Biology subjects

Lingle, C. J.

Publications and source records attributed to Lingle, C. J..

7 recordsLinked to original sources

bioRxiv2025

β2 and β3a regulatory subunits can coassemble in the same BK channels

Ca2+- and voltage-activated BK-type K+ channels are influenced profoundly by associated regulatory subunits, including {beta} subunits (Kcnmb1-4; {beta}1-{beta}4). Although overlap in expression of different BK {beta} subunits occurs in native tissues, whether they can coassemble in the same channel complex is not known. We coexpress {beta}2- and {beta}3a subunits together with BK and, through a combination of macroscopic and single channel recordings, along with quantitative pull-down of tagged subunits, test whether coassembly can occur. We evaluate two models: 1) random mixing in which {beta}2 and {beta}3a subunits co-assemble in the same channels, and 2) segregated in which {beta}2 and {beta}3a are found in separate complexes. Our results support the view that, for {beta}2 and {beta}3a, BK currents arise from the random, independent assembly of both subunits in the same channels. Single channel recordings directly confirm coassembly of {beta}2 and {beta}3a subunits in the same channels. Quantitative biochemical analysis of coexpression of tagged BK subunits also reveals that ternary complexes form.

biophysics↗

bioRxiv2025

Disruption of a side portal pathway permits closed-state inactivation by BK β subunit N-termini

Cytosolic N-termini of several BK channel {beta} regulatory subunits mediate rapid inactivation. However, in contrast to Kv channels, inactivation does not occur via a simple, open channel block mechanism, but involves two steps, an association step in which ion permeation is maintained (O*), then followed by inactivation (I). To produce inactivation, BK {beta} subunit N-termini enter the central cavity through a lateral entry pathway ("side portal") separating the transmembrane pore-gate-domain and cytosolic gating ring. Comparison of BK conformations reveals an aqueous pathway into the central cavity in the open structure, while in the closed structure three sequential basic residues (R329K330K331) in S6 occlude central cavity access. We probed the impact of mutations of the RKK motif (RKK3Q, RKK3E, and RKK3V) on inactivation mediated by the {beta}3a N-terminus. All three RKK-mutated constructs differentially reduce depolarization-activated outward current, prolong {beta}3a-mediated tail current upon repolarization, and produce a persistent inward current at potentials down to -240 mV. With depolarization channels are driven into O*-I inactivated states and, upon repolarization, slow tails and persistent inward currents reflect slow changes in O*-I occupancy. However, evaluation of closed state occupancy prior to depolarization and at the end of slow tails reveals that some fraction of closed states at negative potentials corresponds to resting closed states in voltage-independent equilibrium with N-terminal-occluded closed-states. Thus, disruption of the RKK triplet both stabilizes the {beta}3a-N-terminus in its position of inactivation and permits access of that N-terminus to its blocking position in closed states. SummaryThe role of BK S6 residues R329K330K331 and E321/E324 in {beta} subunit-mediated inactivation is probed. WT R329K330K331 hinders inactivation in closed states, while RKK mutations stabilize inactivated states even under conditions where channels are otherwise closed. E321/E324 mutations do not permit closed-state inactivation.

biophysics↗

bioRxiv2023

High-resolution structures illuminate key principles underlying voltage and LRRC26 regulation of Slo1 channels

Multi-modal regulation of Slo1 channels by membrane voltage, intracellular calcium, and auxiliary subunits enables its pleiotropic physiological functions. Our understanding of how voltage impacts Slo1 conformational dynamics and the mechanisms by which auxiliary subunits, particularly of the LRRC (Leucine Rich Repeat containing) family of proteins, modulate its voltage gating remain unresolved. Here, we used single particle cryo-electron microscopy to determine structures of human Slo1 mutants which functionally stabilize the closed pore (F315A) or the activated voltage-sensor (R207A). Our structures, obtained under calcium-free conditions, reveal that a key step in voltage-sensing by Slo1 involves a rotameric flip of the voltage-sensing charges (R210 and R213) moving them by [~]6 [A] across a hydrophobic gasket. Next we obtained reconstructions of a complex of human Slo1 with the human LRRC26 ({gamma}1) subunit in absence of calcium. Together with extensive biochemical tests, we show that the extracellular domains of {gamma}1 form a ring of interlocked dominos that stabilizes the quaternary assembly of the complex and biases Slo1:{gamma}1 assembly towards high stoichiometric complexes. The transmembrane helix of {gamma}1 is kinked and tightly packed against the Slo1 voltage-sensor. We hypothesize that {gamma}1 subunits exert relatively small effects on early steps in voltage-gating but structurally stabilize non-S4 helices of Slo1 voltage-sensor which energetically facilitate conformational rearrangements that occur late in voltage stimulated transitions.

biophysics↗

bioRxiv2020

Fast inactivation of Na+ current in rat adrenal chromaffin cells involves two independent inactivation pathways

Voltage-dependent sodium (Nav) current in adrenal chromaffin cells (CCs) is rapidly inactivating and TTX-sensitive. The fractional availability of CC Nav current has been implicated in regulation of action potential (AP) frequency and the occurrence of slow-wave burst firing. To ascertain whether features of CC Nav inactivation might influence AP firing, we recorded Nav current in rat CCs, primarily from adrenal medullary slices. A key feature of CC Nav current is that recovery from inactivation, even following brief (5 ms) inactivation steps, exhibits two exponential components of generally similar amplitude. Variations of standard paired pulse protocols support the view that entry into the fast and slower recovery processes result from largely independent, competing inactivation pathways, both of which occur with similar onset times at depolarizing potentials. Over voltages from -120 to -80 mV, faster recovery varies from ~3 to 30 ms, while slower recovery from about 50-400 ms. At strong activation voltages (+0 mV and more positive), the relative entry into slow or fast recovery pathways is similar and independent of voltage. Trains of brief inactivating steps result in cumulative increases in the slower recovery fraction. This supports idea that brief recovery intervals preferentially allow recovery of channels from fast recovery pathways, thereby increasing the fraction of channels in the slow recovery pathway with each subsequent inactivation step. This provides a mechanism whereby differential rates of recovery produce use-dependent accumulation in slower recovery pathways. Consistent with use-dependent accumulation of channels in slow recovery pathways, repetitive AP clamp waveforms at 1-10 Hz frequencies reduce Nav availability to 10-20% of initial amplitude dependent on holding potential. The results indicate that there are two distinct pathways of fast inactivation, one that leads to normal fast recovery and the other with a slower time course, which together are well-suited to mediate use-dependent changes in Nav availability.

biophysics↗

bioRxiv2020

Nav1.3 and fibroblast growth factor homologous factor 14 are primary determinants of the TTX-sensitive sodium current in mouse adrenal chromaffin cells

Adrenal chromaffin cells (CCs) in rodents express a rapidly inactivating, TTX-sensitive sodium current. The current has generally been attributed to Nav1.7, although a possible role for Nav1.3 has also been suggested. Nav channels in rat CCs rapidly inactivate into two separable pathways, which differ in their time course of recovery from inactivation. One population recovers with time constants similar to traditional fast inactivation and the other about 10-fold slower. Inactivation properties suggest that the two pathways result from a single homogenous population of channels. Here we probe the properties and molecular components of the Nav current present in mouse CCs. We first confirm that functional properties of Nav current in rat and mouse cells are generally similar in terms of activation range, steady-state inactivation, and dual pathway fast inactivation. The results then show that all inward Nav current is absent in CCs from Nav1.3 KO mice. Subsequently, in a mouse with KO of fibroblast growth factor homology factor 14 (FGF14), we find that the slow component of recovery from fast inactivation is completely absent in most CCs, with no change in the time constant of fast recovery. Experiments probing the use-dependence of Nav current diminution between WT and FGF14 KO mice directly demonstrate a role of slow recovery from inactivation in determination of Nav current availability. Overall, the results indicate that the FGF14-mediated inactivation is the major determinant in defining use-dependent changes in Nav availability in CCs. We also consider the potential impact that inactivating FGFs with different recovery kinetics can exert on differential use-dependent changes in Nav availability.

biophysics↗

bioRxiv2020

Goblet cell LRRC26 regulates BK channel activation and protects against colitis in mice

Goblet cells (GCs) are specialized cells of the intestinal epithelium contributing critically to mucosal homeostasis. One of the functions of GCs is to produce and secrete MUC2, the mucin that forms the scaffold of the intestinal mucus layer coating the epithelium and separates the luminal pathogens and commensal microbiota from the host tissues. Although a variety of ion channels and transporters are thought to impact on MUC2 secretion, the specific cellular mechanisms that regulate GC function remain incompletely understood. Previously, we demonstrated that leucine-rich-repeat-containing protein 26 (LRRC26), a known regulatory subunit of the Ca2+-and voltage-activated K+ channel (BK channel), localizes specifically to secretory cells within the intestinal tract. Here, utilizing a mouse model in which MUC2 is fluorescently tagged allowing visualization of single GCs in intact colonic crypts, we show that murine colonic GCs have functional LRRC26-associated BK channels. In the absence of LRRC26, BK channels are present in GCs, but are not activated at physiological conditions. In contrast, all tested MUC2-negative cells completely lacked BK channels. Moreover, LRRC26-associated BK channels underlie the BK channel contribution to the resting transepithelial current across mouse distal colonic mucosa. Genetic ablation of either LRRC26 or BK-pore forming -subunit in mice results in a dramatically enhanced susceptibility to colitis induced by dextran sodium sulfate (DSS). These results demonstrate that normal potassium flux through LRRC26-associated BK channels in GCs has protective effects against colitis in mice. SignificanceA primary function of goblet cells (GCs) of the intestinal epithelium is to generate a protective mucus layer lining the intestinal lumen. GC dysfunction is linked to Inflammatory Bowel Disease (IBD). GC mucus secretion is thought to be dependent on contributions of an ensemble of anion and cation fluxes, although understanding remains limited. Here, it is shown in mouse colon that the Ca2+- and voltage-dependent BK-type K+ channel, specifically in association with the LRRC26 regulatory subunit, plays a critical role in normal GC function, protecting mice against chemically-induced colitis. The results demonstrate that normal K+ fluxes mediated by LRRC26-containing BK channels are required for normal GC function, potentially providing insights into the potential role of BK channels in IBD.

physiology↗

bioRxiv2018

BK channel inhibition by strong extracellular acidification

BK-type voltage- and Ca2+-dependent K+ channels are found in a wide range of cells and intracellular organelles. Among different loci, the composition of the extracellular microenvironment, including pH, may differ substantially. For example, it has been reported that BK channels are expressed in lysosomes with their extracellular side facing the strongly acidified lysosomal lumen (pH ~ 4.5). Here we show that BK activation is strongly and reversibly inhibited by extracellular H+, with its conductance-voltage relationship shifted by more than +100 mV at pHO 4. Our results reveal that this inhibition is mainly caused by H+ inhibition of BK voltage-sensor (VSD) activation through three acidic residues on the extracellular side of BK VSD. Given that these key residues (D133, D147, D153) are highly conserved among members in the voltage-dependent cation channel superfamily, the mechanism underlying BK inhibition by extracellular acidification might also be applicable to other members in the family.

biophysics↗