Abstract
study and characterization of unliganded openings is of central significance for the elucidation of gating mechanisms for allosteric ligand-gated ion channels. Unliganded openings have been reported for many channel types, but their low open probability can make it difficult to study their kinetics in detail. Because the large conductance calcium-activated potassium channel mSlo is sensitive to both intracellular calcium and to membrane potential, we have been able to obtain stable unliganded single-channel recordings of mSlo with relatively high opening probability. We have found that the single-channel gating behavior of mSlo is complex, with multiple open and closed states, even when no ligand is present. Our results rule out a Monod-Wyman-Changeux allosteric mechanism with a central voltage-dependent concerted step, and they support the existence of quaternary states with less than the full number of voltage sensors activated, as has been suggested by previous work involving measurements of gating currents.
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- Blatz A. L., Magleby K. L. Correcting single channel data for missed events. Biophys J. 1986 May;49(5):967–980. doi: 10.1016/S0006-3495(86)83725-0. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Blatz A. L., Magleby K. L. Single voltage-dependent chloride-selective channels of large conductance in cultured rat muscle. Biophys J. 1983 Aug;43(2):237–241. doi: 10.1016/S0006-3495(83)84344-6. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Brehm P., Kullberg R., Moody-Corbett F. Properties of non-junctional acetylcholine receptor channels on innervated muscle of Xenopus laevis. J Physiol. 1984 May;350:631–648. doi: 10.1113/jphysiol.1984.sp015222. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Changeux J. P., Edelstein S. J. Allosteric receptors after 30 years. Neuron. 1998 Nov;21(5):959–980. doi: 10.1016/s0896-6273(00)80616-9. [DOI] [PubMed] [Google Scholar]
- Colquhoun D., Sakmann B. Fast events in single-channel currents activated by acetylcholine and its analogues at the frog muscle end-plate. J Physiol. 1985 Dec;369:501–557. doi: 10.1113/jphysiol.1985.sp015912. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Cox D. H., Cui J., Aldrich R. W. Allosteric gating of a large conductance Ca-activated K+ channel. J Gen Physiol. 1997 Sep;110(3):257–281. doi: 10.1085/jgp.110.3.257. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Cui J., Cox D. H., Aldrich R. W. Intrinsic voltage dependence and Ca2+ regulation of mslo large conductance Ca-activated K+ channels. J Gen Physiol. 1997 May;109(5):647–673. doi: 10.1085/jgp.109.5.647. [DOI] [PMC free article] [PubMed] [Google Scholar]
- DiChiara T. J., Reinhart P. H. Redox modulation of hslo Ca2+-activated K+ channels. J Neurosci. 1997 Jul 1;17(13):4942–4955. doi: 10.1523/JNEUROSCI.17-13-04942.1997. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Diaz F., Wallner M., Stefani E., Toro L., Latorre R. Interaction of internal Ba2+ with a cloned Ca(2+)-dependent K+ (hslo) channel from smooth muscle. J Gen Physiol. 1996 Mar;107(3):399–407. doi: 10.1085/jgp.107.3.399. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Edelstein S. J., Changeux J. P. Allosteric proteins after thirty years: the binding and state functions of the neuronal alpha 7 nicotinic acetylcholine receptors. Experientia. 1996 Dec 15;52(12):1083–1090. doi: 10.1007/BF01952106. [DOI] [PubMed] [Google Scholar]
- Edelstein S. J., Changeux J. P. Allosteric transitions of the acetylcholine receptor. Adv Protein Chem. 1998;51:121–184. doi: 10.1016/s0065-3233(08)60652-x. [DOI] [PubMed] [Google Scholar]
- Horn R., Lange K. Estimating kinetic constants from single channel data. Biophys J. 1983 Aug;43(2):207–223. doi: 10.1016/S0006-3495(83)84341-0. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Horrigan F. T., Aldrich R. W. Allosteric voltage gating of potassium channels II. Mslo channel gating charge movement in the absence of Ca(2+). J Gen Physiol. 1999 Aug;114(2):305–336. doi: 10.1085/jgp.114.2.305. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Horrigan F. T., Cui J., Aldrich R. W. Allosteric voltage gating of potassium channels I. Mslo ionic currents in the absence of Ca(2+). J Gen Physiol. 1999 Aug;114(2):277–304. doi: 10.1085/jgp.114.2.277. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Hoshi T., Zagotta W. N., Aldrich R. W. Shaker potassium channel gating. I: Transitions near the open state. J Gen Physiol. 1994 Feb;103(2):249–278. doi: 10.1085/jgp.103.2.249. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Jackson M. B., Imoto K., Mishina M., Konno T., Numa S., Sakmann B. Spontaneous and agonist-induced openings of an acetylcholine receptor channel composed of bovine muscle alpha-, beta- and delta-subunits. Pflugers Arch. 1990 Oct;417(2):129–135. doi: 10.1007/BF00370689. [DOI] [PubMed] [Google Scholar]
- Jackson M. B. Spontaneous openings of the acetylcholine receptor channel. Proc Natl Acad Sci U S A. 1984 Jun;81(12):3901–3904. doi: 10.1073/pnas.81.12.3901. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Koshland D. E., Jr, Némethy G., Filmer D. Comparison of experimental binding data and theoretical models in proteins containing subunits. Biochemistry. 1966 Jan;5(1):365–385. doi: 10.1021/bi00865a047. [DOI] [PubMed] [Google Scholar]
- Liu D. T., Tibbs G. R., Paoletti P., Siegelbaum S. A. Constraining ligand-binding site stoichiometry suggests that a cyclic nucleotide-gated channel is composed of two functional dimers. Neuron. 1998 Jul;21(1):235–248. doi: 10.1016/s0896-6273(00)80530-9. [DOI] [PubMed] [Google Scholar]
- MONOD J., WYMAN J., CHANGEUX J. P. ON THE NATURE OF ALLOSTERIC TRANSITIONS: A PLAUSIBLE MODEL. J Mol Biol. 1965 May;12:88–118. doi: 10.1016/s0022-2836(65)80285-6. [DOI] [PubMed] [Google Scholar]
- Magleby K. L., Pallotta B. S. Burst kinetics of single calcium-activated potassium channels in cultured rat muscle. J Physiol. 1983 Nov;344:605–623. doi: 10.1113/jphysiol.1983.sp014958. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Magleby K. L., Pallotta B. S. Calcium dependence of open and shut interval distributions from calcium-activated potassium channels in cultured rat muscle. J Physiol. 1983 Nov;344:585–604. doi: 10.1113/jphysiol.1983.sp014957. [DOI] [PMC free article] [PubMed] [Google Scholar]
- McManus O. B., Magleby K. L. Accounting for the Ca(2+)-dependent kinetics of single large-conductance Ca(2+)-activated K+ channels in rat skeletal muscle. J Physiol. 1991 Nov;443:739–777. doi: 10.1113/jphysiol.1991.sp018861. [DOI] [PMC free article] [PubMed] [Google Scholar]
- McManus O. B., Magleby K. L. Kinetic states and modes of single large-conductance calcium-activated potassium channels in cultured rat skeletal muscle. J Physiol. 1988 Aug;402:79–120. doi: 10.1113/jphysiol.1988.sp017195. [DOI] [PMC free article] [PubMed] [Google Scholar]
- McManus O. B., Magleby K. L. Kinetic time constants independent of previous single-channel activity suggest Markov gating for a large conductance Ca-activated K channel. J Gen Physiol. 1989 Dec;94(6):1037–1070. doi: 10.1085/jgp.94.6.1037. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Methfessel C., Boheim G. The gating of single calcium-dependent potassium channels is described by an activation/blockade mechanism. Biophys Struct Mech. 1982;9(1):35–60. doi: 10.1007/BF00536014. [DOI] [PubMed] [Google Scholar]
- Moczydlowski E., Latorre R. Gating kinetics of Ca2+-activated K+ channels from rat muscle incorporated into planar lipid bilayers. Evidence for two voltage-dependent Ca2+ binding reactions. J Gen Physiol. 1983 Oct;82(4):511–542. doi: 10.1085/jgp.82.4.511. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Neyton J. A Ba2+ chelator suppresses long shut events in fully activated high-conductance Ca(2+)-dependent K+ channels. Biophys J. 1996 Jul;71(1):220–226. doi: 10.1016/S0006-3495(96)79218-4. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Pallotta B. S., Blatz A. L., Magleby K. L. Recording from calcium-activated potassium channels. Methods Enzymol. 1992;207:194–207. doi: 10.1016/0076-6879(92)07014-f. [DOI] [PubMed] [Google Scholar]
- Pallotta B. S., Magleby K. L., Barrett J. N. Single channel recordings of Ca2+-activated K+ currents in rat muscle cell culture. Nature. 1981 Oct 8;293(5832):471–474. doi: 10.1038/293471a0. [DOI] [PubMed] [Google Scholar]
- Pallotta B. S. N-bromoacetamide removes a calcium-dependent component of channel opening from calcium-activated potassium channels in rat skeletal muscle. J Gen Physiol. 1985 Nov;86(5):601–611. doi: 10.1085/jgp.86.5.601. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Picones A., Korenbrot J. I. Spontaneous, ligand-independent activity of the cGMP-gated ion channels in cone photoreceptors of fish. J Physiol. 1995 Jun 15;485(Pt 3):699–714. doi: 10.1113/jphysiol.1995.sp020763. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Rothberg B. S., Bello R. A., Magleby K. L. Two-dimensional components and hidden dependencies provide insight into ion channel gating mechanisms. Biophys J. 1997 Jun;72(6):2524–2544. doi: 10.1016/S0006-3495(97)78897-0. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Rothberg B. S., Bello R. A., Song L., Magleby K. L. High Ca2+ concentrations induce a low activity mode and reveal Ca2(+)-independent long shut intervals in BK channels from rat muscle. J Physiol. 1996 Jun 15;493(Pt 3):673–689. doi: 10.1113/jphysiol.1996.sp021414. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Rothberg B. S., Magleby K. L. Gating kinetics of single large-conductance Ca2+-activated K+ channels in high Ca2+ suggest a two-tiered allosteric gating mechanism. J Gen Physiol. 1999 Jul;114(1):93–124. doi: 10.1085/jgp.114.1.93. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Rothberg B. S., Magleby K. L. Kinetic structure of large-conductance Ca2+-activated K+ channels suggests that the gating includes transitions through intermediate or secondary states. A mechanism for flickers. J Gen Physiol. 1998 Jun;111(6):751–780. doi: 10.1085/jgp.111.6.751. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Ruiz M. L., Karpen J. W. Single cyclic nucleotide-gated channels locked in different ligand-bound states. Nature. 1997 Sep 25;389(6649):389–392. doi: 10.1038/38744. [DOI] [PubMed] [Google Scholar]
- Schoppa N. E., Sigworth F. J. Activation of shaker potassium channels. I. Characterization of voltage-dependent transitions. J Gen Physiol. 1998 Feb;111(2):271–294. doi: 10.1085/jgp.111.2.271. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Silberberg S. D., Lagrutta A., Adelman J. P., Magleby K. L. Wanderlust kinetics and variable Ca(2+)-sensitivity of dSlo [correction of Drosophila], a large conductance CA(2+)-activated K+ channel, expressed in oocytes. Biophys J. 1996 Jul;71(1):2640–2651. [PMC free article] [PubMed] [Google Scholar]
- Stefani E., Ottolia M., Noceti F., Olcese R., Wallner M., Latorre R., Toro L. Voltage-controlled gating in a large conductance Ca2+-sensitive K+channel (hslo). Proc Natl Acad Sci U S A. 1997 May 13;94(10):5427–5431. doi: 10.1073/pnas.94.10.5427. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Tibbs G. R., Goulding E. H., Siegelbaum S. A. Allosteric activation and tuning of ligand efficacy in cyclic-nucleotide-gated channels. Nature. 1997 Apr 10;386(6625):612–615. doi: 10.1038/386612a0. [DOI] [PubMed] [Google Scholar]
