A Critical Gating Switch at a Modulatory Site in Neuronal Kir3 Channels

Abstract

Inwardly rectifying potassium channels enforce tight control of resting membrane potential in excitable cells. The Kir3.2 channel, a member of the Kir3 subfamily of G-protein-activated potassium channels (GIRKs), plays several roles in the nervous system, including key responsibility in the GABA B pathway of inhibition, in pain perception pathways via opioid receptors, and is also involved in alcoholism. PKC phosphorylation acts on the channel to reduce activity, yet the mechanism is incompletely understood. Using the heterologous Xenopus oocyte system combined with molecular dynamics simulations, we show that PKC modulation of channel activity is dependent on Ser-196 in Kir3.2 such that, when this site is phosphorylated, the channel is less sensitive to PKC inhibition. This reduced inhibition is dependent on an interaction between phospho-Ser (

Dynamic role of the tether helix in PIP2-dependent gating of a G protein–gated potassium channel

“…We next analyzed the rates of current depletion and recovery after Dr-Vsp activation by fitting the decay of current with a single exponential and determining the time constant (tau). These rates correlate with the differences in PIP 2 affinity, where higher-affinity channels typically have a slow depletion and fast recovery ( Bodhinathan and Slesinger, 2013 ; Adney et al, 2015 ; Rjasanow et al, 2015 ). The rate of recovery depends on both the relative affinity of the channel for PIP 2 and the rate of resynthesizing membrane-bound PIP 2 .…”

“…To probe for possible changes in the relative association of PIP 2 with the channel, we studied the effect of reversibly depleting plasma membrane PIP 2 on whole-cell Kir currents using the voltage-activated phosphatase Dr-VSP ( Bodhinathan and Slesinger, 2013 ; Adney et al, 2015 ; Rjasanow et al, 2015 ). Previous studies showed that activation of Dr-Vsp by depolarizing the membrane to +100 mV for varying lengths of time produces a time-dependent, graded depletion of PIP 2 and a corresponding reduction in Kir current ( Bodhinathan and Slesinger, 2013 ).…”

Hydrogen sulfide inhibits Kir2 and Kir3 channels by decreasing sensitivity to the phospholipid phosphatidylinositol 4,5-bisphosphate (PIP2)

“…2 Interactions --A common feature of Kir channels is that they all require the membrane phospholipid phosphatidylinositol (4,5)-bisphosphate (PIP 2 ) to maintain their activity (29). The strength of Kir-PIP 2 interactions determines the sensitivity of Kir channels to several regulatory factors such as pH, protein kinase C, and G proteins (30,31). Kir2.1 channels, with the highest apparent affinity to PIP 2 among the channels tested, was unaffected by NaHS treatment, suggesting that the extent of NaHS effect may depend on the strength of channel-PIP 2 interactions.…”

“…First, Kir3.2* was expressed and purified from Pichia pastoris as described previously (30). We also purchased from Sigma-Aldrich recombinant Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) purified from Escherichia coli.…”

Desensitization and Tolerance of Mu Opioid Receptors on Pontine Kölliker-Fuse Neurons

“…In the LC, the enhancement of opioid-mediated desensitization appears to be specifically due to PKCa (Bailey et al, 2009a,b). PKC can also reduce GIRK activity directly (Stevens et al, 1999;Mao et al, 2004;Adney et al, 2015). The KF highly expresses PKCg (Lein et al, 2007), which could mediate the heterologous reduction in GIRK current after PDBu, but does not enhance opioid-mediated desensitization.…”