The resting and activated conformations of the voltage sensor of Ci-VSP from functional and solvent accessibility determinations

The voltage sensor domain (VSD) is responsible for electromechanical transduction in voltage-gated ion channels and enzymes. In all known VSDs, both architecture and voltage-sensing mechanism are conserved: the positive charged residues (R/K) on the fourth transmembrane segment S4 respond to the voltage change across the membrane, which trigger its own conformation change leading to the response of downstream domain. A wealth of biophysical information on voltage sensors in the last two decades has revealed one of the major functional states - “up” or activated state. However, the structure and functional properties of the “down” or resting state remains controversial. Here, we show electrophysiological and structural studies of the voltage sensor from Ciona intestinalis voltage sensitive phosphatase (Ci-VSP), that point to conformational transitions between the resting and activated conformations of the sensor. The voltage dependence of Ci-VSP mutants, analyzed by gating charge measurement in oocytes, show significant shift in their Q-V relationships along the voltage axis (R217E −60 mV, R217Q −20 mV, WT +60 mV, D136N +130 mV). At 0 mV, these mutants populate different functional states under biochemical conditions: WT and D136N mostly in the “down” state while R217E is mostly in the “up” state. A Ci-VSD biochemical preparation was developed for each of the four mutants and studied by site-directed spin labeling EPR (SDSL-EPR) methods in proteoliposomes. Mobility and accessibility information revealed the secondary structure of transmembrane segments and their positions relative to membrane and each other, suggesting the extend and direction of the motion of S4 between “up” and “down” states. These results are consistent with the down movement of S4 under hyperpolarization and render critical structural information, that allow us to propose a gating mechanism for Ci-VSD

MiRP3 acts as an accessory subunit with the BK potassium channel.

MinK-related peptides (MiRPs) are single-span membrane proteins that assemble with specific voltage-gated K+ (Kv) channel alpha-subunits to establish gating kinetics, unitary conductance, expression level, and pharmacology of the mixed complex. MiRP3 (encoded by the KCNE4 gene) has been shown to alter the behavior of some Kv alpha-subunits in vitro but its natural partners and physiologic functions are unknown. Seeking in vivo partners for MiRP3, immunohistochemistry was used to localize its expression to a unique subcellular site, the apical membrane of renal intercalated cells, where one potassium channel type has been recorded, the calcium- and voltage-gated channel BK. Overlapping staining of these two proteins was found in rabbit intercalated cells, and MiRP3 and BK subunits expressed in tissue culture cells were found to form detergent-stable complexes. Electrophysiologic and biochemical evaluation showed MiRP3 to act on BK to reduce current density in two fashions: shifting the current-voltage relationship to more depolarized voltages in a calcium-dependent fashion ( approximately 10 mV at normal intracellular calcium levels) and accelerating degradation of MiRP3-BK complexes. The findings suggest a role for MiRP3 modulation of BK-dependent urinary potassium excretion

MiRP3 acts as an accessory subunit with the BK potassium channel

MinK-related peptides (MiRPs) are single-span membrane proteins that assemble with specific voltage-gated K+ (Kv) channel α-subunits to establish gating kinetics, unitary conductance, expression level, and pharmacology of the mixed complex. MiRP3 (encoded by the KCNE4 gene) has been shown to alter the behavior of some Kv α-subunits in vitro but its natural partners and physiologic functions are unknown. Seeking in vivo partners for MiRP3, immunohistochemistry was used to localize its expression to a unique subcellular site, the apical membrane of renal intercalated cells, where one potassium channel type has been recorded, the calcium- and voltage-gated channel BK. Overlapping staining of these two proteins was found in rabbit intercalated cells, and MiRP3 and BK subunits expressed in tissue culture cells were found to form detergent-stable complexes. Electrophysiologic and biochemical evaluation showed MiRP3 to act on BK to reduce current density in two fashions: shifting the current-voltage relationship to more depolarized voltages in a calcium-dependent fashion (∼10 mV at normal intracellular calcium levels) and accelerating degradation of MiRP3-BK complexes. The findings suggest a role for MiRP3 modulation of BK-dependent urinary potassium excretion

Structural dynamics in the resting and activated states of the voltage sensor of Ci-VSP from dipolar distance measurements

The mechanism of electromechanical transduction in voltage sensing domains remains controversial. Here, we have probed the conformation of the voltage sensor of Ci-VSP in different functional states by means of EPR-based distance measurements. Ci-VSP is a voltage-sensing phosphatase from Ciona intestinalis. Although it is coupled to a cytoplasmic phosphatase, its voltage-sensing domain (VSD) is homologous to voltage sensors found in voltage-gated ion channels. It therefore serves as an excellent model to study voltage sensor movement independent of the interaction with pore domain. On the basis of voltage dependence of Ci-VSP sensing currents (Q-V curves), it is agreed that, at 0 mV, the S4 of wild-type Ci-VSP is in the resting conformation (down state). The arginine at position 217, located in the extracellular end of S4, has a strong effect on the voltage dependence of Ci-VSP sensing currents. Mutations at arginine 217 with a neutral or negative residue (R217Q or R217E), lead to a large leftward shifts in the Q-V curve so that, at 0 mV, the sensor is in the activated conformation (up state). This provides a unique opportunity to monitor the conformational differences in the VSD between resting and activated states in the absence of membrane potential. We expressed and purified a series of double cysteine mutants in the isolated voltage sensor (S1 to S4) of Ci-VSP in wild-type and R217E backgrounds, and measured distances using CW-based dipolar broadenings (for short distances, 8 to 20 Å) and double electron-electron resonance (DEER) spectroscopy (for longer distances, 20 to 50 Å). Our preliminary analysis of the distance measurements suggest defined conformational differences between resting and activated states of the VSD

The membrane protein MiRP3 regulates Kv4.2 channels in a KChIP-dependent manner.

MiRP3, the single-span membrane protein encoded by KCNE4, is localized by immunofluorescence microscopy to the transverse tubules of murine cardiac myocytes. MiRP3 is found to co-localize with Kv4.2 subunits that contribute to cardiac transient outward potassium currents (I(to)). Whole-cell, voltage-clamp recordings of human MiRP3 and Kv4.2 expressed in a clonal cell line (tsA201) reveal MiRP3 to modulate Kv4.2 current activation, inactivation and recovery from inactivation. MiRP3 shifts the half-maximal voltage for activation (V(1/2)) approximately 20 mV and slows time to peak approximately 100%. In addition, MiRP3 slows inactivation approximately 100%, speeds recovery from inactivation approximately 30%, and enhances restored currents so they 'overshoot' baseline levels. The cytoplasmic accessory subunit KChIP2 also assembles with Kv4.2 in tsA201 cells to increase peak current, shift V(1/2) approximately 5 mV, slow time to peak approximately 10%, slow inactivation approximately 100%, and speed recovery from inactivation approximately 250% without overshoot. Simultaneous expression of all three subunits yields a biophysical profile unlike either accessory subunit alone, abolishes MiRP3-induced overshoot, and allows biochemical isolation of the ternary complex. Thus, regional heterogeneity in cardiac expression of MiRP3, Kv4.2 and KChIP2 in health and disease may establish the local attributes and magnitude of cardiac I(to)