Journal of General Physiology最新文献

Cantu syndrome (CS) is a rare disease caused by gain-of-function (GOF) mutations of Kir6.1 or SUR2 subunits of ATP-sensitive potassium (KATP) channels. CS patients with SUR2 and Kir6.1 variants display a similar constellation of symptoms, including muscle weakness and fatigue. The effects of CS mutations on skeletal muscle KATP channels, and any consequent direct effects on contractility, are currently unclear. Here, we used two knock-in mouse models of CS, respectively, carrying GOF mutations Kir6.1[V65M] or SUR2[A478V], to assess KATP channel properties and contractility in isolated fast-twitch extensor digitorum longus (EDL) and slow-twitch soleus (SOL) muscles. Electrophysiological recordings in isolated myofibers showed normal resting potentials, and excised patch-clamp recordings showed normal KATP channel density in both genotypes, but enhanced Mg-nucleotide activation only in SUR2[A478V] fibers, consistent with muscle KATP channels being formed predominantly as complexes of SUR2A and Kir6.2 subunits. Ex vivo testing of isolated SUR2[A478V], but not Kir6.1[V65M], muscles showed an earlier onset of fatigue and a marked intra-tetanic decline of force compared with littermate controls. Importantly, normal contractile behavior was restored ex vivo and in vivo in SUR2[A478V] muscles in the presence of the FDA-approved KATP channel inhibitor glibenclamide, indicating that the increased fatigue of isolated muscles is a direct consequence of overactive sarcolemmal KATP channels. These results shed light on the pathophysiologic relevance of SUR2-dependent KATP channel subunits in skeletal muscle and highlight their role in fatiguing conditions, as well as identifying potential therapeutic benefit of skeletal muscle KATP inhibition in CS.

The sodium leak channel NALCN, a key regulator of neuronal excitability, associates with three ancillary subunits that are critical for its function: a subunit called FAM155, which interacts with the extracellular regions of the channel, and two cytoplasmic subunits called UNC79 and UNC80. Interestingly, NALCN and FAM155 have orthologous phylogenetic relationships with the fungal calcium channel Cch1 and its subunit Mid1; however, UNC79 and UNC80 have not been reported outside of animals. In this study, we leveraged expanded gene sequence data available for eukaryotes to reexamine the evolutionary origins of NALCN and Cch1 channel subunits. Our analysis corroborates the direct phylogenetic relationship between NALCN and Cch1 and identifies a larger clade of related channels in additional eukaryotic taxa. We also identify homologues of FAM155/Mid1 in Cryptista algae and UNC79 and UNC80 homologues in numerous non-metazoan eukaryotes, including basidiomycete and mucoromycete fungi and the microbial eukaryotic taxa Apusomonadida, Malawimonadida, and Discoba. Furthermore, we find that most major animal lineages, except ctenophores, possess a full complement of NALCN subunits. Comparing structural predictions with the solved structure of the human NALCN complex supports orthologous relationships between metazoan and non-metazoan FAM155/Mid1, UNC79, and UNC80 homologues. Together, our analyses reveal unexpected diversity and ancient eukaryotic origins of NALCN/Cch1 channelosome subunits and raise interesting questions about the functional nature of this channel complex within a broad, eukaryotic context.

Stretch activation (SA) is the delayed increase in force following a rapid stretch and improves muscle performance during repetitive cyclical contractions in insect flight and cardiac muscles. Although historically considered too low to be physiologically relevant in skeletal muscle, our recent work showed that higher phosphate concentrations ([Pi]) increased SA in mouse soleus fibers. These results suggest SA has a role combating fatigue, which increases [Pi], lowers pH, and reduces active calcium concentration ([Ca2+]). To test this, we measured SA during Active, High [Ca2+] Fatigue and Low [Ca2+] Fatigue conditions in myosin heavy chain (MHC) I, IIA, IIX, and IIB fibers from mouse soleus and extensor digitorum longus muscles. In the fast-contracting MHC II fibers, calcium-activated isometric tension (F0) decreased from Active to High [Ca2+] Fatigue to Low [Ca2+] Fatigue, as expected. Remarkably, SA tension (FSA) was not decreased but remained unchanged or increased under High and Low [Ca2+] Fatigue, except for a small decrease in MHC IIB fibers in Low [Ca2+] Fatigue compared with Active. This results in SA's percent contribution to total tension production (FSA/[F0 + FSA]) being much greater (58-114%) under fatiguing conditions in fast-contracting MHC II fibers. The SA tension peak for MHC I fibers was not visibly apparent under either fatigue condition, and the peak was about 20% of MHC II fibers' peaks under active conditions. Our results show SA improves force production under fatiguing conditions in MHC II fibers, which could play an important role in increasing endurance for muscles that are lengthened prior to shortening.

β-cardiac myosin mediates cardiac muscle contraction within the sarcomere by binding to the thin filament in an ATP-powered reaction. This process is highly regulated on a beat-to-beat basis by calcium interactions with the thin filament, but also contractile force is highly regulated by controlling the number of myosins available, resulting in a dynamic reserve. Our goal was to examine the size of this reserve and how it is modulated by cardiac myosin binding protein-C (cMyBP-C). We used single-molecule imaging to determine myosin activity with high spatial resolution by measuring fluorescently tagged ATP molecules binding to and releasing from myosins within the cardiac sarcomere. Three myosin ATPase states were detected: the fastest species was consistent with nonspecific ATP binding to myosin's surface, and the slower two species were consistent with the previously identified DRX and SRX states. The former represents myosins in a state ready to interact with the thin filament, and the latter in a cardiac reserve state with slowed ATPase. We found the cardiac reserve was 46% across the whole sarcomere in porcine myofibrils. Subdividing into the P-, C-, and D-zones revealed the D-zone has the smallest population of reserve heads (44%). Treatment with PKA that phosphorylates cMyBP-C led to a 16% reduction of reserve in the C-zone (where cMyBP-C is found) and a 10% reduction in the P-zone, with an unexpected 15% increase in the D-zone. Interestingly, the changes in SRX myosin head distribution by PKA phosphorylation of cMyBP-C across each subsarcomeric zone mirror the changes we identified in human cardiac myofibrils isolated from a hypertrophic cardiomyopathy patient mutation (MYBPC3-c.772G>A) that exhibits cMyBP-C haploinsufficiency. These results provide novel insights into how the C-zone functions in both porcine and human β-cardiac myosin-containing thick filaments, revealing a possible compensatory change in the D-zone upon altered cMyBP-C phosphorylation and/or haploinsufficiency.

Large conductance calcium-activated potassium channels (BK channels) are unique in their ability to respond to two distinct physiological stimuli: intracellular Ca2+ and membrane depolarization. In neurons, these channels are activated through a coordinated response to both signals; however, for BK channels to respond to physiological voltage changes, elevated concentrations of intracellular Ca2+ (ranging from 1 to 10 μM) are necessary. In many physiological contexts, BK channels are typically localized within nanodomains near Ca2+ sources (∼20-50 nm), such as N-methyl-D-aspartate receptors (NMDARs; encoded by the GRIN genes). Since the direct evidence of NMDAR-BK channel coupling reported by Isaacson and Murphy in 2001 in the olfactory bulb, further studies have identified functional coupling between NMDARs and BK channels in other regions of the brain, emphasizing their importance in neuronal function. Mutations in the genes encoding NMDAR subunits have been directly linked to developmental encephalopathies, including intellectual disability, epilepsy, and autism spectrum features. Specifically, mutations V15M and V618G in the GRIN2B gene, which encodes the GluN2B subunit of NMDARs, are implicated in the pathogenesis of GRIN2B-related neurodevelopmental disorders. Here, we explored the effects of these two GluN2B mutations on NMDAR-BK channel coupling, employing a combination of electrophysiological, biochemical, and imaging techniques. Taken together, our results demonstrate that mutation V618G specifically disrupts NMDAR-BK complex formation, impairing functional coupling, in spite of robust individual channel expression in the membrane. These results provide a potential mechanistic basis for GRIN2B-related pathophysiology and uncover new clues about NMDAR-BK complex formation.

Charybdotoxin (CTX), a peptide neurotoxin derived from the scorpion Leiurus quinquestriatus, binds to the external entrance of open voltage-gated K+ channels (VGKCs) with minimal conformational impact. By occluding the VGKC pore, CTX blocks passive K+ flow-a defining function of these membrane proteins. Due to its mechanistic simplicity and high signal-to-noise ratio, the CTX-VGKC interaction is an ideal system to investigate the molecular details of binding and unbinding. CTX bound to the Shaker VGKC exhibits thermal motion (wobbling) that permits access of external K+ to the channel pore. To test whether this wobbling is part of the reaction pathway during toxin-channel interaction, the energetic role of external K+ was examined in the association and dissociation kinetics. A high-affinity Shaker K427E-VGKC variant was expressed in Xenopus oocytes, and its activity was monitored via two-electrode voltage clamp between ∼10 and ∼30°C. Nanomolar applications of CTX to open and closed channels, in the presence of high external Na+ or high K+ concentrations, were used to measure blockade kinetics at different voltages and temperatures. In high K+, both the dissociation and association rates showed higher activation enthalpies, by ∼15 kJ/mol and ∼25 kJ/mol, respectively, compared with high Na+ conditions. However, the association rates under high Na+ and K+ were equal at ∼20°C, indicating a compensatory K+-induced activation entropy. We propose transient CTX-wobbling intermediates in both directions of the reaction pathway. Such a wobbling intermediate could enhance the diversity of productive collisions during association, increasing the efficacy of the scorpion venom.

The epithelial Na channel (ENaC) is a heterotrimer whose trafficking to the apical membrane is stimulated by aldosterone. Trafficking is associated with proteolytic cleavage of the α and γ subunits. We examined the kinetics of this process to ascertain whether the observed changes could contribute to the most rapid anti-natriuretic effects (within 1-3 h) of hormone administration in rats. Infusion of aldosterone increased the abundance of cleaved αENaC and γENaC with time constants of 2.2 and 2.3 h, respectively. Decreases in full-length γENaC and increases in full-length αENaC occurred more slowly, with time constants of 22 and 17 h. Decreases in aldosterone also caused rapid decreases in cleaved and slower changes in full-length forms. Kinetic modeling suggested that the major effect of aldosterone on γENaC kinetics was on the transition from a full-length, intracellular (I) to a cleaved, membrane-associated (M) population. This rate is relatively slow (0.002-0.01 h-1) compared with rates of degradation of M (∼0.4 h-1) and I (∼0.04 h-1). Short lifetimes (∼1 h) of channels at the surface were confirmed in a mouse collecting duct cell line (mCCD). Lifetimes of full-length forms of α and γENaC were also short in whole-cell extracts of mCCD cells but were much longer in the cytoplasm of mouse tubule suspensions (10-20 h). We conclude that one effect of aldosterone in the kidney is to increase forward trafficking of ENaC to the apical membrane, where rapid degradation from the surface permits fast regulation of apical channel abundance.

Voltage-sensing domains (VSDs) are highly conserved protein modules that regulate the activation of voltage-gated ion channels. In response to membrane depolarization, positive gating charges in the S4 helix of VSDs move across the membrane electric field, which is focused at the hydrophobic constriction site (HCS) in the center of the VSD. This conformational change is translated into opening of the channel gate. Transient interactions of the gating charges with negatively charged countercharges in the adjacent helices are critical for catalyzing this state transition and for determining its voltage dependence and kinetics. However, the mechanism by which the sequential interactions between the multiple gating- and countercharges regulate these properties remains poorly understood. Here, we analyze the state transitions of the first VSD of CaV1.1 using MD simulation of the channel exposed to an electric field and site-directed mutagenesis of gating and countercharges to investigate the role of their interactions in determining the gating properties of CaV1.1. Alanine substitutions of gating charges differentially altered the kinetics or voltage dependence of activation, depending on whether they pass the HCS (R2 and R3) or not (K0, R1, and R4). Alanine substitutions of countercharges differentially altered kinetics and voltage dependence, depending on whether they facilitate the transfer of gating charges across the HCS (E100 and D126), and whether they stabilize the activated (E87, E90, and E140) or the resting state (E100, D126). Thus, our results reveal basic mechanistic principles by which variable interactions between gating charges and countercharges regulate the gating properties of voltage-gated calcium channels.

The function of the heart depends critically on the precise timing and coordination of electrical signals generated by ion channels in cardiac cells. The voltage-gated sodium current (INa) plays a pivotal role in initiating the rapid depolarization that drives each heartbeat. Two important descriptive properties of cardiac INa are its activation and inactivation midpoints, which describe the membrane voltages at which there is a 50% probability of the channel being open or unavailable, respectively. These midpoints determine the voltage range over which sodium channels contribute to the action potential and influence how easily the heart can initiate and propagate electrical signals. Because even small shifts in these kinetic parameters can affect excitability, conduction, and arrhythmia risk, they are commonly used to characterize the effects of drugs, mutations, and disease states.