Hydrophobic residues in S1 modulate enzymatic function and voltage sensing in voltage-sensing phosphatase.

The voltage-sensing domain (VSD) is a four-helix modular protein domain that converts electrical signals into conformational changes, leading to open pores and active enzymes. In most voltage-sensing proteins, the VSDs do not interact with one another, and the S1-S3 helices are considered mainly scaffolding, except in the voltage-sensing phosphatase (VSP) and the proton channel (Hv). To investigate its contribution to VSP function, we mutated four hydrophobic amino acids in S1 to alanine (F127, I131, I134, and L137), individually or in combination. Most of these mutations shifted the voltage dependence of activity to higher voltages; however, not all substrate reactions were the same. The kinetics of enzymatic activity were also altered, with some mutations significantly slowing down dephosphorylation. The voltage dependence of VSD motions was consistently shifted to lower voltages and indicated a second voltage-dependent motion. Additionally, none of the mutations broke the VSP dimer, indicating that the S1 impact could stem from intra- and/or intersubunit interactions. Lastly, when the same mutations were introduced into a genetically encoded voltage indicator, they dramatically altered the optical readings, making some of the kinetics faster and shifting the voltage dependence. These results indicate that the S1 helix in VSP plays a critical role in tuning the enzyme's conformational response to membrane potential transients and influencing the function of the VSD.

S1 hydrophobic residues modulate voltage sensing phosphatase enzymatic function and voltage sensing.

The voltage sensing domain (VSD) is a four-helix modular protein domain that converts electrical signals into conformational changes, leading to open pores and active enzymes. In most voltage sensing proteins, the VSDs do not interact with one another and the S1-S3 helices are considered mainly as scaffolding. The two exceptions are the voltage sensing phosphatase (VSP) and the proton channel (Hv). VSP is a voltage-regulated enzyme and Hvs are channels that only have VSDs. To investigate the S1 contribution to VSP function, we individually mutated four hydrophobic amino acids in S1 to alanine (F127, I131, I134 and L137). We also combined these mutations to generate quadruple mutation designated S1-Q. Most of these mutations shifted the voltage dependence of activity to higher voltages though interestingly, not all substrate reactions were the same. The kinetics of enzymatic activity were also altered with some mutations significantly slowing down dephosphorylation. The voltage dependence of VSD motions were consistently shifted to lower voltages and indicated a second voltage dependent motion. Co-immunoprecipitation demonstrated that none of the mutations broke the VSP dimer indicating that the S1 impact could stem from intrasubunit and/or intersubunit interactions. Lastly, when the same alanine mutations were introduced into a genetically encoded voltage indicator, they dramatically altered the optical readings, making some of the kinetics faster and shifting the voltage dependence. These results indicate that the S1 helix in VSP plays a critical role in tuning the enzymes conformational response to membrane potential transients and influencing the function of the VSD.

Diagnostic and predictive values of thirst, angiotensin II, and vasopressin during trauma resuscitation.

BACKGROUND: Thirst perception involves neurochemical signals attributed to acute elevation of arginine vasopressin (AVP) and angiotensin II (AT2) levels, and may accompany acute hemorrhage. OBJECTIVE: To determine whether thirst or plasma AVP or AT2 levels predict hemorrhagic shock, injury severity, or outcome in trauma patients at initial presentation. METHODS: This was a prospective case series of adult subjects presenting as trauma activations to an urban level I trauma center. Subjects were included if they were alert and nonintoxicated. During resuscitation, subjects were queried for thirst perception using binary and continuous data formats employing a 100-mm nonhatched visual analog scale. Blood for AT2 and AVP assessment was obtained during initial laboratory collection. Other data were abstracted retrospectively from our trauma registry. Crude and stratified analyses (blunt and penetrating trauma) assessed the correlation of thirst, AVP, and AT2 to the initial shock index, base deficit, blood transfusion requirement, admission, and Injury Severity Score (ISS). Our institutional review board (IRB) granted a waiver of informed consent. RESULTS: Of 105 subjects, the average age was 35 years (95% confidence interval [CI] 32 to 38), with 31% penetrating trauma. For AVP, there was no difference in thirst perception between subjects with normal (59 mm, 95% CI 47 to 71) versus elevated (63 mm, 95% CI 56 to 70) plasma levels. For AT2, results were likewise insignificant for normal (63 mm, 95% CI 56 to 70) versus elevated (58 mm, 95% CI 46 to 70) plasma levels. Thirst, AT2 level, and AVP level demonstrated no correlation to shock index, base deficit, transfusion requirement, hospital admission, or ISS. CONCLUSION: The results of this study imply that thirst severity and AVP and AT2 plasma levels are not reliable predictors of impending hemorrhagic shock, injury severity, or outcome. The presence or absence of severe thirst should not be employed as a primary marker for dismissing or suspecting incipient shock.