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Hydrophobic interactions between the voltage sensor and pore mediate inactivation in Kv11.1 channels.

Perry MD, Wong S, Ng CA, Vandenberg JI - J. Gen. Physiol. (2013)

Bottom Line: Crucially in Kv11.1 channels, inactivation gating occurs much more rapidly, and over a distinct range of voltages, compared with activation gating.Combining REFER analysis with double mutant cycle analysis, we provide evidence for a hydrophobic interaction between residues on the S4 and S5 helices.Based on a Kv11.1 channel homology model, we propose that this hydrophobic interaction forms the basis of an intersubunit coupling between the voltage sensor and pore domain that is an important mediator of inactivation gating.

View Article: PubMed Central - HTML - PubMed

Affiliation: Molecular Cardiology and Biophysics Division, Victor Chang Cardiac Research Institute, Darlinghurst, NSW 2010, Australia.

ABSTRACT
Kv11.1 channels are critical for the maintenance of a normal heart rhythm. The flow of potassium ions through these channels is controlled by two voltage-regulated gates, termed "activation" and "inactivation," located at opposite ends of the pore. Crucially in Kv11.1 channels, inactivation gating occurs much more rapidly, and over a distinct range of voltages, compared with activation gating. Although it is clear that the fourth transmembrane segments (S4), within each subunit of the tetrameric channel, are important for controlling the opening and closing of the activation gate, their role during inactivation gating is much less clear. Here, we use rate equilibrium free energy relationship (REFER) analysis to probe the contribution of the S4 "voltage-sensor" helix during inactivation of Kv11.1 channels. Contrary to the important role that charged residues play during activation gating, it is the hydrophobic residues (Leu529, Leu530, Leu532, and Val535) that are the key molecular determinants of inactivation gating. Within the context of an interconnected multi-domain model of Kv11.1 inactivation gating, our REFER analysis indicates that the S4 helix and the S4-S5 linker undergo a conformational rearrangement shortly after that of the S5 helix and S5P linker, but before the S6 helix. Combining REFER analysis with double mutant cycle analysis, we provide evidence for a hydrophobic interaction between residues on the S4 and S5 helices. Based on a Kv11.1 channel homology model, we propose that this hydrophobic interaction forms the basis of an intersubunit coupling between the voltage sensor and pore domain that is an important mediator of inactivation gating.

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Measurement of inactivation kinetics for WT and V535S Kv11.1 channels. (A) Voltage protocols used to measure the rates of onset of inactivation (i) and the rates of recovery from inactivation (ii), with key regions for the measurement of current highlighted in black. (B and C) Representative families of current traces measuring the rates of onset of inactivation (i) and rates of recovery from inactivation (ii) for WT (B) and V535S (C) Kv11.1 channels. Highlighted in bold are current traces at 0 mV (i) and −130 mV (ii) to aid comparison. (D) Chevron plots of the logarithm of the observed rates for onset of (open squares) and recovery from (closed squares) inactivation for WT (i) and V535S (ii) channels, plotted against voltage. Solid black lines are a fit of Eq. 1 (see Materials and methods), whereas the solid gray line in (ii) indicates kobs,V for WT channels to aid comparison. Dashed lines indicate the derived unidirectional rate constants for the onset of (kinact,V) and recovery from (krec,V) inactivation, with values at 0 mV indicated by arrows. The equilibrium constant for inactivation (Keq) at 0 mV was calculated by: Keq,0 = kinact,0/krec,0 (Eq. 2 in Materials and methods).
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fig1: Measurement of inactivation kinetics for WT and V535S Kv11.1 channels. (A) Voltage protocols used to measure the rates of onset of inactivation (i) and the rates of recovery from inactivation (ii), with key regions for the measurement of current highlighted in black. (B and C) Representative families of current traces measuring the rates of onset of inactivation (i) and rates of recovery from inactivation (ii) for WT (B) and V535S (C) Kv11.1 channels. Highlighted in bold are current traces at 0 mV (i) and −130 mV (ii) to aid comparison. (D) Chevron plots of the logarithm of the observed rates for onset of (open squares) and recovery from (closed squares) inactivation for WT (i) and V535S (ii) channels, plotted against voltage. Solid black lines are a fit of Eq. 1 (see Materials and methods), whereas the solid gray line in (ii) indicates kobs,V for WT channels to aid comparison. Dashed lines indicate the derived unidirectional rate constants for the onset of (kinact,V) and recovery from (krec,V) inactivation, with values at 0 mV indicated by arrows. The equilibrium constant for inactivation (Keq) at 0 mV was calculated by: Keq,0 = kinact,0/krec,0 (Eq. 2 in Materials and methods).

Mentions: Oocytes were impaled with glass capillary micropipettes that had access resistances in the range of 0.3 to 1.0 MΩ. Currents were recorded from oocytes using a Geneclamp500B two-electrode voltage-clamp amplifier interfaced to a PC via a Digidata 1440 (Molecular Devices). Signals were filtered at 2 kHz and digitized at 5–10 kHz. Voltage-clamp protocols are shown as insets in Fig. 1 A. In all protocols, the cells were held at −90 mV before any voltage steps. After each experiment, the offset potential was checked, and if it exceeded ±5 mV, the recordings were discarded. Data acquisition and analysis were performed using pCLAMP (version 10.2; Molecular Devices), Excel (Microsoft) and Prism (version 6; GraphPad) software. All parameter values were calculated as mean ± SEM for n experiments, where n denotes the number of different oocytes studied for each construct.


Hydrophobic interactions between the voltage sensor and pore mediate inactivation in Kv11.1 channels.

Perry MD, Wong S, Ng CA, Vandenberg JI - J. Gen. Physiol. (2013)

Measurement of inactivation kinetics for WT and V535S Kv11.1 channels. (A) Voltage protocols used to measure the rates of onset of inactivation (i) and the rates of recovery from inactivation (ii), with key regions for the measurement of current highlighted in black. (B and C) Representative families of current traces measuring the rates of onset of inactivation (i) and rates of recovery from inactivation (ii) for WT (B) and V535S (C) Kv11.1 channels. Highlighted in bold are current traces at 0 mV (i) and −130 mV (ii) to aid comparison. (D) Chevron plots of the logarithm of the observed rates for onset of (open squares) and recovery from (closed squares) inactivation for WT (i) and V535S (ii) channels, plotted against voltage. Solid black lines are a fit of Eq. 1 (see Materials and methods), whereas the solid gray line in (ii) indicates kobs,V for WT channels to aid comparison. Dashed lines indicate the derived unidirectional rate constants for the onset of (kinact,V) and recovery from (krec,V) inactivation, with values at 0 mV indicated by arrows. The equilibrium constant for inactivation (Keq) at 0 mV was calculated by: Keq,0 = kinact,0/krec,0 (Eq. 2 in Materials and methods).
© Copyright Policy - openaccess
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3753607&req=5

fig1: Measurement of inactivation kinetics for WT and V535S Kv11.1 channels. (A) Voltage protocols used to measure the rates of onset of inactivation (i) and the rates of recovery from inactivation (ii), with key regions for the measurement of current highlighted in black. (B and C) Representative families of current traces measuring the rates of onset of inactivation (i) and rates of recovery from inactivation (ii) for WT (B) and V535S (C) Kv11.1 channels. Highlighted in bold are current traces at 0 mV (i) and −130 mV (ii) to aid comparison. (D) Chevron plots of the logarithm of the observed rates for onset of (open squares) and recovery from (closed squares) inactivation for WT (i) and V535S (ii) channels, plotted against voltage. Solid black lines are a fit of Eq. 1 (see Materials and methods), whereas the solid gray line in (ii) indicates kobs,V for WT channels to aid comparison. Dashed lines indicate the derived unidirectional rate constants for the onset of (kinact,V) and recovery from (krec,V) inactivation, with values at 0 mV indicated by arrows. The equilibrium constant for inactivation (Keq) at 0 mV was calculated by: Keq,0 = kinact,0/krec,0 (Eq. 2 in Materials and methods).
Mentions: Oocytes were impaled with glass capillary micropipettes that had access resistances in the range of 0.3 to 1.0 MΩ. Currents were recorded from oocytes using a Geneclamp500B two-electrode voltage-clamp amplifier interfaced to a PC via a Digidata 1440 (Molecular Devices). Signals were filtered at 2 kHz and digitized at 5–10 kHz. Voltage-clamp protocols are shown as insets in Fig. 1 A. In all protocols, the cells were held at −90 mV before any voltage steps. After each experiment, the offset potential was checked, and if it exceeded ±5 mV, the recordings were discarded. Data acquisition and analysis were performed using pCLAMP (version 10.2; Molecular Devices), Excel (Microsoft) and Prism (version 6; GraphPad) software. All parameter values were calculated as mean ± SEM for n experiments, where n denotes the number of different oocytes studied for each construct.

Bottom Line: Crucially in Kv11.1 channels, inactivation gating occurs much more rapidly, and over a distinct range of voltages, compared with activation gating.Combining REFER analysis with double mutant cycle analysis, we provide evidence for a hydrophobic interaction between residues on the S4 and S5 helices.Based on a Kv11.1 channel homology model, we propose that this hydrophobic interaction forms the basis of an intersubunit coupling between the voltage sensor and pore domain that is an important mediator of inactivation gating.

View Article: PubMed Central - HTML - PubMed

Affiliation: Molecular Cardiology and Biophysics Division, Victor Chang Cardiac Research Institute, Darlinghurst, NSW 2010, Australia.

ABSTRACT
Kv11.1 channels are critical for the maintenance of a normal heart rhythm. The flow of potassium ions through these channels is controlled by two voltage-regulated gates, termed "activation" and "inactivation," located at opposite ends of the pore. Crucially in Kv11.1 channels, inactivation gating occurs much more rapidly, and over a distinct range of voltages, compared with activation gating. Although it is clear that the fourth transmembrane segments (S4), within each subunit of the tetrameric channel, are important for controlling the opening and closing of the activation gate, their role during inactivation gating is much less clear. Here, we use rate equilibrium free energy relationship (REFER) analysis to probe the contribution of the S4 "voltage-sensor" helix during inactivation of Kv11.1 channels. Contrary to the important role that charged residues play during activation gating, it is the hydrophobic residues (Leu529, Leu530, Leu532, and Val535) that are the key molecular determinants of inactivation gating. Within the context of an interconnected multi-domain model of Kv11.1 inactivation gating, our REFER analysis indicates that the S4 helix and the S4-S5 linker undergo a conformational rearrangement shortly after that of the S5 helix and S5P linker, but before the S6 helix. Combining REFER analysis with double mutant cycle analysis, we provide evidence for a hydrophobic interaction between residues on the S4 and S5 helices. Based on a Kv11.1 channel homology model, we propose that this hydrophobic interaction forms the basis of an intersubunit coupling between the voltage sensor and pore domain that is an important mediator of inactivation gating.

Show MeSH