Jiusheng Yan

LRRC26 auxiliary protein allows BK channel activation at resting voltage without calcium

Large-conductance, voltage- and calcium-activated potassium (BK, or KCa1.1) channels are ubiquitously expressed in electrically excitable and non-excitable cells, either as α-subunit (BKα) tetramers or together with tissue specific auxiliary β-subunits (β1–β4). Activation of BK channels typically requires coincident membrane depolarization and elevation in free cytosolic Ca2+ concentration ([Ca2+]i), which are not physiological conditions for most non-excitable cells. Here we present evidence that in non-excitable LNCaP prostate cancer cells, BK channels can be activated at negative voltages without rises in [Ca2+]i through their complex with an auxiliary protein, leucine-rich repeat (LRR)-containing protein 26 (LRRC26). LRRC26 modulates the gating of a BK channel by enhancing the allosteric coupling between voltage-sensor activation and the channel’s closed–open transition. This finding reveals a novel auxiliary protein of a voltage-gated ion channel that gives an unprecedentedly large negative shift (∼−140 mV) in voltage dependence and provides a molecular basis for activation of BK channels at physiological voltages and calcium levels in non-excitable cells.

BK potassium channel modulation by leucine-rich repeat-containing proteins

Molecular diversity of ion channel structure and function underlies variability in electrical signaling in nerve, muscle, and nonexcitable cells. Regulation by variable auxiliary subunits is a major mechanism to generate tissue- or cell-specific diversity of ion channel function. Mammalian large-conductance, voltage- and calcium-activated potassium channels (BK, KCa1.1) are ubiquitously expressed with diverse functions in different tissues or cell types, consisting of the pore-forming, voltage- and Ca2+-sensing α-subunits (BKα), either alone or together with the tissue-specific auxiliary β-subunits (β1–β4). We recently identified a leucine-rich repeat (LRR)-containing membrane protein, LRRC26, as a BK channel auxiliary subunit, which causes an unprecedented large negative shift (∼140 mV) in voltage dependence of channel activation. Here we report a group of LRRC26 paralogous proteins, LRRC52, LRRC55, and LRRC38 that potentially function as LRRC26-type auxiliary subunits of BK channels. LRRC52, LRRC55, and LRRC38 produce a marked shift in the BK channel’s voltage dependence of activation in the hyperpolarizing direction by ∼100 mV, 50 mV, and 20 mV, respectively, in the absence of calcium. They along with LRRC26 show distinct expression in different human tissues: LRRC26 and LRRC38 mainly in secretory glands, LRRC52 in testis, and LRRC55 in brain. LRRC26 and its paralogs are structurally and functionally distinct from the β-subunits and we designate them as a γ family of the BK channel auxiliary proteins, which potentially regulate the channel’s gating properties over a spectrum of different tissues or cell types.

Profiling the Phospho-status of the BKCa Channel α Subunit in Rat Brain Reveals Unexpected Patterns and Complexity

Molecular diversity of ion channel structure and function underlies variability in electrical signaling in nerve, muscle, and non-excitable cells. Protein phosphorylation and alternative splicing of pre-mRNA are two important mechanisms to generate structural and functional diversity of ion channels. However, systematic mass spectrometric analyses of in vivo phosphorylation and splice variants of ion channels in native tissues are largely lacking. Mammalian large-conductance calcium-activated potassium (BKCa) channels are tetramers of α subunits (BKα) either alone or together with β subunits, exhibit exceptionally large single channel conductance, and are dually activated by membrane depolarization and intracellular Ca2+. The cytoplasmic C terminus of BKα is subjected to extensive pre-mRNA splicing and, as predicted by several algorithms, offers numerous phospho-acceptor amino acids. Here we use nanoflow liquid chromatography tandem mass spectrometry on BKCa channels affinity-purified from rat brain to analyze in vivo BKα phosphorylation and splicing. We found 7 splice variations and identified as many as 30 Ser/Thr in vivo phosphorylation sites; most of which were not predicted by commonly used algorithms. Of the identified phosphosites 23 are located in the C terminus, four were found on splice insertions. Electrophysiological analyses of phospho- and dephosphomimetic mutants transiently expressed in HEK-293 cells suggest that phosphorylation of BKα differentially modulates the voltage- and Ca2+-dependence of channel activation. These results demonstrate that the pore-forming subunit of BKCa channels is extensively phosphorylated in the mammalian brain providing a molecular basis for the regulation of firing pattern and excitability through dynamic modification of BKα structure and function.

BK channel opening involves side-chain reorientation of multiple deep-pore residues

Significance The BK calcium-activated potassium channels are important regulators of electrical and calcium signaling in many types of tissue. BK channel activation involves complex conformational changes. We investigated the molecular details of such conformational changes with scanning mutagenesis and electrophysiological measurements. We found multiple pore residues that reorient their side chains during channel opening. These findings bridge the gap between our knowledge about the static X-ray structure of ion channel pores and knowledge about their functional dynamics. Three deep-pore locations, L312, A313, and A316, were identified in a scanning mutagenesis study of the BK (Ca2+-activated, large-conductance K+) channel S6 pore, where single aspartate substitutions led to constitutively open mutant channels (L312D, A313D, and A316D). To understand the mechanisms of the constitutive openness of these mutant channels, we individually mutated these three sites into the other 18 amino acids. We found that charged or polar side-chain substitutions at each of the sites resulted in constitutively open mutant BK channels, with high open probability at negative voltages, as well as a loss of voltage and Ca2+ dependence. Given the fact that multiple pore residues in BK displayed side-chain hydrophilicity-dependent constitutive openness, we propose that BK channel opening involves structural rearrangement of the deep-pore region, where multiple residues undergo conformational changes that may increase the exposure of their side chains to the polar environment of the pore.

Molecular basis for differential modulation of BK channel voltage-dependent gating by auxiliary γ subunits

The marked difference in the efficacy of BKγ subunits in shifting the voltage dependence of BK activation depends mainly on the TM segment and a neighboring intracellular cluster of positively charged amino acids.

The single transmembrane segment determines the modulatory function of the BK channel auxiliary γ subunit

Researchers identify regions of tissue-specific γ subunits that are responsible for regulating the activity of calcium-activated potassium channels.

Closed state-coupled C-type inactivation in BK channels

Significance Ion channels regulate ion flow by opening and closing their pore gates. K+ channels commonly possess two pore gates, one at the intracellular end for fast channel activation/deactivation and the other at the selectivity filter for slow C-type inactivation/recovery. The large-conductance calcium-activated potassium (BK) channel lacks a classic intracellular bundle-crossing activation gate and normally show no C-type inactivation. We found conditions to induce BK channels into a C-inactivated state and observed a unique closed state-coupled C-type inactivation, which is contrary to the commonly observed open state-coupled C-type inactivation in other K+ channels. We propose that the activation gate of BK channels may exist at or near the selectivity filter and that the channel’s normal closing may represent an early conformational stage of C-type inactivation. Ion channels regulate ion flow by opening and closing their pore gates. K+ channels commonly possess two pore gates, one at the intracellular end for fast channel activation/deactivation and the other at the selectivity filter for slow C-type inactivation/recovery. The large-conductance calcium-activated potassium (BK) channel lacks a classic intracellular bundle-crossing activation gate and normally show no C-type inactivation. We hypothesized that the BK channel’s activation gate may spatially overlap or coexist with the C-type inactivation gate at or near the selectivity filter. We induced C-type inactivation in BK channels and studied the relationship between activation/deactivation and C-type inactivation/recovery. We observed prominent slow C-type inactivation/recovery in BK channels by an extreme low concentration of extracellular K+ together with a Y294E/K/Q/S or Y279F mutation whose equivalent in Shaker channels (T449E/K/D/Q/S or W434F) caused a greatly accelerated rate of C-type inactivation or constitutive C-inactivation. C-type inactivation in most K+ channels occurs upon sustained membrane depolarization or channel opening and then recovers during hyperpolarized membrane potentials or channel closure. However, we found that the BK channel C-type inactivation occurred during hyperpolarized membrane potentials or with decreased intracellular calcium ([Ca2+]i) and recovered with depolarized membrane potentials or elevated [Ca2+]i. Constitutively open mutation prevented BK channels from C-type inactivation. We concluded that BK channel C-type inactivation is closed state-dependent and that its extents and rates inversely correlate with channel-open probability. Because C-type inactivation can involve multiple conformational changes at the selectivity filter, we propose that the BK channel’s normal closing may represent an early conformational stage of C-type inactivation.

Molecular determinants of Ca2+ sensitivity at the intersubunit interface of the BK channel gating ring

The large-conductance calcium-activated K+ (BK) channel contains two intracellular tandem Ca2+-sensing RCK domains (RCK1 and RCK2), which tetramerize into a Ca2+ gating ring that regulates channel opening by conformational expansion in response to Ca2+ binding. Interestingly, the gating ring’s intersubunit assembly interface harbors the RCK2 Ca2+-binding site, known as the Ca2+ bowl. The gating ring’s assembly interface is made in part by intersubunit coordination of a Ca2+ ion between the Ca2+ bowl and an RCK1 Asn residue, N449, and by apparent intersubunit electrostatic interactions between E955 in RCK2 and R786 and R790 in the RCK2 of the adjacent subunit. To understand the role of the intersubunit assembly interface in Ca2+ gating, we performed mutational analyses of these putative interacting residues in human BK channels. We found that N449, despite its role in Ca2+ coordination, does not set the channel’s Ca2+ sensitivity, whereas E955 is a determinant of Ca2+ sensitivity, likely through intersubunit electrostatic interactions. Our findings provide evidence that the intersubunit assembly interface contains molecular determinants of Ca2+-sensitivity in BK channels.

Glutamate-activated BK channel complexes formed with NMDA receptors

Significance Large-conductance BK channels are dually activated by voltage and Ca2+ and play a powerful integrative role in regulating cellular excitability and Ca2+ signaling in neurons. However, BK channels have a requirement of high intracellular free Ca2+ concentrations for activation under physiological conditions, and the Ca2+ sources for their activation are not well understood. In this work, we establish that BK channels physically form protein complexes with Ca2+-permeable NMDA receptors via their obligatory BKα and GluN1 subunits. The activation mechanism and function of postsynaptic BK channels at synapses remain largely unknown. We found that postsynaptic BK channels in medial perforant path-dentate gyrus granule cell synapses are activated by NMDA receptor-mediated Ca2+ influx and modulate excitatory synaptic transmission. The large-conductance calcium- and voltage-activated K+ (BK) channel has a requirement of high intracellular free Ca2+ concentrations for its activation in neurons under physiological conditions. The Ca2+ sources for BK channel activation are not well understood. In this study, we showed by coimmunopurification and colocalization analyses that BK channels form complexes with NMDA receptors (NMDARs) in both rodent brains and a heterologous expression system. The BK–NMDAR complexes are broadly present in different brain regions. The complex formation occurs between the obligatory BKα and GluN1 subunits likely via a direct physical interaction of the former’s intracellular S0–S1 loop with the latter’s cytosolic regions. By patch-clamp recording on mouse brain slices, we observed BK channel activation by NMDAR-mediated Ca2+ influx in dentate gyrus granule cells. BK channels modulate excitatory synaptic transmission via functional coupling with NMDARs at postsynaptic sites of medial perforant path-dentate gyrus granule cell synapses. A synthesized peptide of the BKα S0–S1 loop region, when loaded intracellularly via recording pipette, abolished the NMDAR-mediated BK channel activation and effect on synaptic transmission. These findings reveal the broad expression of the BK–NMDAR complexes in brain, the potential mechanism underlying the complex formation, and the NMDAR-mediated activation and function of postsynaptic BK channels in neurons.

Relationship between auxiliary gamma subunits and mallotoxin on BK channel modulation

The large-conductance, calcium- and voltage-activated K+(BK) channel consists of the pore-forming α subunits (BKα) and auxiliary subunits. The auxiliary γ1-3 subunits potently modulate the BK channel by shifting its voltage-dependence of channel activation toward the hyperpolarizing direction by approximately 145 mV (γ1), 100 mV (γ2), and 50 mV (γ3). Mallotoxin is a potent small-molecule BK channel activator. We analyzed the relationship between mallotoxin and the γ subunits in their BK channel-activating effects in membrane patches excised from HEK-293 cells. We found that mallotoxin, when applied extracellularly, shifted the half-activation voltage (V1/2) of BKα channels by −72 mV. The channel-activating effect of mallotoxin was greatly attenuated in the presence of the γ1, γ2, or γ3 subunit, with resultant ΔV1/2 (+/− mallotoxin) values of −9, −28, or −15 mV, respectively. Most examined γ1 mutant subunits antagonized mallotoxin’s channel-activating effect in a manner that was largely dependent on its own modulatory function. However, mallotoxin caused an irreversible functional and structural disengagement of the γ1-F273S mutant from BK channels. We infer that the auxiliary γ subunit effectively interferes with mallotoxin on BK channel modulation via either a direct steric competition or an indirect allosteric influence on mallotoxin’s binding and action on BKα.