In vitro and in vivo phosphorylation of the Cav2.3 voltage-gated R-type calcium channel

During the recording of whole cell currents from stably transfected HEK-293 cells, the decline of currents carried by the recombinant human Cav2.3+β3 channel subunits is related to adenosine triphosphate (ATP) depletion after rupture of the cells. It reduces the number of functional channels and leads to a progressive shift of voltage-dependent gating to more negative potentials (Neumaier F., et al., 2018). Both effects can be counteracted by hydrolysable ATP, whose protective action is almost completely prevented by inhibition of serine/threonine but not tyrosine or lipid kinases. These findings indicate that ATP promotes phosphorylation of either the channel or an associated protein, whereas dephosphorylation during cell dialysis results in run-down. Protein phosphorylation is required for Ca_v2.3 channel function and could directly influence the normal features of current carried by these channels. Therefore, results from in vitro and in vivo phosphorylation of Ca_v2.3 are summarized to come closer to a functional analysis of structural variations in Ca_v2.3 splice variants.     

Cooperative regulation of Cav1.2 channels by intracellular Mg2+, the proximal C-terminal EF-hand,and the distal C-terminal domain

L-type Ca²⁺ currents conducted by Ca_v1.2 channels initiate excitation–contraction coupling in cardiac myocytes. Intracellular Mg²⁺ (Mg_i) inhibits the ionic current of Ca_v1.2 channels. Because Mg_i is altered in ischemia and heart failure, its regulation of Ca_v1.2 channels is important in understanding cardiac pathophysiology. Here, we studied the effects of Mg_i on voltage-dependent inactivation (VDI) of Ca_v1.2 channels using Na⁺ as permeant ion to eliminate the effects of permeant divalent cations that engage the Ca²⁺-dependent inactivation process. We confirmed that increased Mg_i reduces peak ionic currents and increases VDI of Ca_v1.2 channels in ventricular myocytes and in transfected cells when measured with Na⁺ as permeant ion. The increased rate and extent of VDI caused by increased Mg_i were substantially reduced by mutations of a cation-binding residue in the proximal C-terminal EF-hand, consistent with the conclusion that both reduction of peak currents and enhancement of VDI result from the binding of Mg_i to the EF-hand (K_D ≈ 0.9 mM) near the resting level of Mg_i in ventricular myocytes. VDI was more rapid for L-type Ca²⁺ currents in ventricular myocytes than for Ca_v1.2 channels in transfected cells. Coexpression of Ca_vβ_2b subunits and formation of an autoinhibitory complex of truncated Ca_v1.2 channels with noncovalently bound distal C-terminal domain (DCT) both increased VDI in transfected cells, indicating that the subunit structure of the Ca_v1.2 channel greatly influences its VDI. The effects of noncovalently bound DCT on peak current amplitude and VDI required Mg_i binding to the proximal C-terminal EF-hand and were prevented by mutations of a key divalent cation-binding amino acid residue. Our results demonstrate cooperative regulation of peak current amplitude and VDI of Ca_v1.2 channels by Mg_i, the proximal C-terminal EF-hand, and the DCT, and suggest that conformational changes that regulate VDI are propagated from the DCT through the proximal C-terminal EF-hand to the channel-gating mechanism.     

A genetic screen for dihydropyridine (DHP)-resistant worms reveals new residues required for DHP-blockage of mammalian calcium channels

Dihydropyridines (DHPs) are L-type calcium channel (Ca_v1) blockers prescribed to treat several diseases including hypertension. Ca_v1 channels normally exist in three states: a resting closed state, an open state that is triggered by membrane depolarization, followed by a non-conducting inactivated state that is triggered by the influx of calcium ions, and a rapid change in voltage. DHP binding is thought to alter the conformation of the channel, possibly by engaging a mechanism similar to voltage dependent inactivation, and locking a calcium ion in the pore, thereby blocking channel conductance. As a Ca_v1 channel crystal structure is lacking, the current model of DHP action has largely been achieved by investigating the role of candidate Ca_v1 residues in mediating DHP-sensitivity. To better understand DHP-block and identify additional Ca_v1 residues important for DHP-sensitivity, we screened 440,000 randomly mutated Caenorhabditis elegans genomes for worms resistant to DHP-induced growth defects. We identified 30 missense mutations in the worm Ca_v1 pore-forming (α₁) subunit, including eleven in conserved residues known to be necessary for DHP-binding. The remaining polymorphisms are in eight conserved residues not previously associated with DHP-sensitivity. Intriguingly, all of the worm mutants that we analyzed phenotypically exhibited increased channel activity. We also created orthologous mutations in the rat α_1C subunit and examined the DHP-block of current through the mutant channels in culture. Six of the seven mutant channels examined either decreased the DHP-sensitivity of the channel and/or exhibited significant residual current at DHP concentrations sufficient to block wild-type channels. Our results further support the idea that DHP-block is intimately associated with voltage dependent inactivation and underscores the utility of C. elegans as a screening tool to identify residues important for DHP interaction with mammalian Ca_v1 channels.     

The HOOK region of β subunits controls gating of voltage-gated Ca2+ channels by electrostatically interacting with plasma membrane

Recently, we showed that the HOOK region of the β2 subunit electrostatically interacts with the plasma membrane and regulates the current inactivation and phosphatidylinositol 4,5-bisphosphate (PIP₂) sensitivity of voltage-gated Ca²⁺ (Ca_V) 2.2 channels. Here, we report that voltage-dependent gating and current density of the Ca_V2.2 channels are also regulated by the HOOK region of the β2 subunit. The HOOK region can be divided into 3 domains: S (polyserine), A (polyacidic), and B (polybasic). We found that the A domain shifted the voltage-dependent inactivation and activation of Ca_V2.2 channels to more hyperpolarized and depolarized voltages, respectively, whereas the B domain evoked these responses in the opposite directions. In addition, the A domain decreased the current density of the Ca_V2.2 channels, while the B domain increased it. Together, our data demonstrate that the flexible HOOK region of the β2 subunit plays an important role in determining the overall Ca_V channel gating properties.     

Contrasting Effects of Cd<Superscript>2+</Superscript> and Co<Superscript>2+</Superscript> on the Blocking/Unblocking of Human Ca<Subscript>v</Subscript>3 Channels

Inorganic ions have been used widely to investigate biophysical properties of high voltage-activated calcium channels (HVA: Ca_v1 and Ca_v2 families). In contrast, such information regarding low voltage-activated calcium channels (LVA: Ca_v3 family) is less documented. We have studied the blocking effect of Cd²⁺, Co²⁺ and Ni²⁺ on T-currents expressed by human Ca_v3 channels: Ca_v3.1, Ca_v3.2, and Ca_v3.3. With the use of the whole-cell configuration of the patch-clamp technique, we have recorded Ca²⁺ (2 mM) currents from HEK−293 cells stably expressing recombinant T-type channels. Cd²⁺ and Co²⁺ block was 2- to 3-fold more potent for Ca_v3.2 channels (EC₅₀ = 65 and 122 μM, respectively) than for the other two LVA channel family members. Current-voltage relationships indicate that Co²⁺ and Ni²⁺ shift the voltage dependence of Ca_v3.1 and Ca_v3.3 channels activation to more positive potentials. Interestingly, block of those two Ca_v3 channels by Co²⁺ and Ni²⁺ was drastically increased at extreme negative voltages; in contrast, block due to Cd²⁺ was significantly decreased. This unblocking effect was slightly voltage-dependent. Tail-current analysis reveals a differential effect of Cd²⁺ on Ca_v3.3 channels, which can not close while the pore is occupied with this metal cation. The results suggest that metal cations affect differentially T-type channel activity by a mechanism involving the ionic radii of inorganic ions and structural characteristics of the channels pore.     

Alternative Splicing Generates a Novel Truncated Cav1.2 Channel in Neonatal Rat Heart

L-type Ca_v1.2 Ca²⁺ channel undergoes extensive alternative splicing, generating functionally different channels. Alternatively spliced Ca_v1.2 Ca²⁺ channels have been found to be expressed in a tissue-specific manner or under pathological conditions. To provide a more comprehensive understanding of alternative splicing in Ca_v1.2 channel, we systematically investigated the splicing patterns in the neonatal and adult rat hearts. The neonatal heart expresses a novel 104-bp exon 33L at the IVS3-4 linker that is generated by the use of an alternative acceptor site. Inclusion of exon 33L causes frameshift and C-terminal truncation. Whole-cell electrophysiological recordings of Ca_v1.2_33L channels expressed in HEK 293 cells did not detect any current. However, when co-expressed with wild type Ca_v1.2 channels, Ca_v1.2_33L channels reduced the current density and altered the electrophysiological properties of the wild type Ca_v1.2 channels. Interestingly, the truncated 3.5-domain Ca_v1.2_33L channels also yielded a dominant negative effect on Ca_v1.3 channels, but not on Ca_v3.2 channels, suggesting that Ca_vβ subunits is required for Ca_v1.2_33L regulation. A biochemical study provided evidence that Ca_v1.2_33L channels enhanced protein degradation of wild type channels via the ubiquitin-proteasome system. Although the physiological significance of the Ca_v1.2_33L channels in neonatal heart is still unknown, our report demonstrates the ability of this novel truncated channel to modulate the activity of the functional Ca_v1.2 channels. Moreover, the human Ca_v1.2 channel also contains exon 33L that is developmentally regulated in heart. Unexpectedly, human exon 33L has a one-nucleotide insertion that allowed in-frame translation of a full Ca_v1.2 channel. An electrophysiological study showed that human Ca_v1.2_33L channel is a functional channel but conducts Ca²⁺ ions at a much lower level.     

Gating consequences of charge neutralization of arginine residues in the S4 segment of K(v)7.2, an epilepsy-linked K+ channel subunit

The K_v7.2 subunits are the main molecular determinants of the M-current, a widespread K⁺ current regulating neuronal excitability. Mutations in the K_v7.2 gene cause benign familial neonatal seizures, an autosomally inherited human epilepsy. The benign familial neonatal seizure-causing mutations include those at arginine residues at positions 207 and 214 in the S₄ segment of K_v7.2. In this study, each of the six S₄ arginines was individually replaced with neutral glutamines, and the functional properties of mutant channels were studied by whole-cell and single-channel voltage-clamp measurements. The results obtained suggest that each S₄ arginine residue plays a relevant role in the voltage-dependent gating of K_v7.2 channels. In particular, a decreased positive charge at the N-terminal end of S₄ stabilized the activated state of the voltage-sensor, whereas positive-charge neutralization at the C-terminal end of S₄ favored the resting conformation. Strikingly, neutralization of a single arginine at position 201 was sufficient to cause a significant loss of voltage dependence in channel activation. Moreover, by comparing the functional properties of glutamine versus tryptophan substitution, we found steric bulk to play a relevant role at position 207, but not at position 214, in which the main functional effect of this disease-causing mutation seems to be a consequence of the loss of the positive charge.     

Structural and biophysical determinants of single CaV3.1 and CaV3.2 T-type calcium channel inhibition by N2O

We investigated the biophysical mechanism of inhibition of recombinant T-type calcium channels Ca_V3.1 and Ca_V3.2 by nitrous oxide (N₂O). To identify functionally important channel structures, chimeras with reciprocal exchange of the N-terminal domains I and II and C-terminal domains III and IV were examined. In whole-cell recordings N₂O significantly inhibited Ca_V3.2, and – less pronounced – Ca_V3.1. A Ca_V3.2-prevalent inhibition of peak currents was also detected in cell-attached multi-channel patches. In cell-attached patches containing ≤3 channels N₂O reduced average peak current of Ca_V3.2 by decreasing open probability and open time duration. Effects on Ca_V3.1 were smaller and mediated by a reduced fraction of sweeps containing channel activity. Without drug, single Ca_V3.1 channels were significantly less active than Ca_V3.2. Chimeras revealed that domains III and IV control basal gating properties. Domains I and II, in particular a histidine residue within Ca_V3.2 (H191), are responsible for the subtype-prevalent N₂O inhibition. Our study demonstrates the biophysical (open times, open probability) and structural (domains I and II) basis of action of N₂O on Ca_V3.2. Such a fingerprint of single channels can help identifying the molecular nature of native channels. This is exemplified by a characterization of single channels expressed in human hMTC cells as functional homologues of recombinant Ca_V3.1.     

CaV3.1 channel pore pseudo-symmetry revealed by selectivity filter mutations in its domains I/II

There is growing evidence indicating that the pore structure of voltage-gated ion channels (VGICs) influences gating besides their conductance. Regarding low voltage-activated (LVA) Ca²⁺ channels, it has been demonstrated that substitutions of the pore aspartate (D) by a glutamate (D-to-E substitution) in domains III and IV alter channel gating properties such as a positive shift in the channel activation voltage dependence. In the present report, we evaluated the effects of E-to-D substitution in domains I and II on the Ca_V3.1 channel gating properties. Our results indicate that substitutions in these two domains differentially modify the gating properties of Ca_V3.1 channels. The channel with a single mutation in domain I (DEDD) presented slower activation and faster inactivation kinetics and a slower recovery from inactivation, as compared with the WT channel. In contrast, the single mutant in domain II (EDDD) presented a small but significant negative shift of activation voltage dependence with faster activation and slower inactivation kinetics. Finally, the double mutant channel (DDDD) presented somehow intermediate properties with respect to the two single mutants but with fastest deactivation kinetics. Overall, our results indicate that single amino acid modification of the selectivity filter of LVA Ca²⁺ channels in distinct domains differentially influence their gating properties, supporting a pore pseudo-symmetry.     

Impact of gating modulation in CaV1.3 L-type calcium channels

Ca_V1.3 L-type channels control inner hair cell (IHC) sensory and sinoatrial node (SAN) function, and excitability in central neurons by means of their low-voltage activation and inactivation properties. In SAN cells Ca_V1.3 inward calcium current (I_Ca) inactivates rapidly whereas in IHCs inactivation is slow. A candidate suggested in slowing Ca_V1.3 channel inactivation is the presynaptically located ribbon-synapse protein RIM that is expressed in immature IHCs in presynaptic compartments also expressing Ca_V1.3 channels. Ca_V1.3 channel gating is also modulated by an intramolecular C-terminal mechanism. This mechanism was elicited during analysis of human C-terminal splice variants that differ in the length of their C-terminus and that modulates the channel’s negative activation range and slows calcium-dependent inactivation.     

Functional properties of the Cav1.2 calcium channel activated by calmodulin in the absence of α2δ subunits

Voltage-activated Ca_v1.2 calcium channels require association of the pore-forming α_1C subunit with accessory Ca_vβ and α₂δ subunits. Binding of a single calmodulin (CaM) to α_1C supports Ca²⁺-dependent inactivation (CDI). The human Ca_v1.2 channel is silent in the absence of Ca_vβ and/or α₂δ. Recently, we found that coexpression of exogenous CaM (CaM_ex) supports plasma membrane targeting, gating facilitation and CDI of the channel in the absence of Ca_vβ. Here we discovered that CaM_ex and its Ca²⁺-insensitive mutant (CaM₁₂₃₄) rendered active α_1C/Ca_vβ channel in the absence of α₂δ. Coexpression of CaM_ex with α_1C and β_2d in calcium-channel-free COS-1 cells recovered gating of the channel and supported CDI. Voltage-dependence of activation was shifted by ≈ +40 mV to depolarization potentials. The calcium current reached maximum at +40 mV (20 mM Ca²⁺) and exhibited approximately 3 times slower activation and 5 times slower inactivation kinetics compared to the wild-type channel. Furthermore, both CaM_ex and CaM₁₂₃₄ accelerated recovery from inactivation and induced facilitation of the calcium current by strong depolarization prepulse, the properties absent from the human vascular/neuronal Ca_v1.2 channel. The data suggest a previously unknown action of CaM that in the presence of Ca_vβ translates into activation of the α₂δ-deficient calcium channel and alteration of its properties.     

Sialic acids attached to N- and O-glycans within the Nav1.4 D1S5–S6 linker contribute to channel gating

Background

Voltage-gated Na⁺ channels (Na_v) are responsible for the initiation and conduction of neuronal and muscle action potentials. Na_v gating can be altered by sialic acids attached to channel N-glycans, typically through isoform-specific electrostatic mechanisms.

Methods

Using two sets of Chinese Hamster Ovary cell lines with varying abilities to glycosylate glycoproteins, we show for the first time that sialic acids attached to O-glycans and N-glycans within the Na_v1.4 D1S5–S6 linker modulate Na_v gating.

Results

All measured steady-state and kinetic parameters were shifted to more depolarized potentials under conditions of essentially no sialylation. When sialylation of only N-glycans or of only O-glycans was prevented, the observed voltage-dependent parameter values were intermediate between those observed under full versus no sialylation. Immunoblot gel shift analyses support the biophysical data.

Conclusions

The data indicate that sialic acids attached to both N- and O-glycans residing within the Na_v1.4 D1S5-S6 linker modulate channel gating through electrostatic mechanisms, with the relative contribution of sialic acids attached to N- versus O-glycans on channel gating being similar.

General significance

Protein N- and O-glycosylation can modulate ion channel gating simultaneously. These data also suggest that environmental, metabolic, and/or congenital changes in glycosylation that impact sugar substrate levels, could lead, potentially, to changes in Na_v sialylation and gating that would modulate AP waveforms and conduction.     

Regulation of Neuronal Cav3.1 Channels by Cyclin-Dependent Kinase 5 (Cdk5)

Low voltage-activated (LVA) T-type Ca²⁺ channels activate in response to subthreshold membrane depolarizations and therefore represent an important source of Ca²⁺ influx near the resting membrane potential. In neurons, these proteins significantly contribute to control relevant physiological processes including neuronal excitability, pacemaking and post-inhibitory rebound burst firing. Three subtypes of T-type channels (Ca_v3.1 to Ca_v3.3) have been identified, and using functional expression of recombinant channels diverse studies have validated the notion that T-type Ca²⁺ channels can be modulated by various endogenous ligands as well as by second messenger pathways. In this context, the present study reveals a previously unrecognized role for cyclin-dependent kinase 5 (Cdk5) in the regulation of native T-type channels in N1E-115 neuroblastoma cells, as well as recombinant Ca_v3.1channels heterologously expressed in HEK-293 cells. Cdk5 and its co-activators play critical roles in the regulation of neuronal differentiation, cortical lamination, neuronal cell migration and axon outgrowth. Our results show that overexpression of Cdk5 causes a significant increase in whole cell patch clamp currents through T-type channels in N1E-115 cells, while siRNA knockdown of Cdk5 greatly reduced these currents. Consistent with this, overexpression of Cdk5 in HEK-293 cells stably expressing Ca_v3.1channels upregulates macroscopic currents. Furthermore, using site-directed mutagenesis we identified a major phosphorylation site at serine 2234 within the C-terminal region of the Ca_v3.1subunit. These results highlight a novel role for Cdk5 in the regulation of T-type Ca²⁺ channels.     

Phospholemman Modulates the Gating of Cardiac L-Type Calcium Channels

Ca²⁺ entry through L-type calcium channels (Ca_V1.2) is critical in shaping the cardiac action potential and initiating cardiac contraction. Modulation of Ca_V1.2 channel gating directly affects myocyte excitability and cardiac function. We have found that phospholemman (PLM), a member of the FXYD family and regulator of cardiac ion transport, coimmunoprecipitates with Ca_V1.2 channels from guinea pig myocytes, which suggests PLM is an endogenous modulator. Cotransfection of PLM in HEK293 cells slowed Ca_V1.2 current activation at voltages near the threshold for activation, slowed deactivation after long and strong depolarizing steps, enhanced the rate and magnitude of voltage-dependent inactivation (VDI), and slowed recovery from inactivation. However, Ca²⁺-dependent inactivation was not affected. Consistent with slower channel closing, PLM significantly increased Ca²⁺ influx via Ca_V1.2 channels during the repolarization phase of a human cardiac action potential waveform. Our results support PLM as an endogenous regulator of Ca_V1.2 channel gating. The enhanced VDI induced by PLM may help protect the heart under conditions such as ischemia or tachycardia where the channels are depolarized for prolonged periods of time and could induce Ca²⁺ overload. The time and voltage-dependent slowed deactivation could represent a gating shift that helps maintain Ca²⁺ influx during the cardiac action potential waveform plateau phase.     

Structural Determinants of the High Affinity Extracellular Zinc Binding Site on Cav3.2 T-type Calcium Channels

Ca_v3.2 T-type channels contain a high affinity metal binding site for trace metals such as copper and zinc. This site is occupied at physiologically relevant concentrations of these metals, leading to decreased channel activity and pain transmission. A histidine at position 191 was recently identified as a critical determinant for both trace metal block of Ca_v3.2 and modulation by redox agents. His¹⁹¹ is found on the extracellular face of the Ca_v3.2 channel on the IS3-S4 linker and is not conserved in other Ca_v3 channels. Mutation of the corresponding residue in Ca_v3.1 to histidine, Gln¹⁷², significantly enhances trace metal inhibition, but not to the level observed in wild-type Ca_v3.2, implying that other residues also contribute to the metal binding site. The goal of the present study is to identify these other residues using a series of chimeric channels. The key findings of the study are that the metal binding site is composed of a Asp-Gly-His motif in IS3–S4 and a second aspartate residue in IS2. These results suggest that metal binding stabilizes the closed conformation of the voltage-sensor paddle in repeat I, and thereby inhibits channel opening. These studies provide insight into the structure of T-type channels, and identify an extracellular motif that could be targeted for drug development.     

Properties of Na+ currents conducted by a skeletal muscle L-type Ca2+ channel pore mutant (SkEIIIK)

Four glutamate residues residing at corresponding positions within the four conserved membrane-spanning repeats of L-type Ca²⁺ channels are important structural determinants for the passage of Ca²⁺ across the selectivity filter. Mutation of the critical glutamate in Repeat III in the a_1S subunit of the skeletal L-type channel (Ca_v1.1) to lysine virtually eliminates passage of Ca²⁺ during step depolarizations. In this study, we examined the ability of this mutant Ca_v1.1 channel (SkEIIIK) to conduct inward Na⁺ current. When 150 mM Na⁺ was present as the sole monovalent cation in the bath solution, dysgenic (Ca_v1.1 null) myotubes expressing SkEIIIK displayed slowly-activating, non-inactivating, nifedipine-sensitive inward currents with a reversal potential (45.6 ± 2.5 mV) near that expected for Na⁺. Ca²⁺ block of SkEIIIK-mediated Na⁺ current was revealed by the substantial enhancement of Na⁺ current amplitude after reduction of Ca²⁺ in the external recording solution from 10 mM to near physiological 1 mM. Inward SkEIIIK-mediated currents were potentiated by either ±Bay K 8644 (10 mM) or 200-ms depolarizing prepulses to +90 mV. In contrast, outward monovalent currents were reduced by ±Bay K 8644 and were unaffected by strong depolarization, indicating a preferential potentiation of inward Na⁺ currents through the mutant Ca_v1.1 channel. Taken together, our results show that SkEIIIK functions as a non-inactivating, junctionally-targeted Na⁺ channel when Na⁺ is the sole monvalent cation present and urge caution when interpreting the impact of mutations designed to ablate Ca²⁺ permeability mediated by Ca_V channels on physiological processes that extend beyond channel gating and permeability.     

Regulation of L-type CaV1.3 channel activity and insulin secretion by the cGMP-PKG signaling pathway

cGMP is a second messenger widely used in the nervous system and other tissues. One of the major effectors for cGMP is the serine/threonine protein kinase, cGMP-dependent protein kinase (PKG), which catalyzes the phosphorylation of a variety of proteins including ion channels. Previously, it has been shown that the cGMP-PKG signaling pathway inhibits Ca²⁺ currents in rat vestibular hair cells and chromaffin cells. This current allegedly flow through voltage-gated Ca_V1.3L-type Ca²⁺ channels, and is important for controlling vestibular hair cell sensory function and catecholamine secretion, respectively. Here, we show that native L-type channels in the insulin-secreting RIN-m5F cell line, and recombinant Ca_V1.3 channels heterologously expressed in HEK-293 cells, are regulatory targets of the cGMP-PKG signaling cascade. Our results indicate that the Ca_Vα₁ ion-conducting subunit of the Ca_V1.3 channels is highly expressed in RIN-m5F cells and that the application of 8-Br-cGMP, a membrane-permeable analogue of cGMP, significantly inhibits Ca²⁺ macroscopic currents and impair insulin release stimulated with high K⁺. In addition, KT-5823, a specific inhibitor of PKG, prevents the current inhibition generated by 8-Br-cGMP in the heterologous expression system. Interestingly, mutating the putative phosphorylation sites to residues resistant to phosphorylation showed that the relevant PKG sites for Ca_V1.3 L-type channel regulation centers on two amino acid residues, Ser793 and Ser860, located in the intracellular loop connecting the II and III repeats of the Ca_Vα₁ pore-forming subunit of the channel. These findings unveil a novel mechanism for how the cGMP-PKG signaling pathway may regulate Ca_V1.3 channels and contribute to regulate insulin secretion.     

Voltage-dependent Gating of Single Wild-Type and S4 Mutant KAT1 Inward Rectifier Potassium Channels

The voltage-dependent gating mechanism of KAT1 inward rectifier potassium channels was studied using single channel current recordings from Xenopus oocytes injected with KAT1 mRNA. The inward rectification properties of KAT1 result from an intrinsic gating mechanism in the KAT1 channel protein, not from pore block by an extrinsic cation species. KAT1 channels activate with hyperpolarizing potentials from −110 through −190 mV with a slow voltage-dependent time course. Transitions before first opening are voltage dependent and account for much of the voltage dependence of activation, while transitions after first opening are only slightly voltage dependent. Using burst analysis, transitions near the open state were analyzed in detail. A kinetic model with multiple closed states before first opening, a single open state, a single closed state after first opening, and a closed-state inactivation pathway accurately describes the single channel and macroscopic data. Two mutations neutralizing charged residues in the S4 region (R177Q and R176L) were introduced, and their effects on single channel gating properties were examined. Both mutations resulted in depolarizing shifts in the steady state conductance–voltage relationship, shortened first latencies to opening, decreased probability of terminating bursts, and increased burst durations. These effects on gating were well described by changes in the rate constants in the kinetic model describing KAT1 channel gating. All transitions before the open state were affected by the mutations, while the transitions after the open state were unaffected, implying that the S4 region contributes to the early steps in gating for KAT1 channels.