Journal of General Physiology最新文献

The recently characterized honeybee CaV4 channel is a high-voltage-activated Ca2+ channel ortholog to the DSC1 channel identified in Drosophila. While sequence similarities to NaV channels are obvious, permeation properties and current kinetics are more closely aligned with those of CaV channels. CaV4 exhibits a distinctive cation-dependent inactivation pattern, a hallmark of Ca2+ channel behavior, and nonetheless displays sensitivity to a Na+ channel-specific regulator, veratrine. Calcium channel facilitation is a phenomenon whereby the probability of calcium channel opening increases with successive depolarization pulses, resulting in an enhanced Ca2+ influx during repetitive or sustained electrical activity. In this study, we have identified an additional specific property of CaV4 in the form of an atypical voltage-dependent facilitation of the Ca2+ or Ba2+ currents by strong pre-depolarizations or prepulses (pPs). This physiologically relevant phenomenon, known as pP-induced facilitation (PiF), is subject to positive regulation by the amplitude of the pP but to negative regulation by its duration. It produces a hyperpolarizing shift of the I-V curve without any change in the reversal potential and macroscopic or single channel conductance. PiF is thus more pronounced for small depolarizations and almost absent when channels reach their maximal open probability. A mutation that affects the inactivation of the CaV4 channel prevents the occurrence of PiF. This previously undocumented form of facilitation appears exclusive to CaV4 channels. A strong pP may lock CaV4 channels in a pre-open state, rendering them more susceptible to activation and thereby shifting the activation curve toward more negative potentials. This, in turn, would accelerate channel opening and increase current amplitude. Lastly, we show that the inactivation particle of CaV4 (MFLT sequence, equivalent to the IFMT motif in human NaV, or MFMT in Apis NaV channel), in addition to its role in the initiation of the voltage-dependent inactivation, also modulates PiF.

The regulation of the hyperpolarization-activated cyclic nucleotide-gated 4 (HCN4) channels in pacemaker myocytes is essential for maintaining physiological cardiac rhythm. HCN4 dysfunctional behavior is among the major factors contributing to sinus node disease, a primary cause of pacemaker implantation. Previous work has shown that AMP-activated protein kinase (AMPK) activation leads to sinus bradycardia, a process attributable to cardiac remodeling that involves a decrease in HCN4 membrane expression, but the mechanism underlying this event remains unclear. We show here that AMPK can act as a posttranslational effector by phosphorylating Ser1157 at the C terminus of HCN4, a modification associated with a decrease in HCN4 membrane expression contributing to altered electrophysiological properties of cardiac pacemaker cells. Furthermore, we provide evidence that AMPK is constitutively activated in aged, but not young, mice, correlating with an increased development of intrinsic bradycardia. These findings support the view that AMPK is a key player in cardiac rhythm regulation and provide new insights into the molecular mechanisms underlying age-related changes in cardiac rhythm regulation.

JGP study (Dapino and Curti. https://doi.org/10.1085/jgp.202513883) reveals that the D-type K+ current regulates coincidence detection by shaping electrical transmission between pairs of MesV neurons.

Voltage-gated sodium (NaV) channels are vital regulators of electrical activity in excitable cells. Given their importance in physiology, NaV channels are key therapeutic targets for treating numerous conditions, yet developing subtype-selective drugs remains challenging due to the high sequence and structural conservation among NaV subtypes. Recent advances in cryo-electron microscopy have resolved most human NaV channels, providing valuable insights into their structure and function. However, limitations persist in fully capturing the complex conformational states that underlie NaV channel gating and modulation. This study explores the capability of AlphaFold2 to sample multiple NaV channel conformations and assess AlphaFold Multimer's accuracy in modeling interactions between the NaV α-subunit and its protein partners, including auxiliary β-subunits and calmodulin. We enhance conformational sampling to explore NaV channel conformations using a subsampled multiple sequence alignment approach and varying the number of recycles. Our results demonstrate that AlphaFold2 models multiple NaV channel conformations, including those observed in experimental structures, states that have not been described experimentally, and potential intermediate states. Correlation and clustering analyses uncover coordinated domain behavior and recurrent state ensembles. Furthermore, AlphaFold Multimer models NaV complexes with auxiliary β-subunits and calmodulin with high accuracy, and the presence of protein partners significantly alters both the modeled conformational landscape of the NaV α-subunit and the coupling between its functional states. These findings highlight the potential of deep learning-based methods to expand our understanding of NaV channel structure, gating, and modulation, while also underscoring the limitations of predicted models that remain hypotheses until validated by experimental data.

The presence of significant amounts of the transition metal, manganese, is essential for living cells where it is bound to some intracellular enzymes. The free (i.e., unbound) Mn2+ concentration in both extracellular and intracellular space is tightly regulated and thought to be considerably lower than the free calcium ion (Ca2+) concentration. Mn2+ can pass through plasmalemma Ca2+ ion channels, but under normal circumstances due to channel selectivity and relative concentrations, this event is rare. But when extracellular Mn2+ is increased to mM levels, significant Mn2+ influx occurs through Ca2+ channels in the plasma membrane and intracellular manganese levels increase above normal physiological levels. Mn2+ ions also have the property of binding to and quenching the fluorescence of fluorophores. This property can be used to detect Mn2+ influx and is the basis of the use of raised extracellular Mn2+ in experiments designed to detect pathways for Ca2+ influx. This commentary features the manganese quench technique as used in a recently published article and discusses in detail the potential consequences for the intracellular Ca2+ handling when intracellular Mn2+ is increased, as it now competes to a greater extent than normal with Ca2+ for intracellular buffers.

Among the three classes of voltage-gated Ca2+ channels (CaV1, CaV2, and CaV3), CaV3 T-type channels are drug targets for disorders, including epilepsy and pain. Antagonists such as Z944 and ML218 are highly selective for CaV3 compared with the CaV1.2 L-type channel, but whether they have additional activity on other CaV1 subtypes is unknown. Here, we investigated the effects of Z944 and ML218 on the CaV1.4 channel, which regulates neurotransmitter release from retinal photoreceptors. In HEK293T cells transfected with CaV1.4 and the auxiliary β2x13 and α2δ-4 subunits, Z944 and ML218 inhibited Ca2+ currents with IC50 values of ∼30 and 2 µM, respectively. Structure-based modeling combined with functional studies revealed the importance of a cluster of methionine residues, particularly M1004, within the DHP-binding site for the effects of ML218. Compared with mutation of a conserved threonine (T1007) that is required for DHP sensitivity of CaV1 channels, mutation of M1004 had a 10-fold greater impact in diminishing the potency of ML218. CaV1.2 was significantly less sensitive to ML218 inhibition (IC50 ∼37 µM) than CaV1.4, which could not be attributed to a valine in place of M1004 in CaV1.2. We conclude that ML218 and Z944 are dual CaV1/CaV3 modulators of CaV1.4 and should be used with caution when dissecting the contributions of CaV3 channels in tissues where CaV1.4 is expressed.

Electrical synapses mediated by gap junctions are widespread in the mammalian brain, playing essential roles in neural circuit function. Beyond their role synchronizing neuronal activity, they also support complex computations such as coincidence detection-a circuit mechanism in which differences in input timing are encoded by the firing rates of coupled neurons, enabling preferential responses to synchronous over temporally dispersed inputs. Electrical coupling allows each neuron to act as a current sink for its partner during independent depolarizations, thereby reducing excitability. In contrast, synchronous inputs across the network minimize voltage differences through gap junctions, reducing current shunting and increasing spiking probability. However, the contribution of intrinsic neuronal properties to coincidence detection remains poorly understood. Here, we investigated this issue in the mesencephalic trigeminal (MesV) nucleus of mice, a structure composed of somatically coupled neurons. Using whole-cell recordings and pharmacological tools, we examined the role of the D-type K+ current (ID), finding that it critically shapes both the intrinsic electrophysiological properties of MesV neurons and the dynamics of electrical synaptic transmission. Its fast activation kinetics and subthreshold voltage range of activation make ID a key determinant of transmission strength and timing. Furthermore, the ID, likely mediated by Kv1 subunits, is expressed at the soma and the axon initial segment. Finally, we characterized two key parameters of coincidence detection-precision (time window for effective input summation) and gain (differential response to coincident versus dispersed inputs)-finding that ID enhances precision by accelerating membrane repolarization and reduces the gain by limiting neuronal excitability.

KdpFABC is an ATP-dependent membrane complex that enables prokaryotes to maintain potassium homeostasis under potassium-limited conditions. It features a unique hybrid mechanism combining a channel-like selectivity filter in KdpA with the ATP-driven transport functionality of KdpB. A key unresolved question is whether K+ ions translocate through the inter-subunit tunnel as a queue of ions or individually within a hydrated environment. Using molecular dynamics simulations, metadynamics, anomalous X-ray scattering, and biochemical assays, we demonstrate that the tunnel is predominantly occupied by water molecules rather than multiple K+ ions. Our results identify only one stable intermediate binding site for K+ within the tunnel, apart from the canonical sites in KdpA and KdpB. Free energy calculations reveal a substantial barrier (∼22 kcal/mol) at the KdpA-KdpB interface, making spontaneous K+ translocation unlikely. Furthermore, mutagenesis and functional assays confirm previous findings that Phe232 at this interface plays a key role in coupling ATP hydrolysis to K+ transport. These findings challenge previous models containing a continuous wire of K+ ions through the tunnel and suggest the existence of an as-yet unidentified intermediate state or mechanistic detail that facilitates K+ movement into KdpB.

Ca2+- and voltage-activated BK-type K+ channels are influenced profoundly by associated regulatory subunits, including β subunits (Kcnmb1-4; β1-β4). Although overlap in expression of different BK β subunits occurs in native tissues, whether they can coassemble in the same channel complex is not known. We coexpress β2 and β3a subunits together with BK α 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 β2 and β3a subunits coassemble in the same channels, and (2) segregation in which β2 and β3a are found in separate complexes. Our results support the view that, for β2 and β3a, BK currents arise from the random, independent assembly of both subunits in the same channels. Single-channel recordings directly confirm coassembly of β2 and β3a subunits in the same channels. Quantitative biochemical analysis of coexpression of tagged β2, β3a, and BK α subunits also reveals that β2:β3a:α ternary complexes form.