- Email: [email protected]
Behind the Scenes of CLC Gating: Deriving the Voltage Dependence of Membrane Proteins by Admittance Measurements
Biophysical Journal Volume 107 September 2014 1261–1262
1261
New and Notable Behind the Scenes of CLC Gating: Deriving the Voltage Dependence of Membrane Proteins by Admittance Measurements Christoph Fahlke* Institute of Complex Systems, Zellula¨re Biophysik (ICS-4), Forschungszentrum Ju¨lich, Ju¨lich, Germany
CLC channels and transporters are expressed in virtually every living cell and fulfill a number of housekeeping functions such as stabilizing the resting potential of skeletal muscles, controlling renal salt excretion, and regulating [Cl] and pH in diverse cell organelles (1). The physiological importance of the CLC family is emphasized by the existence of various human diseases associated with mutations in genes encoding CLC channels or transporters. The CLC family encompasses anion channels and secondary-active Cl/Hþ exchangers (2), and thus contains proteins that are mediators of thermodynamically different transport processes. Despite these principal differences in function, all known CLC isoforms seem to be regulated by complex voltage-dependent gating. The importance of this regulation is illustrated by a large number of disease-associated mutations that specifically modify the voltage-dependence of various members of the CLC family. Gating of CLC channels and transporters are both related to conformational changes that underlie the coupled exchange cycle in the isoforms belonging to the transporter branch. High-resolution, three-dimensional structures of pro- and eukaryotic CLC transporters in different conformations provide insights into the structural rearrangement underly-
Submitted July 31, 2014, and accepted for publication August 4, 2014. *Correspondence: [email protected] Editor: Ian Forster. Ó 2014 by the Biophysical Society 0006-3495/14/09/1261/2 $2.00
ing coupled transport as well as the channel and transporter gating. This structural information revealed the existence of a highly conserved glutamate side chain at the extracellular side that can either project into the aqueous external solution or occupy two of three anion-binding sites in the CLC anion selectivity filter (3,4). This glutamate side chain—often referred to as ‘‘gating glutamate’’—is apparently moving in two sequential steps from the outside into the anion conduction pathway of the protein (Fig. 1 A). Surprisingly, there are no functional data that demonstrate the existence of such sequential steps in mammalian CLC proteins. Gating of CLC transporters is perfectly well described with a simple two-state gating scheme. The nonlinear capacitances associated with activation gating are also monophasic and change upon voltage steps with monoexponential time dependences. This inconsistency is now addressed in an exciting new study by Grieschat and Alekov in this issue of the Biophysical Journal (5), and it is used to provide novel insights into the mechanisms underlying CLC channel/transporter gating. CLC transporters do not only display pronounced time- and voltage-dependence (6,7) but also exhibit prominent gating charge movements (8,9) that are reminiscent of gating currents of voltage-gated cation channels. Grieschat and Alekov (5) used these charge movements to study two mutants of the human ClC-5 exchanger by applying lock-in-based admittance measurements. Both mutations, K210R and E201D, perturb the motility of the external gate of the CLC transporter and lead to the appearance of two well-separated peaks in the voltagedependent capacitances. The novel biphasic voltage dependence suggests that activation of mutant ClC-5 is not executed in one single step, but includes several distinct substeps. These steps depend on the concentrations of the permeant anion Cl. Changes in internal but not external [Cl] affected the
apparent gating charge responsible for the activation of ClC-5. This asymmetry led the authors to conclude that internal anions directly contribute to the voltage sensitivity of the gating process. The different behavior of the two investigated mutations was attributed to a derailed activation sequence with lost synchronization between two intrinsically voltage-dependent steps—the sequential occupation of two stable conformations by the gating glutamate and the knock-off of Cl ions from the internal anion binding site (Fig. 1 B). Although the nature of processes that couple the conformational state of the gating glutamate and the anion occupancy of the internal binding site was not clarified, the reported findings convincingly demonstrate the discrete nature of gating processes in mammalian CLC transporters. The findings of Grieschat and Alekov (5) provide novel important insights into the mechanisms of voltagedependent gating in the CLC family. CLC proteins lack an obvious endogenous voltage sensor, and the origin of voltage-dependent channel gating has been debated for many years. Gating was suggested to be entirely conferred by the permeant anion; or by protons; or by the movement of charged amino-acid side chains. The reported data now suggest that all these processes jointly contribute to voltagedependent CLC channel gating. In particular, proteins of the CLC family appear to sense voltage by both movements of the gating glutamate as well as by anion binding/unbinding reactions. The study not only provides exciting results about a highly important family of transport proteins, but also illustrates how lock-in based admittance measurements (10,11) can provide novel insights into complex conformational changes in membrane proteins. Admittance measurements can be used to separate nonlinear terms in
http://dx.doi.org/10.1016/j.bpj.2014.08.002
1262
Fahlke
complex function of many other ion channels, coupled transporters, or pumps.
A
REFERENCES 1. Jentsch, T. J. 2008. CLC chloride channels and transporters: from genes to protein structure, pathology and physiology. Crit. Rev. Biochem. Mol. Biol. 43:3–36. 2. Accardi, A., and C. Miller. 2004. Secondary active transport mediated by a prokaryotic homologue of ClC Cl channels. Nature. 427:803–807.
B
3. Dutzler, R., E. B. Campbell, and R. MacKinnon. 2003. Gating the selectivity filter in ClC chloride channels. Science. 300:108–112. 4. Feng, L., E. B. Campbell, ., R. MacKinnon. 2010. Structure of a eukaryotic CLC transporter defines an intermediate state in the transport cycle. Science. 330:635–641. FIGURE 1 Movement of the gating glutamate and anion permeation in WT and mutant ClC-5. Gating charge movement (gc) consists of two components gc1 and gc2. In WT ClC-5 (A), the first component (gc1) arises from binding of the gating glutamate to the external Cl binding site Sext and the simultaneous unbinding of one internal Cl ion. The value gc2 is conferred by the movement of the gating glutamate to the central Cl binding site Scen and unbinding of another internally bound Cl. K201R and E211D disrupt the synchronization of gating glutamate movement and unbinding of the internal anion (B). To see this figure in color, go online.
the membrane capacitance that reflect movements of charges within the transmembrane electric field, and which depend on the responses of the investigated protein to voltage. In an oversimplified view, the nonlinear capacitance produced by voltage-dependent conformational changes is proportional to the first derivative of the electric charge that is moved during the underlying gating process. Mathematically, the ability of the derivative to detect not only local minima and maxima of a function, but also inflection points, makes such measurements more sensitive to experimentally separate individual steps within more complex gating
Biophysical Journal 107(6) 1261–1262
schemes, even when the steps do not exhibit strongly differing voltage dependences. Similar strategies might prove advantageous to explore the gating sequence of any arbitrary protein when an increased resolution of its voltage dependence is desired. A huge number of novel transport proteins with complex properties have been identified in recent years that await biophysical investigation and interpretation. Some of us will use the methods elegantly applied in this study, and we are all looking forward to studies that apply high-resolution electrical recordings to understand the
5. Grieschat, M., and A. K. Alekov. 2014. Multiple discrete transitions underlie voltagedependent activation in CLC Cl/Hþ antiporters. Biophys. J. 107:L13–L15. 6. Zdebik, A. A., G. Zifarelli, ., M. Pusch. 2008. Determinants of anion-proton coupling in mammalian endosomal CLC proteins. J. Biol. Chem. 283:4219–4227. 7. Alekov, A. K., and C. Fahlke. 2009. Channellike slippage modes in the human anion/ proton exchanger ClC-4. J. Gen. Physiol. 133:485–496. 8. Smith, A. J., and J. D. Lippiat. 2010. Voltage-dependent charge movement associated with activation of the CLC-5 2Cl/1Hþ exchanger. FASEB J. 24:3696–3705. 9. Guzman, R. E., M. Grieschat, ., A. K. Alekov. 2013. ClC-3 is an intracellular chloride/ proton exchanger with large voltage-dependent nonlinear capacitance. ACS Chem. Neurosci. 4:994–1003. 10. Fidler, N., and J. M. Fernandez. 1989. Phase tracking: an improved phase detection technique for cell membrane capacitance measurements. Biophys. J. 56:1153–1162. 11. Gillis, K. D. 2000. Admittance-based measurement of membrane capacitance using the EPC-9 patch-clamp amplifier. Pflugers Arch. 439:655–664.
1261
New and Notable Behind the Scenes of CLC Gating: Deriving the Voltage Dependence of Membrane Proteins by Admittance Measurements Christoph Fahlke* Institute of Complex Systems, Zellula¨re Biophysik (ICS-4), Forschungszentrum Ju¨lich, Ju¨lich, Germany
CLC channels and transporters are expressed in virtually every living cell and fulfill a number of housekeeping functions such as stabilizing the resting potential of skeletal muscles, controlling renal salt excretion, and regulating [Cl] and pH in diverse cell organelles (1). The physiological importance of the CLC family is emphasized by the existence of various human diseases associated with mutations in genes encoding CLC channels or transporters. The CLC family encompasses anion channels and secondary-active Cl/Hþ exchangers (2), and thus contains proteins that are mediators of thermodynamically different transport processes. Despite these principal differences in function, all known CLC isoforms seem to be regulated by complex voltage-dependent gating. The importance of this regulation is illustrated by a large number of disease-associated mutations that specifically modify the voltage-dependence of various members of the CLC family. Gating of CLC channels and transporters are both related to conformational changes that underlie the coupled exchange cycle in the isoforms belonging to the transporter branch. High-resolution, three-dimensional structures of pro- and eukaryotic CLC transporters in different conformations provide insights into the structural rearrangement underly-
Submitted July 31, 2014, and accepted for publication August 4, 2014. *Correspondence: [email protected] Editor: Ian Forster. Ó 2014 by the Biophysical Society 0006-3495/14/09/1261/2 $2.00
ing coupled transport as well as the channel and transporter gating. This structural information revealed the existence of a highly conserved glutamate side chain at the extracellular side that can either project into the aqueous external solution or occupy two of three anion-binding sites in the CLC anion selectivity filter (3,4). This glutamate side chain—often referred to as ‘‘gating glutamate’’—is apparently moving in two sequential steps from the outside into the anion conduction pathway of the protein (Fig. 1 A). Surprisingly, there are no functional data that demonstrate the existence of such sequential steps in mammalian CLC proteins. Gating of CLC transporters is perfectly well described with a simple two-state gating scheme. The nonlinear capacitances associated with activation gating are also monophasic and change upon voltage steps with monoexponential time dependences. This inconsistency is now addressed in an exciting new study by Grieschat and Alekov in this issue of the Biophysical Journal (5), and it is used to provide novel insights into the mechanisms underlying CLC channel/transporter gating. CLC transporters do not only display pronounced time- and voltage-dependence (6,7) but also exhibit prominent gating charge movements (8,9) that are reminiscent of gating currents of voltage-gated cation channels. Grieschat and Alekov (5) used these charge movements to study two mutants of the human ClC-5 exchanger by applying lock-in-based admittance measurements. Both mutations, K210R and E201D, perturb the motility of the external gate of the CLC transporter and lead to the appearance of two well-separated peaks in the voltagedependent capacitances. The novel biphasic voltage dependence suggests that activation of mutant ClC-5 is not executed in one single step, but includes several distinct substeps. These steps depend on the concentrations of the permeant anion Cl. Changes in internal but not external [Cl] affected the
apparent gating charge responsible for the activation of ClC-5. This asymmetry led the authors to conclude that internal anions directly contribute to the voltage sensitivity of the gating process. The different behavior of the two investigated mutations was attributed to a derailed activation sequence with lost synchronization between two intrinsically voltage-dependent steps—the sequential occupation of two stable conformations by the gating glutamate and the knock-off of Cl ions from the internal anion binding site (Fig. 1 B). Although the nature of processes that couple the conformational state of the gating glutamate and the anion occupancy of the internal binding site was not clarified, the reported findings convincingly demonstrate the discrete nature of gating processes in mammalian CLC transporters. The findings of Grieschat and Alekov (5) provide novel important insights into the mechanisms of voltagedependent gating in the CLC family. CLC proteins lack an obvious endogenous voltage sensor, and the origin of voltage-dependent channel gating has been debated for many years. Gating was suggested to be entirely conferred by the permeant anion; or by protons; or by the movement of charged amino-acid side chains. The reported data now suggest that all these processes jointly contribute to voltagedependent CLC channel gating. In particular, proteins of the CLC family appear to sense voltage by both movements of the gating glutamate as well as by anion binding/unbinding reactions. The study not only provides exciting results about a highly important family of transport proteins, but also illustrates how lock-in based admittance measurements (10,11) can provide novel insights into complex conformational changes in membrane proteins. Admittance measurements can be used to separate nonlinear terms in
http://dx.doi.org/10.1016/j.bpj.2014.08.002
1262
Fahlke
complex function of many other ion channels, coupled transporters, or pumps.
A
REFERENCES 1. Jentsch, T. J. 2008. CLC chloride channels and transporters: from genes to protein structure, pathology and physiology. Crit. Rev. Biochem. Mol. Biol. 43:3–36. 2. Accardi, A., and C. Miller. 2004. Secondary active transport mediated by a prokaryotic homologue of ClC Cl channels. Nature. 427:803–807.
B
3. Dutzler, R., E. B. Campbell, and R. MacKinnon. 2003. Gating the selectivity filter in ClC chloride channels. Science. 300:108–112. 4. Feng, L., E. B. Campbell, ., R. MacKinnon. 2010. Structure of a eukaryotic CLC transporter defines an intermediate state in the transport cycle. Science. 330:635–641. FIGURE 1 Movement of the gating glutamate and anion permeation in WT and mutant ClC-5. Gating charge movement (gc) consists of two components gc1 and gc2. In WT ClC-5 (A), the first component (gc1) arises from binding of the gating glutamate to the external Cl binding site Sext and the simultaneous unbinding of one internal Cl ion. The value gc2 is conferred by the movement of the gating glutamate to the central Cl binding site Scen and unbinding of another internally bound Cl. K201R and E211D disrupt the synchronization of gating glutamate movement and unbinding of the internal anion (B). To see this figure in color, go online.
the membrane capacitance that reflect movements of charges within the transmembrane electric field, and which depend on the responses of the investigated protein to voltage. In an oversimplified view, the nonlinear capacitance produced by voltage-dependent conformational changes is proportional to the first derivative of the electric charge that is moved during the underlying gating process. Mathematically, the ability of the derivative to detect not only local minima and maxima of a function, but also inflection points, makes such measurements more sensitive to experimentally separate individual steps within more complex gating
Biophysical Journal 107(6) 1261–1262
schemes, even when the steps do not exhibit strongly differing voltage dependences. Similar strategies might prove advantageous to explore the gating sequence of any arbitrary protein when an increased resolution of its voltage dependence is desired. A huge number of novel transport proteins with complex properties have been identified in recent years that await biophysical investigation and interpretation. Some of us will use the methods elegantly applied in this study, and we are all looking forward to studies that apply high-resolution electrical recordings to understand the
5. Grieschat, M., and A. K. Alekov. 2014. Multiple discrete transitions underlie voltagedependent activation in CLC Cl/Hþ antiporters. Biophys. J. 107:L13–L15. 6. Zdebik, A. A., G. Zifarelli, ., M. Pusch. 2008. Determinants of anion-proton coupling in mammalian endosomal CLC proteins. J. Biol. Chem. 283:4219–4227. 7. Alekov, A. K., and C. Fahlke. 2009. Channellike slippage modes in the human anion/ proton exchanger ClC-4. J. Gen. Physiol. 133:485–496. 8. Smith, A. J., and J. D. Lippiat. 2010. Voltage-dependent charge movement associated with activation of the CLC-5 2Cl/1Hþ exchanger. FASEB J. 24:3696–3705. 9. Guzman, R. E., M. Grieschat, ., A. K. Alekov. 2013. ClC-3 is an intracellular chloride/ proton exchanger with large voltage-dependent nonlinear capacitance. ACS Chem. Neurosci. 4:994–1003. 10. Fidler, N., and J. M. Fernandez. 1989. Phase tracking: an improved phase detection technique for cell membrane capacitance measurements. Biophys. J. 56:1153–1162. 11. Gillis, K. D. 2000. Admittance-based measurement of membrane capacitance using the EPC-9 patch-clamp amplifier. Pflugers Arch. 439:655–664.