Qiong-Yao Tang

Gating of a G protein-sensitive Mammalian Kir3.1 Prokaryotic Kir Channel Chimera in Planar Lipid Bilayers

Kir3 channels control heart rate and neuronal excitability through GTP-binding (G) protein and phosphoinositide signaling pathways. These channels were the first characterized effectors of the βγ subunits of G proteins. Because we currently lack structures of complexes between G proteins and Kir3 channels, their interactions leading to modulation of channel function are not well understood. The recent crystal structure of a chimera between the cytosolic domain of a mammalian Kir3.1 and the transmembrane region of a prokaryotic KirBac1.3 (Kir3.1 chimera) has provided invaluable structural insight. However, it was not known whether this chimera could form functional K+ channels. Here, we achieved the functional reconstitution of purified Kir3.1 chimera in planar lipid bilayers. The chimera behaved like a bona fide Kir channel displaying an absolute requirement for PIP2 and Mg2+-dependent inward rectification. The channel could also be blocked by external tertiapin Q. The three-dimensional reconstruction of the chimera by single particle electron microscopy revealed a structure consistent with the crystal structure. Channel activity could be stimulated by ethanol and activated G proteins. Remarkably, the presence of both activated Gα and Gβγ subunits was required for gating of the channel. These results confirm the Kir3.1 chimera as a valid structural and functional model of Kir3 channels.

Phosphatidylinositol 4,5-Bisphosphate Activates Slo3 Currents and Its Hydrolysis Underlies the Epidermal Growth Factor-induced Current Inhibition

The Slo3 gene encodes a high conductance potassium channel, which is activated by both voltage and intracellular alkalinization. Slo3 is specifically expressed in mammalian sperm cells, where it gives rise to pH-dependent outwardly rectifying K+ currents. Sperm Slo3 is the main current responsible for the capacitation-induced hyperpolarization, which is required for the ensuing acrosome reaction, an exocytotic process essential for fertilization. Here we show that in intact spermatozoa and in a heterologous expression system, the activation of Slo3 currents is regulated by phosphatidylinositol 4,5-bisphosphate (PIP2). Depletion of endogenous PIP2 in inside-out macropatches from Xenopus oocytes inhibited heterologously expressed Slo3 currents. Whole-cell recordings of sperm Slo3 currents or of Slo3 channels co-expressed in Xenopus oocytes with epidermal growth factor receptor, demonstrated that stimulation by epidermal growth factor (EGF) could inhibit channel activity in a PIP2-dependent manner. High concentrations of PIP2 in the patch pipette not only resulted in a strong increase in sperm Slo3 current density but also prevented the EGF-induced inhibition of this current. Mutation of positively charged residues involved in channel-PIP2 interactions enhanced the EGF-induced inhibition of Slo3 currents. Overall, our results suggest that PIP2 is an important regulator for Slo3 activation and that receptor-mediated hydrolysis of PIP2 leads to inhibition of Slo3 currents both in native and heterologous expression systems.

Structural Determinants of Phosphatidylinositol 4,5-Bisphosphate (PIP2) Regulation of BK Channel Activity through the RCK1 Ca2+ Coordination Site

Background: PIP2 has been reported to enhance Ca2+-driven gating, but the molecular determinants of this interplay are not known. Results: PIP2 interacts with specific basic residues and enhances Ca2+ gating through the αA-KDRDD-αB structural elements. Conclusion: The RCK1 Ca2+-binding site is coupled to PIP2. Significance: PIP2 is a key element in the regulation of BK channel activity. Big or high conductance potassium (BK) channels are activated by voltage and intracellular calcium (Ca2+). Phosphatidylinositol 4,5-bisphosphate (PIP2), a ubiquitous modulator of ion channel activity, has been reported to enhance Ca2+-driven gating of BK channels, but a molecular understanding of this interplay or even of the PIP2 regulation of this channels activity remains elusive. Here, we identify structural determinants in the KDRDD loop (which follows the αA helix in the RCK1 domain) to be responsible for the coupling between Ca2+ and PIP2 in regulating BK channel activity. In the absence of Ca2+, RCK1 structural elements limit channel activation through a decrease in the channels PIP2 apparent affinity. This inhibitory influence of BK channel activation can be relieved by mutation of residues that (a) connect either the RCK1 Ca2+ coordination site (Asp367 or its flanking basic residues in the KDRDD loop) to the PIP2-interacting residues (Lys392 and Arg393) found in the αB helix or (b) are involved in hydrophobic interactions between the αA and αB helix of the RCK1 domain. In the presence of Ca2+, the RCK1-inhibitory influence of channel-PIP2 interactions and channel activity is relieved by Ca2+ engaging Asp367. Our results demonstrate that, along with Ca2+ and voltage, PIP2 is a third factor critical to the integral control of BK channel activity.

SLO-2 isoforms with unique Ca2+- and voltage-dependence characteristics confer sensitivity to hypoxia in C. elegans

Slo channels are large conductance K+ channels that display marked differences in their gating by intracellular ions. Among them, the Slo1 and C. elegans SLO-2 channels are gated by calcium (Ca2+), while mammalian Slo2 channels are activated by both sodium (Na+) and chloride (Cl−). Here, we report that SLO-2 channels, SLO-2a and a novel N-terminal variant isoform, SLO-2b, are activated by Ca2+ and voltage, but in contrast to previous reports they do not exhibit Cl− sensitivity. Most importantly, SLO-2 provides a unique case in the Slo family for sensing Ca2+ with the high-affinity Ca2+ regulatory site in the RCK1 but not the RCK2 domain, formed through interactions with residues E319 and E487 (that correspond to D362 and E535 of Slo1, respectively). The SLO-2 RCK2 domain lacks the Ca2+ bowl structure and shows minimal Ca2+ dependence. In addition, in contrast to SLO-1, SLO-2 loss-of-function mutants confer resistance to hypoxia in C. elegans. Thus, the C. elegans SLO-2 channels possess unique biophysical and functional properties.

A unique alkaline pH-regulated and fatty acid-activated tandem pore domain potassium channel (K2P) from a marine sponge

SUMMARY A cDNA encoding a potassium channel of the two-pore domain family (K2P, KCNK) of leak channels was cloned from the marine sponge Amphimedon queenslandica. Phylogenetic analysis indicated that AquK2P cannot be placed into any of the established functional groups of mammalian K2P channels. We used the Xenopus oocyte expression system, a two-electrode voltage clamp and inside-out patch clamp electrophysiology to determine the physiological properties of AquK2P. In whole cells, non-inactivating, voltage-independent, outwardly rectifying K+ currents were generated by external application of micromolar concentrations of arachidonic acid (AA; EC50 ∼30 μmol l–1), when applied in an alkaline solution (≥pH 8.0). Prior activation of channels facilitated the pH-regulated, AA-dependent activation of AquK2P but external pH changes alone did not activate the channels. Unlike certain mammalian fatty-acid-activated K2P channels, the sponge K2P channel was not activated by temperature and was insensitive to osmotically induced membrane distortion. In inside-out patch recordings, alkalinization of the internal pH (pKa 8.18) activated the AquK2P channels independently of AA and also facilitated activation by internally applied AA. The gating of the sponge K2P channel suggests that voltage-independent outward rectification and sensitivity to pH and AA are ancient and fundamental properties of animal K2P channels. In addition, the membrane potential of some poriferan cells may be dynamically regulated by pH and AA.

Regulatory Effect of General Anesthetics on Activity of Potassium Channels

General anesthesia is an unconscious state induced by anesthetics for surgery. The molecular targets and cellular mechanisms of general anesthetics in the mammalian nervous system have been investigated during past decades. In recent years, K+ channels have been identified as important targets of both volatile and intravenous anesthetics. This review covers achievements that have been made both on the regulatory effect of general anesthetics on the activity of K+ channels and their underlying mechanisms. Advances in research on the modulation of K+ channels by general anesthetics are summarized and categorized according to four large K+ channel families based on their amino-acid sequence homology. In addition, research achievements on the roles of K+ channels in general anesthesia in vivo, especially with regard to studies using mice with K+ channel knockout, are particularly emphasized.

C. Elegans Slo-2b uses its RCK1 Domain as a Ca2+ Sensor and does not Exhibit Cl- Dependence

Slo-2 channels play an important role in the adaption of neuronal firing rates and have been implicated in protection against ischemia. Slo-2 channels belong to the family of high-conductance potassium channels but their gating mechanism is unique and has been reported to exhibit species differences. The rat Slo2 (Slack) channel is activated by Na+ and Cl-, whereas the C.elegans Slo-2a has been reported to be sensitive to Ca2+ and Cl-. Here, we report isolation of a novel isoform of the C. elegans channel Slo-2b (F08b12.3c) that was cloned from ESTs (YK1522e1, YK1193) of C.elegans, which has a distinct N-terminal region (by 18 amino acids) compared to the previously reported Slo-2a (F08b12.3b) (Yuan et al, 2000). This new clone shows voltage- and Ca2+-activated macroscopic currents when expressed in Xenopus oocytes. We find that the C. elegans Slo-2b channel isoform exhibits a unitary conductance consistent with Slo-2a but it is not activated by Cl-. Furthermore, the current characteristics of Slo-2b can be described well by the Horrigan-Aldrich model, which had been developed to describe Slo1 current properties. Mutagenesis screening revealed that the Slo-2b channel with mutation of a critical Glu residue in the RCK1 domain largely controls Ca2+ sensitivity. In contrast, mutations of negatively charged residues around the region corresponding to the Na+ sensitive site of Slack channels in the RCK2 domain do not affect Ca2+ sensitivity of the Slo-2b channel. Thus, we conclude that the Ca2+ sensor of the Slo-2b in the RCK1 domain is largely sufficient to confer Ca2+-sensitivity to the Slo-2b channel isoform.

An Epilepsy/Dyskinesia-Associated Mutation in BK Channel Enhanced Channel-PIP2 Apparent Affinity

Large conductance, Ca2+- and voltage-gated BK (Slo1) channels are critical for neuronal functions. A previous study has shown that BK channels are regulated by membrane phosphatidylinositol 4,5-bisphosphate (PIP2). However, the mechanism of such regulation remains largely unknown. Here we show that an Asp-to-Gly mutation (D369G) associated with the human syndrome of generalized epilepsy and paroxysmal dyskinesia (GEPD) enhanced channel-PIP2 sensitivity in BK channels. With 106 mM Ca2+ in the bath, the inside-out patch G-V curve of D369G was leftward shifted by ∼ −28mV, suggesting enhanced channel activity for the mutated channel. Following depletion of endogenous PIP2 by poly-lysine, PIP2 activated D369G in a dose- and voltage-dependent manner. The EC50 for diC8-PIP2 activation of D369G decreased ∼4-fold compared to the WT channel, suggesting an enhanced channel-PIP2 interaction. Structural models of the BK channel place D369 near the membrane, where it could interact directly with PIP2. To study the mechanism of BK channel regulation by PIP2, we also used neomycin, a polycation that binds PIP2, as an indirect assay of channel-PIP2 affinity. We tested whether the D362G and D367G mutants, sites previously shown to be involved in BK Ca2+-sensitivity and located closely to D369, also affected channel-PIP2 affinity. Our results show that the IC50 values for neomycin effects on both D362G and D367G were largely decreased (∼100-fold), indicating that the mutations weakened channel-PIP2 interaction. In contrast, the D369G mutation increased the IC50 for neomycin in a voltage-dependent manner, suggesting an enhanced channel-PIP2 interaction. Taken together, these results suggested that mutation of the negatively charged residues D362 and D367, which lowers Ca2+ affinity, also decreases channel-PIP2 affinity, while the mutant D369G, which increases Ca2+ affinity also enhanced channel-PIP2 interaction. Thus Ca2+ and PIP2 affinities are interrelated down to the single site interaction level.