Use of voltage clamp fluorimetry in understanding potassium channel gating: a review of Shaker fluorescence data

A.J. Horne; D. Fedida

doi:10.1139/y09-024

Use of voltage clamp fluorimetry in understanding potassium channel gating: a review of Shaker fluorescence data

Canadian Journal of Physiology and Pharmacology ◽

10.1139/y09-024 ◽

2009 ◽

Vol 87 (6) ◽

pp. 411-418 ◽

Cited By ~ 11

Author(s):

A.J. Horne ◽

D. Fedida

Keyword(s):

Potassium Channels ◽

Potassium Channel ◽

Voltage Clamp ◽

Channel Gating ◽

Specific Protein ◽

Channel Activation ◽

Shaker Potassium Channel ◽

Inactivation Gating ◽

Nanosecond Time Resolution ◽

Shaker Potassium Channels

Voltage clamp fluorimetry (VCF) utilizes fluorescent probes that covalently bind to cysteine residues introduced into proteins and emit light as a function of their environment. Measurement of this emitted light during membrane depolarization reveals changes in the emission level as the environment of the labelled residue changes. This allows for the correlation of channel gating events with movement of specific protein moieties, at nanosecond time resolution. Since the pioneering use of this technique to investigate Shaker potassium channel activation movements, VCF has become an invaluable technique used to understand ion channel gating. This review summarizes the theory and some of the data on the application of the VCF technique. Although its usage has expanded beyond voltage-gated potassium channels and VCF is now used in a number of other voltage- and ligand-gated channels, we will focus on studies conducted in Shaker potassium channels, and what they have told us about channel activation and inactivation gating.

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Activation-inactivation of potassium channels and development of the potassium-channel spike in internally perfused squid giant axons.

The Journal of General Physiology ◽

10.1085/jgp.78.1.43 ◽

1981 ◽

Vol 78 (1) ◽

pp. 43-61 ◽

Cited By ~ 17

Author(s):

I Inoue

Keyword(s):

Potassium Channels ◽

Potassium Channel ◽

Voltage Clamp ◽

Time Constant ◽

Equilibrium Potential ◽

Giant Axons ◽

Dependent Inactivation ◽

Voltage Dependent ◽

Time Courses ◽

Calcium Permeability

A spike that is the result of calcium permeability through potassium channels was separated from the action potential is squid giant axons internally perfused with a 30 mM NaF solution and bathed in a 100 mM CaCl2 solution by blocking sodium channels with tetrodotoxin. Currents through potassium channels were studied under voltage clamp. The records showed a clear voltage-dependent inactivation of the currents. The inactivation was composed of at least two components; one relatively fast, having a time constant of 20--30 ms, and the other very slow, having a time constant of 5--10 s. Voltage clamp was carried out with a variety of salt compositions in both the internal and external solutions. A similar voltage-dependent inactivation, also composed of the two components, was recognized in all the current through potassium channels. Although the direction and intensity of current strongly depended on the salt composition of the solutions, the time-courses of these currents at corresponding voltages were very similar. These results strongly suggest that the inactivation of the currents in attributable to an essential, dynamic property of potassium channels themselves. Thus, the generation of a potassium-channel spike can be understood as an event that occurs when the equilibrium potential across the potassium channel becomes positive.

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Uncharged S4 Residues and Cooperativity in Voltage-dependent Potassium Channel Activation

The Journal of General Physiology ◽

10.1085/jgp.111.3.421 ◽

1998 ◽

Vol 111 (3) ◽

pp. 421-439 ◽

Cited By ~ 131

Author(s):

Catherine J. Smith-Maxwell ◽

Jennifer L. Ledwell ◽

Richard W. Aldrich

Keyword(s):

Amino Acids ◽

Amino Acid ◽

Potassium Channel ◽

Voltage Dependence ◽

Channel Gating ◽

Ionic Currents ◽

Channel Activation ◽

Functional Changes ◽

Activation Pathway ◽

Voltage Dependent

Substitution of the S4 of Shaw into Shaker alters cooperativity in channel activation by slowing a cooperative transition late in the activation pathway. To determine the amino acids responsible for the functional changes in Shaw S4, we created several mutants by substituting amino acids from Shaw S4 into Shaker. The S4 amino acid sequences of Shaker and Shaw S4 differ at 11 positions. Simultaneous substitution of just three noncharged residues from Shaw S4 into Shaker (V369I, I372L, S376T; ILT) reproduces the kinetic and voltage-dependent properties of Shaw S4 channel activation. These substitutions cause very small changes in the structural and chemical properties of the amino acid side chains. In contrast, substituting the positively charged basic residues in the S4 of Shaker with neutral or negative residues from the S4 of Shaw S4 does not reproduce the shallow voltage dependence or other properties of Shaw S4 opening. Macroscopic ionic currents for ILT could be fit by modifying a single set of transitions in a model for Shaker channel gating (Zagotta, W.N., T. Hoshi, and R.W. Aldrich. 1994. J. Gen. Physiol. 103:321–362). Changing the rate and voltage dependence of a final cooperative step in activation successfully reproduces the kinetic, steady state, and voltage-dependent properties of ILT ionic currents. Consistent with the model, ILT gating currents activate at negative voltages where the channel does not open and, at more positive voltages, they precede the ionic currents, confirming the existence of voltage-dependent transitions between closed states in the activation pathway. Of the three substitutions in ILT, the I372L substitution is primarily responsible for the changes in cooperativity and voltage dependence. These results suggest that noncharged residues in the S4 play a crucial role in Shaker potassium channel gating and that small steric changes in these residues can lead to large changes in cooperativity within the channel protein.

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Voltage clamp fluorimetry studies of mammalian voltage-gated K+ channel gating

Biochemical Society Transactions ◽

10.1042/bst0351080 ◽

2007 ◽

Vol 35 (5) ◽

pp. 1080-1082 ◽

Cited By ~ 14

Author(s):

T.W. Claydon ◽

D. Fedida

Keyword(s):

Ion Channel ◽

Potassium Channel ◽

Real Time ◽

Voltage Clamp ◽

Conformational Changes ◽

Channel Gating ◽

K Channel ◽

Voltage Gated ◽

Kv Channels ◽

Health And Disease

VCF (voltage clamp fluorimetry) provides a powerful technique to observe real-time conformational changes that are associated with ion channel gating. The present review highlights the insights such experiments have provided in understanding Kv (voltage-gated potassium) channel gating, with particular emphasis on the study of mammalian Kv1 channels. Further applications of VCF that would contribute to our understanding of the modulation of Kv channels in health and disease are also discussed.

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Amino Acid Substitution within the S2 and S4 Transmembrane Segments in Shaker Potassium Channel Modulates Channel Gating

Biochemical and Biophysical Research Communications ◽

10.1006/bbrc.2000.3369 ◽

2000 ◽

Vol 275 (3) ◽

pp. 720-724 ◽

Cited By ~ 2

Author(s):

Myeong Hyeon Wang ◽

Uhtaek Oh ◽

Hae Ik Rhee

Keyword(s):

Amino Acid ◽

Potassium Channel ◽

Amino Acid Substitution ◽

Channel Gating ◽

Shaker Potassium Channel ◽

Transmembrane Segments

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Investigating the Putative Glycine Hinge in Shaker Potassium Channel

The Journal of General Physiology ◽

10.1085/jgp.200509287 ◽

2005 ◽

Vol 126 (3) ◽

pp. 213-226 ◽

Cited By ~ 67

Author(s):

Shinghua Ding ◽

Lindsey Ingleby ◽

Christopher A. Ahern ◽

Richard Horn

Keyword(s):

Potassium Channel ◽

Functional Expression ◽

Shaker Potassium Channel ◽

Glycine Residue ◽

Shaker Channels ◽

Channel Opening ◽

Activation Gating ◽

Voltage Dependent ◽

Shaker Potassium Channels

The crystal structure of an open potassium channel reveals a kink in the inner helix that lines the pore (Jiang, Y.X., A. Lee, J.Y. Chen, M. Cadene, B.T. Chait, and R. MacKinnon. 2002. Nature 417:523–526). The putative hinge point is a highly conserved glycine residue. We examined the role of the homologous residue (Gly466) in the S6 transmembrane segment of Shaker potassium channels. The nonfunctional alanine mutant G466A will assemble, albeit poorly, with wild-type (WT) subunits, suppressing functional expression. To test if this glycine residue is critical for activation gating, we did a glycine scan along the S6 segment in the background of G466A. Although all of these double mutants lack the higher-level glycosylation that is characteristic of mature Shaker channels, one (G466A/V467G) is able to generate voltage-dependent potassium current. Surface biotinylation shows that functional and nonfunctional constructs containing G466A express at comparable levels in the plasma membrane. Compared with WT channels, the shifted-glycine mutant has impairments in voltage-dependent channel opening, including a right-shifted activation curve and a decreased rate of activation. The double mutant has relatively normal open-channel properties, except for a decreased affinity for intracellular blockers, a consequence of the loss of the side chain of Val467. Control experiments with the double mutants M440A/G466A and G466A/V467A suggest that the flexibility provided by Gly466 is more important for channel function than its small size. Our results support roles for Gly466 both in biogenesis of the channel and as a hinge in activation gating.

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Role of the S4 in Cooperativity of Voltage-dependent Potassium Channel Activation

The Journal of General Physiology ◽

10.1085/jgp.111.3.399 ◽

1998 ◽

Vol 111 (3) ◽

pp. 399-420 ◽

Cited By ~ 116

Author(s):

Catherine J. Smith-Maxwell ◽

Jennifer L. Ledwell ◽

Richard W. Aldrich

Keyword(s):

Potassium Channel ◽

Amino Acid Sequences ◽

Channel Activation ◽

Shaker Potassium Channel ◽

Activation Pathway ◽

Activation Gating ◽

Voltage Dependent ◽

Rate Limiting ◽

Cooperative Transition

Charged residues in the S4 transmembrane segment of voltage-gated cation channels play a key role in opening channels in response to changes in voltage across the cell membrane. However, the molecular mechanism of channel activation is not well understood. To learn more about the role of the S4 in channel gating, we constructed chimeras in which S4 segments from several divergent potassium channels, Shab, Shal, Shaw, and Kv3.2, were inserted into a Shaker potassium channel background. These S4 donor channels have distinctly different voltage-dependent gating properties and S4 amino acid sequences. None of the S4 chimeras have the gating behavior of their respective S4 donor channels. The conductance–voltage relations of all S4 chimeras are shifted to more positive voltages and the slopes are decreased. There is no consistent correlation between the nominal charge content of the S4 and the slope of the conductance–voltage relation, suggesting that the mutations introduced by the S4 chimeras may alter cooperative interactions in the gating process. We compared the gating behavior of the Shaw S4 chimera with its parent channels, Shaker and Shaw, in detail. The Shaw S4 substitution alters activation gating profoundly without introducing obvious changes in other channel functions. Analysis of the voltage-dependent gating kinetics suggests that the dominant effect of the Shaw S4 substitution is to alter a single cooperative transition late in the activation pathway, making it rate limiting. This interpretation is supported further by studies of channels assembled from tandem heterodimer constructs with both Shaker and Shaw S4 subunits. Activation gating in the heterodimer channels can be predicted from the properties of the homotetrameric channels only if it is assumed that the mutations alter a cooperative transition in the activation pathway rather than independent transitions.

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A Mutation in S6 of Shaker Potassium Channels Decreases the K+ Affinity of an Ion Binding Site Revealing Ion–Ion Interactions in the Pore

The Journal of General Physiology ◽

10.1085/jgp.112.2.243 ◽

1998 ◽

Vol 112 (2) ◽

pp. 243-257 ◽

Cited By ~ 48

Author(s):

Eva M. Ogielska ◽

Richard W. Aldrich

Keyword(s):

Potassium Channels ◽

Potassium Channel ◽

Binding Site ◽

Sodium Current ◽

High Potassium ◽

Independent Evidence ◽

Channel Pore ◽

Shaker Potassium Channel ◽

Ion Interactions ◽

Barium Block

Under physiological conditions, potassium channels are extraordinarily selective for potassium over other ions. However, in the absence of potassium, certain potassium channels can conduct sodium. Sodium flux is blocked by the addition of low concentrations of potassium. Potassium affinity, and therefore the ability to block sodium current, varies among potassium channel subtypes (Korn, S.J., and S.R. Ikeda. 1995. Science. 269:410–412; Starkus, J.G., L. Kuschel, M.D. Rayner, and S.H. Heinemann. 1997. J. Gen. Physiol. 110:539–550). The Shaker potassium channel conducts sodium poorly in the presence of very low (micromolar) potassium due to its high potassium affinity (Starkus, J.G., L. Kuschel, M.D. Rayner, and S.H. Heinemann. 1997. J. Gen. Physiol. 110:539–550; Ogielska, E.M., and R.W. Aldrich. 1997. Biophys. J. 72:A233 [Abstr.]). We show that changing a single residue in S6, A463C, decreases the apparent internal potassium affinity of the Shaker channel pore from the micromolar to the millimolar range, as determined from the ability of potassium to block the sodium currents. Independent evidence that A463C decreases the apparent affinity of a binding site in the pore comes from a study of barium block of potassium currents. The A463C mutation decreases the internal barium affinity of the channel, as expected if barium blocks current by binding to a potassium site in the pore. The decrease in the apparent potassium affinity in A463C channels allows further study of possible ion interactions in the pore. Our results indicate that sodium and potassium can occupy the pore simultaneously and that multiple occupancy results in interactions between ions in the channel pore.

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