMed Scopus (174) Google Scholar). Despite the presumed structural similarity to K+ channels, direct evidence regarding the topology of HCN channels is relatively sparse. Inspection of the primary sequences of HCN channels (1Gauss R. Seifert R. Kaupp U.B. Nature. 1998; 393: 583-587Crossref PubMed Scopus (374) Google Scholar, 2Ludwig A. Zong X. Jeglitsch M. Hofmann F. Biel M. Nature. 1998; 393: 587-591Crossref PubMed Scopus (779) Google Scholar, 3Santoro 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 (577) Google Scholar) reveals the presence of a total of five endogenous cysteines in the transmembrane domains: three appear to be close to the extracellular side (Cys303, Cys318, and Cys347 in the S5, S5-P, and P segments, respectively; HCN1 numbering), whereas the remaining two are likely to be cytoplasmic or buried (S5 Cys298 and S6 Cys374) (Fig.1). It is known that the cysteine sulfhydryl, in its reduced form, may readily undergo chemical reactions (such as alkylation, acylation, and arylation) or even cross-link with a second nearby cysteine to form a disulfide bridge (7Benitah J.P. Tomaselli G.F. Marbán E. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 7392-7396Crossref PubMed Scopus (57) Google Scholar, 8Tsushima R.G. Li R.A. Backx P.H. J. Gen. Physiol. 1997; 110: 59-72Crossref PubMed Scopus (57) Google Scholar). These properties render free cysteinyl the most chemically reactive amino acid side chain (9Creighton T. Proteins: Structures and Molecular Properties. 5th Ed. W. H. Freeman & Co., New York1997Google Scholar). When exposed to the aqueous phase, cysteinyls can be selectively and covalently modified by hydrophilic sulfhydryl-reactive agents (10Karlin A. Akabas M.H. Methods Enzymol. 1998; 293: 123-145Crossref PubMed Scopus (541) Google Scholar). Indeed, engineering cysteines into proteins, followed by the assessment of their accessibility pattern using a variety of sulfhydryl-reactive probes with different physical and chemical properties (i.e. cysteine scanning mutagenesis), has been a proven approach to study the structure-function relationships of numerous ion channels, receptors, and proteins (7Benitah J.P. Tomaselli G.F. Marbán E. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 7392-7396Crossref PubMed Scopus (57) Google Scholar, 8Tsushima R.G. Li R.A. Backx P.H. J. Gen. Physiol. 1997; 110: 59-72Crossref PubMed Scopus (57) Google Scholar, 10Karlin A. Akabas M.H. Methods Enzymol. 1998; 293: 123-145Crossref PubMed Scopus (541) Google Scholar, 11Li R.A. Velez P. Chiamvimonvat N. Tomaselli G.F. Marbán E. J. Gen. Physiol. 2000; 115: 81-92Crossref PubMed Scopus (32) Google Scholar, 12Perez-Garcia M.T. Chiamvimonvat N. Marbán E. Tomaselli G.F. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 300-304Crossref PubMed Scopus (107) Google Scholar, 13Lu Q. Miller C. Science. 1995; 268: 304-307Crossref PubMed Scopus (172) Google Scholar, 14Akabas M.H. Stauffer D.A. Xu M. Karlin A. Science. 1992; 258: 307-310Crossref PubMed Scopus (595) Google Scholar, 15Akabas M.H. Karlin A. Biochemistry. 1995; 34: 12496-12500Crossref PubMed Scopus (159) Google Scholar, 16Akabas M.H. Kaufmann C. Archdeacon P. Karlin A. Neuron. 1994; 13: 919-927Abstract Full Text PDF PubMed Scopus (356) Google Scholar, 17Sun Z.P. Akabas M.H. Goulding E.H. Karlin A. Siegelbaum S.A. Neuron. 1996; 16: 141-149Abstract Full Text Full Text PDF PubMed Scopus (105) Google Scholar). The presence of endogenous cysteines in HCN channels that are putatively exposed to the extracellular milieu raises the possibility that wild-type (WT) 1The abbreviations used for: WT, wild-type; MTS, methanethiosulfonate; MTSEA, methanethiosulfonate ethylammonium; MTSES, methanethiosulfonate ethylsulfonate; DTT, dithiothreitol. 1The abbreviations used for: WT, wild-type; MTS, methanethiosulfonate; MTSEA, methanethiosulfonate ethylammonium; MTSES, methanethiosulfonate ethylsulfonate; DTT, dithiothreitol. channels are susceptible to modifications by sulfhydryl-reactive compounds, which in turn provide an excellent opportunity for localizing structurally and functionally important channel domains. In this report, we examined the effects of the hydrophilic covalent sulfhydryl modifiers methanethiosulfonate ethylammonium (MTSEA) and methanethiosulfonate ethylsulfonate (MTSES), which are positively and negatively charged, respectively, on the functions of HCN1 channels. We found that WT HCN1 channels were indeed modified by these agents. Using site-directed mutagenesis, we identified the native cysteine in the S5-P linker (i.e. Cys318) as the residue responsible for the post-translational changes caused by these agents. Novel insights into the structure-function relationships of HCN channels from these results are discussed. We are indebted to Dr. Eduardo Marbán for guidance, encouragement, and generous support and for critical reading of the manuscript. We also thank Peihong Dong for constructing the channel mutants.
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