Rocheleau JM and Kobertz WR (2008), KCNE peptides differently affect voltage sensor...

XB-ART-36986

J Gen Physiol 2008 Jan 01;1311:59-68. doi: 10.1085/jgp.200709816.

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KCNE peptides differently affect voltage sensor equilibrium and equilibration rates in KCNQ1 K+ channels.

Rocheleau JM , Kobertz WR .

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KCNQ1 voltage-gated K(+) channels assemble with the family of KCNE type I transmembrane peptides to afford membrane-embedded complexes with diverse channel gating properties. KCNQ1/KCNE1 complexes generate the very slowly activating cardiac I(Ks) current, whereas assembly with KCNE3 produces a constitutively conducting complex involved in K(+) recycling in epithelia. To determine whether these two KCNE peptides influence voltage sensing in KCNQ1 channels, we monitored the position of the S4 voltage sensor in KCNQ1/KCNE complexes using cysteine accessibility experiments. A panel of KCNQ1 S4 cysteine mutants was expressed in Xenopus oocytes, treated with the membrane-impermeant cysteine-specific reagent 2-(trimethylammonium) ethyl methanethiosulfonate (MTSET), and the voltage-dependent accessibility of each mutant was determined. Of these S4 cysteine mutants, three (R228C, G229C, I230C) were modified by MTSET only when KCNQ1 was depolarized. We then employed these state-dependent residues to determine how assembly with KCNE1 and KCNE3 affects KCNQ1 voltage sensor equilibrium and equilibration rates. In the presence of KCNE1, MTSET modification rates for the majority of the cysteine mutants were approximately 10-fold slower, as was recently reported to indicate that the kinetics of the KCNQ1 voltage sensor are slowed by KCNE1 (Nakajo, K., and Y. Kubo. 2007 J. Gen. Physiol. 130:269-281). Since MTS modification rates reflect an amalgam of reagent accessibility, chemical reactivity, and protein conformational changes, we varied the depolarization pulse duration to determine whether KCNE1 slows the equilibration rate of the voltage sensors. Using the state-dependent cysteine mutants, we determined that MTSET modification rates were essentially independent of depolarization pulse duration. These results demonstrate that upon depolarization the voltage sensors reach equilibrium quickly in the presence of KCNE1 and the slow gating of the channel complex is not due to slowly moving voltage sensors. In contrast, all cysteine substitutions in the S4 of KCNQ1/KCNE3 complexes were freely accessible to MTSET independent of voltage, which is consistent with KCNE3 shifting the voltage sensor equilibrium to favor the active state at hyperpolarizing potentials. In total, these results suggest that KCNE peptides differently modulate the voltage sensor in KCNQ1 K(+) channels.

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Species referenced: Xenopus
Genes referenced: kcne1 kcne3 kcnq1

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	Figure 1. Cartoon models depicting possible mechanisms for KCNE1 and KCNE3 modulation of KCNQ1 channels (A) In Q1/E1 channel complexes, slow gating arises from either the slow transition of S4 voltage-sensing domains (orange with positive charges) from resting to active positions, or from slow activation gate opening. (B) Q1/E3 complexes are open at hyperpolarizing potentials (denoted by negative charges along the cytoplasmic membrane) because S4 voltage sensors are uncoupled from the opening of the gate (left), or because the equilibrium of voltage sensors is shifted to favor the activated state (right). (C) TEVC recordings of Q1 channels alone and partnered with E1 and E3 peptides in ND96 solution (Materials and methods). Oocytes were held at −80 mV, and currents were elicited from 4-s command voltages from −100 mV to +40 mV in 20-mV increments. Scale bars represent 0.5 μA and 0.5 s. Dashed line indicates zero current.
	Figure 2. S4 cysteine substitutions in KCNQ1 show state-dependent MTSET modification. (A) TEVC recordings of representative Q1 channels with cysteine substitutions in S4 expressed in Xenopus oocytes before and after MTSET modification. Oocytes were held at −80 mV, and currents were elicited from 4-s command voltages from −100 mV to +40 mV in 20-mV increments. Scale bars represent 0.5 μA and 0.5 s. Dashed line indicates zero current. (B) Change in current monitored over time using 40-mV test pulses with continuous perfusion of MTSET. For negative controls, 800 μM MTSET was used for Q1 and Q1/E3; 1600 μM for Q1/E1 and the data were plotted on the same y-axis scale as the cysteine mutants and are separated by line hatches. Open circles represent the “open” protocol where channels were depolarized for 11% of total time; filled squares: “closed” protocol, 0.6% of total time. Currents from the open and closed protocols were measured ∼5 ms before the end of the shortest depolarizing pulse and were normalized to the maximal change in current for comparison. Curves were fit to single or double exponentials to calculate reaction rate constants (Table I). (C) Comparison of MTSET modification rates for Q1 S4 cysteine substitutions in the open (open circles) or closed (filled squares) protocols. The gray bar gives the fold-change in rate between the open and closed protocols. X-out open circles indicate no observed change of current using open protocol; X-out squares are an estimate of reaction rate in the closed protocol based on the extent of modification determined by switching to the open protocol. Data were averaged from three to six oocytes ± SEM.
	Figure 3. The S4 voltage sensor reaches equilibrium quickly in KCNQ1/KCNE1 complexes upon depolarization. (A) TEVC recordings from R228C/E1 complexes expressed in Xenopus oocytes before and after MTSET modification. Oocytes were held at −80 mV and currents were elicited from 4-s command voltages from −100 mV to +40 mV in 20-mV increments. Gray dotted lines denote the amount of current from a 40-mV depolarization at 0.5, 2, and 4 s. Scale bars represent 0.5 μA and 0.5 s. Dashed line indicates zero current. (B) Change in current for R228C/E1 monitored over time using 40-mV test pulses with continuous perfusion of 400 μM MTSET. In the “open” protocol (open circles) channels were depolarized 11% of the total time; “closed” protocol (filled squares) 0.6% of the total time. Shifting to the open protocol (arrow) after ∼1,000 s shows the completion of MTSET modification. Currents from the open and closed protocols were normalized to the maximal change in current for comparison. (C) Pulse duration has no effect on the rate of MTSET modification of R228C/E1. Representative plots from the MTSET reaction with R228C/E1 using 0.1, 0.5, 2, or 4 s 40-mV pulses, where the total depolarization time was kept constant (inset). The total MTSET exposure time is plotted versus normalized current at the end of the depolarization. Filled diamonds indicate the interpulse interval required to reset voltage sensors between pulses when no MTSET was added (900-ms interval for 100-ms pulse). (D) Comparison of R228C, R228C/E1, and I230C/E1 in pulse duration experiments. Black symbols represent modification by MTSET, red symbols modification by MTSES. Data were averaged from three to six oocytes ± SEM.
	Figure 4. The equilibrium of S4 voltage sensors is shifted to favor the active state in KCNQ1/KCNE3 complexes. (A) TEVC recordings from A226C/E3, R228C/E3, and I230C/E3 complexes expressed in Xenopus oocytes before and after MTSET modification. Oocytes were held at −80 mV and currents were elicited from 4-s command voltages from −100 to + 40 mV in 20-mV increments. Scale bars represent 0.5 μA and 0.5 s. Dashed line indicates zero current. (B) Change in current monitored over time using 40-mV test pulses with continuous perfusion of 400 μM MTSET. In the “open” protocol (open circles), the channel complexes were depolarized 11% of the total time; “closed protocol” (filled squares) 0.6% of the total time. Currents from the open and closed protocols were normalized to the maximal change in current for comparison. Curves were fit to monoexponential time courses and the reaction rates are tabulated in Table I. (C) Comparison of MTSET modification rates for Q1/E3 S4 cysteine substitutions in the open (open circles) or closed (filled squares) protocols. The gray bar gives the fold-change in rate between the open and closed protocols. X-out open circles indicate no observed change of current using open protocol. Data were averaged from three to five oocytes ± SEM. (D) The rate of MTSET reaction with I230C/E3 channel complexes is independent of voltage. Oocytes were held at −80 mV, and for each voltage, 4-s pulses were followed by a −30 mV tail pulse (100 ms), which was used to monitor the change in current upon MTSET application. The modification reaction time constant from single exponential fits is plotted for each voltage potential. Data were averaged from three to four oocytes ± SEM.

References [+] :

Aggarwal, Contribution of the S4 segment to gating charge in the Shaker K+ channel. 1996, Pubmed, Xenbase