A Kir6.2 pore mutation causes inactivation of ATP-sensitive potassium channels by disrupting PIP2-dependent gating.

Jeremy D Bushman,Qing Zhou,Show-Ling Shyng,Jorge Arreola

doi:10.1371/journal.pone.0063733

Abstract

In the absence of intracellular nucleotides, ATP-sensitive potassium (KATP) channels exhibit spontaneous activity via a phosphatidylinositol-4,5-bisphosphate (PIP2)-dependent gating process. Previous studies show that stability of this activity requires subunit-subunit interactions in the cytoplasmic domain of Kir6.2; selective mutagenesis and disease mutations at the subunit interface result in time-dependent channel inactivation. Here, we report that mutation of the central glycine in the pore-lining second transmembrane segment (TM2) to proline in Kir6.2 causes KATP channel inactivation. Unlike C-type inactivation, a consequence of selectivity filter closure, in many K+ channels, the rate of inactivation in G156P channels was insensitive to changes in extracellular ion concentrations or ion species fluxing through the pore. Instead, the rate of G156P inactivation decreased with exogenous application of PIP2 and increased when PIP2-channel interaction was inhibited with neomycin or poly-L-lysine. These findings indicate the G156P mutation reduces the ability of PIP2 to stabilize the open state of KATP channels, similar to mutations in the cytoplasmic domain that produce inactivation. Consistent with this notion, when PIP2-dependent open state stability was substantially increased by addition of a second gain-of-function mutation, G156P inactivation was abolished. Importantly, bath application and removal of Mg2+-free ATP or a nonhydrolyzable analog of ATP, which binds to the cytoplasmic domain of Kir6.2 and causes channel closure, recover G156P channel from inactivation, indicating crosstalk between cytoplasmic and transmembrane domains. The G156P mutation provides mechanistic insight into the structural and functional interactions between the pore and cytoplasmic domains of Kir6.2 during gating.

Highlights

Rectifying potassium (Kir) channels are expressed in a wide variety of cell types where they regulate membrane excitability in response to diverse signals [1]
We present evidence that this pore mutation decreases channel open state stability by disrupting a gate controlled by PIP2 which binds the cytoplasmic domains of the channel
Fixed cells were pre-blocked in phosphate-buffered saline (PBS) plus 0.1% bovine serum albumin (BSA) for 30 min, incubated with the M2 mouse monoclonal anti-FLAG antibody (10 mg/ml, Sigma) for 1 h to label f-sulfonylurea receptor 1 (SUR1), washed 3620 min in PBS plus 0.1% BSA, incubated with a horseradish peroxidaseconjugated anti-mouse secondary antibody (Jackson ImmunoResearch, Inc., 1:1000 dilution) for 30 min, and washed again 4630 min in PBS plus 0.1% BSA

Summary

Introduction

Rectifying potassium (Kir) channels are expressed in a wide variety of cell types where they regulate membrane excitability in response to diverse signals [1]. ATP-sensitive potassium (KATP) channels composed of Kir6.2 and sulfonylurea receptor 1 (SUR1) play a critical role in controlling insulin secretion and neuronal excitability [2,3,4]. The majority of evidence to date suggests a model in which a gate located near the helix bundle crossing where the four inner helices converge, commonly referred to as the ‘‘lower’’ gate, is sensitive to PIP2 and ATP regulation [9,10,11]. A gate located near the selectivity filter, referred to as the ‘‘upper’’ gate, controls the ligand-independent fast gating observed in single channel kinetics [12]

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Results

Conclusion