Villalba-Galea CA et al. (2009), Coupling between the voltage-sensing and phosph...

XB-ART-40446

J Gen Physiol 2009 Jul 01;1341:5-14. doi: 10.1085/jgp.200910215.

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Coupling between the voltage-sensing and phosphatase domains of Ci-VSP.

Villalba-Galea CA , Miceli F , Taglialatela M , Bezanilla F .

Abstract
The Ciona intestinalis voltage sensor-containing phosphatase (Ci-VSP) shares high homology with the phosphatidylinositol phosphatase enzyme known as PTEN (phosphatase and tensin homologue deleted on chromosome 10). We have taken advantage of the similarity between these proteins to inquire about the coupling between the voltage sensing and the phosphatase domains in Ci-VSP. Recently, it was shown that four basic residues (R11, K13, R14, and R15) in PTEN are critical for its binding onto the membrane, required for its catalytic activity. Ci-VSP has three of the basic residues of PTEN. Here, we show that when R253 and R254 (which are the homologues of R14 and R15 in PTEN) are mutated to alanines in Ci-VSP, phosphatase activity is disrupted, as revealed by a lack of effect on the ionic currents of KCNQ2/3, where current decrease is a measure of phosphatase activity. The enzymatic activity was not rescued by the introduction of lysines, indicating that the binding is an arginine-specific interaction between the phosphatase binding domain and the membrane, presumably through the phosphate groups of the phospholipids. We also found that the kinetics and steady-state voltage dependence of the S4 segment movement are affected when the arginines are not present, indicating that the interaction of R253 and R254 with the membrane, required for the catalytic action of the phosphatase, restricts the movement of the voltage sensor.

PubMed ID: 19564425
PMC ID: PMC2712979
Article link: J Gen Physiol
Grant support: [+]

Species referenced: Xenopus laevis
Genes referenced: kcnq2 kcnq3 krt15 pten tbx2 tns1

Article Images: [+] show captions

	Figure 1. (A) Cartoon representation of the structure of Ci-VSP. The N terminus of Ci-VSP spans four transmembrane segments forming the VSD, and the fourth segment (S4 segment) of the VSD is connected to the PD by the PBM. (B) The proposed PBM of Ci-VSP shares 50% identity with the first 16 amino acids of the PTEN. (C) Homology model of the PD of Ci-VSP. This homology model was created by the online package 3D-jigsaw using residues 240–576 of Ci-VSP (http:// bmm.cancerresearchuk.org/∼3djigsaw/). The obtained model comprises residues 253–574. (D) Crystal structure of PTEN. The crystal structure of PTEN is constituted by residues 14–351 (Lee et al., 1999). Arginines 253 and 254 of Ci-VSP (C) and 14 and 15 of PTEN (D) are displayed in red. These residues are implicated in the formation of the putative PBM in these proteins. Cysteine 363 of Ci-VSP and 124 in PTEN are displayed in blue. These residues are essential for the activity of the catalytic site.
	Figure 2. Sensing currents from oocytes expressing different mutants of Ci-VSP. Sensing currents were recorded by applying voltage pulses of 400 ms, ranging from −100 to 140 mV. The HP was −60 mV. (A) Sensing currents for the phosphatase-inactivated mutant Ci-VSP C363S. When pulsing to positive potentials from the HP, all the mutants, R253A-R254A-C363S (B), R245Q-R246Q-C363S (C), and R245A-R246A (D), displayed sensing currents similar to those observed with the mutant C363S. In contrast, during the repolarization to the HP, all the PBM mutants displayed faster OFF sensing currents.
	Figure 3. Average time constant of the OFF sensing current of different mutants of Ci-VSP. (A) The OFF sensing current recorded from the PBM mutants (open symbols) R253A-R254A-C363S (n = 5), R253K-R254K-C363S (n = 4), R245Q-R246Q-C363S (n = 4), and R245A-R246A (n = 4) were significantly faster than those recorded from the C363S mutant (filled squares; n = 5) for all potentials tested above +20 mV (HP = −60 mV). (B) The mutant R245Q-R246Q C363S displayed slower OFF sensing current than those from the mutant R253A-R254A-C363S for pulses above +100 mV.
	Figure 4. Phosphatase activity of Ci-VSP monitored by the activity of the potassium-selective, voltage-, and PI(4,5)P2-dependent channels KCNQ2 and KCNQ3. (A) Currents of KCNQ2/3 expressed without Ci-VSP. (B) When KCNQ2/3 were expressed with the WT form of Ci-VSP, a strong decrease in the activity was observed at potentials above +20 mV. (C) Coexpression with the mutant R253A-R254A caused a slight decrease in the current of KCNQ2/3, only observable at very positive potentials. (D) Likewise, coexpression with the mutant R245A-R246A caused an initial decrease in the current of the channels at potentials above +80 mV. However, the effect on the ionic current was less profound than that observed with the mutant R253A-R254A. (E) Transient initial currents were not observed when mRNA coding for Ci-VSP was not injected in the oocytes along with the mRNA for KCNQ2/3. (F–H) In contrast, transient initial currents were observed in the presence of the phosphatase. These currents are the sensing currents of Ci-VSP. Pulses range from −100 to +100 mV. HP = −90 mV. For A–C and E–G, the interval between pulses was 10 mV. For D and H, it was 20 mV.
	Figure 5. Block of ionic current from oocytes expressing KCNQ2/KCNQ3 alone (A and C) and coexpressed with Ci-VSP R253A-R254A (B and D). (A) The K+ currents of KCNQ2/3 when depolarizing the oocytes. (B) Coexpression of the mutant R253A-R254A and KCNQ2/3 yielded K+ currents and, in addition, sensing currents from Ci-VSP. (C) Oocyte expressing KCQ2/3 alone shows no currents after replacing K+ for TEA+ in the internal and external solutions during cut-open recording. (D) In contrast, when KCNQ2/3 was coexpressed with the R253A-R254A mutant of Ci-VSP, TEA treatment showed the transient current characteristic of the sensing currents of Ci-VSP. Pulses range from −100 to +100 mV. HP = −90 mV.
	Figure 6. (A) The ionic current through KCNQ2/3 was not decreased after depolarization when coexpressed with the mutant R253K-R254K. (B) Ci-VSP is shown to be expressed because its sensing currents can be observed immediately after depolarization. (C) The phosphatase-inactivated mutant R253K-R254K-C363S was expressed alone to study sensing currents. The R253K-R254K-C363S mutant exhibits ON sensing currents that look very similar to those observed from the C363S mutant, although the OFF currents did not display the typical change in mode, observed with the C363S mutant (see Fig. 3). Pulses range from −100 to +100 mV. HP = −90 mV for ionic currents and −60 mV for sensing currents.
	Figure 7. The activity of the Ci-VSP expressed as the average of the amplitude of the KCNQ2/3 current after 1.5 s of depolarization normalized to the maximum current. When compared with the C363S mutant, the only values significantly different are those from the R253A-R254A-C363S mutant and the R245A-R246A mutant for potentials above +100 and +80 mV, respectively.
	Figure 8. The mutants G214C-C363S and G214C-R253A-R254A-C363S were expressed in oocytes and labeled with TMRM. Changes in the fluorescence emission of TMRM observed from the mutant G214C-R253A-R254A-C363S (B) were slower than those observed from the mutant G214C-C363S (A) when a conditioning pulse to +80 mV was applied for 5 s to produce the relaxation of the VSD. The differences in the time constant of the fluorescence were significant for all the potentials tested below −10 mV (C), reaching up to fourfold below −100 mV. The voltage dependence of the normalized amplitude of the fluorescence change showed no difference between G214C-C363S (D; filled squares) and G214C-R253A-R254A-C363S (D; open squares) when the HP = −60 mV. However, the amplitude of the fluorescence change from the G214C-R253A-R254A-C363S mutant showed a clear shift toward negative potentials (D; open circles) with respect to the G214C-C363S mutant (D; filled circles) when the membrane was conditioned to +80 mV for 5 s. Likewise, the voltage dependence of the charge movement displays a small shift toward positive potentials for the mutant G214C-R253A-R254A-C363S (E; open squares) with respect to the mutant G214C-C363S (E; filled squares). Prepulsing the membrane to +80 mV for 5 s produces a deeper relaxation of the VSD of the mutant G214C-R253A-R254A-C363S, as reflected by a larger shift toward negative potentials (E; open circles) with respect to the shift observed in the mutant G214C-C363S (E; filled circles).

References [+] :

Campbell, Allosteric activation of PTEN phosphatase by phosphatidylinositol 4,5-bisphosphate. 2003, Pubmed