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Negative and positive temperature dependence of potassium leak in MscS mutants: Implications for understanding
Piotr Koprowski1, Malgorzata A Sliwinska1, Andrzej Kubalski1
1Department of Cell Biology, The Nencki Institute of Experimental Biology, 02-093 Warsaw, Poland.
Abstract:
Bacterial mechanosensitive channel of small conductance (MscS) is a protein, whose activity is modulated by membrane tension, voltage and cytoplasmic crowding. MscS is a homoheptamer and each monomer consists of three transmembrane helices (TM1-3). Hydrophobic pore of the channel is made of TM3s surrounded by peripheral TM1/2s. MscS gating is a complex process, which involves opening and inactivation in response to the increase of membrane tension. A number of MscS mutants were isolated. Among them mutants affecting gating have been found including gain-of-function (GOF) and loss-of-function (LOF) that open at lower or at higher thresholds, respectively. Previously, using an in vivo screen we isolated multiple MscS mutants that leak potassium and some of them were GOF or LOF. Here we show that for a subset of these mutants K+ leak is negatively (NTD) or positively (PTD) temperature dependent. We show that temperature reliance of these mutants does not depend on how MS gating is affected by a particular mutation. Instead, we argue that NTD or PTD leak is due to the opposite allosteric coupling of the structures that determine the temperature dependence to the channel gate. In PTD mutants an increased hydration of the pore vestibule is directly coupled to the increase in the channel conductance. In NTD mutants, at higher temperatures an increased hydration of peripheral structures leads to complete separation of TM3 and a pore collapse.
Insights
Bacterial mechanosensitive channel of small conductance (MscS) mutants exhibit temperature-dependent potassium leak. This leak is linked to altered pore hydration, not gating mechanisms, revealing new insights into MscS channel regulation.
Area of Science:
- Biophysics
- Molecular Biology
- Membrane Protein Research
Background:
- Bacterial mechanosensitive channel of small conductance (MscS) activity is regulated by membrane tension, voltage, and cytoplasmic crowding.
- MscS is a homoheptameric protein with a pore formed by transmembrane helices (TM3s) and peripheral helices (TM1/2s).
- MscS gating involves complex opening and inactivation in response to membrane tension, with identified gain-of-function (GOF) and loss-of-function (LOF) mutants.
Purpose of the Study:
- To investigate the temperature dependence of potassium (K+) leak in previously isolated MscS mutants.
- To elucidate the relationship between temperature reliance, MscS gating, and channel structure.
- To understand the allosteric mechanisms underlying negative (NTD) and positive (PTD) temperature dependence in MscS mutants.
Main Methods:
- Utilized an in vivo screen to isolate MscS mutants with altered potassium leak.
- Characterized a subset of these mutants for their temperature-dependent K+ leak (NTD and PTD).
- Analyzed the structural basis for temperature reliance in relation to channel gating and hydration.
Main Results:
- A subset of MscS mutants displayed either negatively (NTD) or positively (PTD) temperature-dependent K+ leak.
- The observed temperature dependence was independent of whether the mutation caused GOF or LOF gating.
- PTD mutants showed increased pore vestibule hydration coupled to conductance, while NTD mutants exhibited peripheral hydration leading to pore collapse at higher temperatures.
Conclusions:
- Temperature reliance in MscS mutants is governed by allosteric coupling of temperature-sensing structures to the channel gate, not directly by gating alterations.
- Altered hydration patterns in specific regions of the MscS channel dictate the temperature-dependent nature of K+ leak.
- This study provides novel insights into the allosteric regulation and structural dynamics of MscS channels.
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