KCNQ1基因及其编码钾通道的研究概况

本文介绍了KCNQ1基因和其编码的通道蛋白的结构、生理学功能及电生理特点。归纳了围绕KCNQ1基因的热点研究方向和成果,并展望了未来的研究趋势。对基础医学上关心的KCNQ1基因突变与疾病的关系,及相关的病理药理学研究做了较为详细的介绍。     

Down-regulation of T-type Cav3.2 channels by hyperpolarization-activated cyclic nucleotide-gated channel 1 (HCN1): Evidence of a signaling complex

Formation of complexes between ion channels is important for signal processing in the brain. Here we investigate the biochemical and biophysical interactions between HCN1 channels and Cav3.2 T-type channels. We found that HCN1 co-immunoprecipitated with Cav3.2 from lysates of either mouse brain or tsA-201 cells, with the HCN1 N-terminus associating with the Cav3.2 N-terminus. Cav3.2 channel activity appeared to be functionally regulated by HCN1. The expression of HCN1 induced a decrease in Cav3.2 Ba²⁺ influx (I_Ba²⁺) along with altered channel kinetics and a depolarizing shift in activation gating. However, a reciprocal regulation of HCN1 by Cav3.2 was not observed. This study highlights a regulatory role of HCN1 on Cav3.2 voltage-dependent properties, which are expected to affect physiologic functions such as synaptic transmission and cellular excitability.     

Characterization of auto-regulation of the human cardiac α1 subunit of the L-type calcium channel: Importance of the C-terminus

The carboxyl terminal of the L-type calcium channel _1C subunit comprises approximately one third of the primary structure of the ₁ subunit (> 700 amino acids residues). This region is sensitive to limited posttranslational processing. In heart and brain the _1C subunits are found to be truncated but the C-terminal domain remains functionally present. Based on our previous data we hypothesized that the distal C-terminus (approximately residues 1650–1950) harbors an important, predominantly inhibitory domain. We generated C-terminal-truncated _1C mutants, and after expressing them in combination with a ₃ subunit in HEK-293 cells, electrophysiological experiments were carried out. In order to dissect the important inhibitory part of the C-terminus, trypsin was dialyzed into the cells. The data provide evidence that there are multiple residues within the inhibitory domain that are crucial to the inhibitory process as well as to the enhancement of expressed current by intracellular application of proteases. In addition, the expression of the chimeric mutant _1C1673-DRK₁ demonstrated that the C-terminal is specific for the heart channel.     

Separation of Gating Properties from Permeation and Block in mslo Large Conductance Ca-activated K+ Channels

In this and the following paper we have examined the kinetic and steady-state properties of macroscopic mslo Ca-activated K⁺ currents in order to interpret these currents in terms of the gating behavior of the mslo channel. To do so, however, it was necessary to first find conditions by which we could separate the effects that changes in Ca²⁺ concentration or membrane voltage have on channel permeation from the effects these stimuli have on channel gating. In this study we investigate three phenomena which are unrelated to gating but are manifest in macroscopic current records: a saturation of single channel current at high voltage, a rapid voltage-dependent Ca²⁺ block, and a slow voltage-dependent Ba²⁺ block. Where possible methods are described by which these phenomena can be separated from the effects that changes in Ca²⁺ concentration and membrane voltage have on channel gating. Where this is not possible, some assessment of the impact these effects have on gating parameters determined from macroscopic current measurements is provided. We have also found that without considering the effects of Ca²⁺ and voltage on channel permeation and block, macroscopic current measurements suggest that mslo channels do not reach the same maximum open probability at all Ca²⁺ concentrations. Taking into account permeation and blocking effects, however, we find that this is not the case. The maximum open probability of the mslo channel is the same or very similar over a Ca²⁺ concentration range spanning three orders of magnitude indicating that over this range the internal Ca²⁺ concentration does not limit the ability of the channel to be activated by voltage.     

Allosteric voltage gating of potassium channels II. Mslo channel gating charge movement in the absence of Ca(2+).

Large-conductance Ca(2+)-activated K(+) channels can be activated by membrane voltage in the absence of Ca(2+) binding, indicating that these channels contain an intrinsic voltage sensor. The properties of this voltage sensor and its relationship to channel activation were examined by studying gating charge movement from mSlo Ca(2+)-activated K(+) channels in the virtual absence of Ca(2+) (<1 nM). Charge movement was measured in response to voltage steps or sinusoidal voltage commands. The charge-voltage relationship (Q-V) is shallower and shifted to more negative voltages than the voltage-dependent open probability (G-V). Both ON and OFF gating currents evoked by brief (0.5-ms) voltage pulses appear to decay rapidly (tau(ON) = 60 microseconds at +200 mV, tau(OFF) = 16 microseconds at -80 mV). However, Q(OFF) increases slowly with pulse duration, indicating that a large fraction of ON charge develops with a time course comparable to that of I(K) activation. The slow onset of this gating charge prevents its detection as a component of I(gON), although it represents approximately 40% of the total charge moved at +140 mV. The decay of I(gOFF) is slowed after depolarizations that open mSlo channels. Yet, the majority of open channel charge relaxation is too rapid to be limited by channel closing. These results can be understood in terms of the allosteric voltage-gating scheme developed in the preceding paper (Horrigan, F.T., J. Cui, and R.W. Aldrich. 1999. J. Gen. Physiol. 114:277-304). The model contains five open (O) and five closed (C) states arranged in parallel, and the kinetic and steady-state properties of mSlo gating currents exhibit multiple components associated with C-C, O-O, and C-O transitions.     

Coupling between voltage sensor activation,Ca2+ binding and channel opening in large conductance (BK) potassium channels

To determine how intracellular Ca(2+) and membrane voltage regulate the gating of large conductance Ca(2+)-activated K(+) (BK) channels, we examined the steady-state and kinetic properties of mSlo1 ionic and gating currents in the presence and absence of Ca(2+) over a wide range of voltage. The activation of unliganded mSlo1 channels can be accounted for by allosteric coupling between voltage sensor activation and the closed (C) to open (O) conformational change (Horrigan, F.T., and R.W. Aldrich. 1999. J. Gen. Physiol. 114:305-336; Horrigan, F.T., J. Cui, and R.W. Aldrich. 1999. J. Gen. Physiol. 114:277-304). In 0 Ca(2+), the steady-state gating charge-voltage (Q(SS)-V) relationship is shallower and shifted to more negative voltages than the conductance-voltage (G(K)-V) relationship. Calcium alters the relationship between Q-V and G-V, shifting both to more negative voltages such that they almost superimpose in 70 microM Ca(2+). This change reflects a differential effect of Ca(2+) on voltage sensor activation and channel opening. Ca(2+) has only a small effect on the fast component of ON gating current, indicating that Ca(2+) binding has little effect on voltage sensor activation when channels are closed. In contrast, open probability measured at very negative voltages (less than -80 mV) increases more than 1,000-fold in 70 microM Ca(2+), demonstrating that Ca(2+) increases the C-O equilibrium constant under conditions where voltage sensors are not activated. Thus, Ca(2+) binding and voltage sensor activation act almost independently, to enhance channel opening. This dual-allosteric mechanism can reproduce the steady-state behavior of mSlo1 over a wide range of conditions, with the assumption that activation of individual Ca(2+) sensors or voltage sensors additively affect the energy of the C-O transition and that a weak interaction between Ca(2+) sensors and voltage sensors occurs independent of channel opening. By contrast, macroscopic I(K) kinetics indicate that Ca(2+) and voltage dependencies of C-O transition rates are complex, leading us to propose that the C-O conformational change may be described by a complex energy landscape.     

Cysteine modification alters voltage- and Ca(2+)-dependent gating of large conductance (BK) potassium channels

The Ca(2+)-activated K+ (BK) channel alpha-subunit contains many cysteine residues within its large COOH-terminal tail domain. To probe the function of this domain, we examined effects of cysteine-modifying reagents on channel gating. Application of MTSET, MTSES, or NEM to mSlo1 or hSlo1 channels changed the voltage and Ca2+ dependence of steady-state activation. These reagents appear to modify the same cysteines but have different effects on function. MTSET increases I(K) and shifts the G(K)-V relation to more negative voltages, whereas MTSES and NEM shift the G(K)-V in the opposite direction. Steady-state activation was altered in the presence or absence of Ca2+ and at negative potentials where voltage sensors are not activated. Combinations of [Ca2+] and voltage were also identified where P(o) is not changed by cysteine modification. Interpretation of our results in terms of an allosteric model indicate that cysteine modification alters Ca2+ binding and the relative stability of closed and open conformations as well as the coupling of voltage sensor activation and Ca2+ binding and to channel opening. To identify modification-sensitive residues, we examined effects of MTS reagents on mutant channels lacking one or more cysteines. Surprisingly, the effects of MTSES on both voltage- and Ca(2+)-dependent gating were abolished by replacing a single cysteine (C430) with alanine. C430 lies in the RCK1 (regulator of K+ conductance) domain within a series of eight residues that is unique to BK channels. Deletion of these residues shifted the G(K)-V relation by > -80 mV. Thus we have identified a region that appears to strongly influence RCK domain function, but is absent from RCK domains of known structure. C430A did not eliminate effects of MTSET on apparent Ca2+ affinity. However an additional mutation, C615S, in the Haem binding site reduced the effects of MTSET, consistent with a role for this region in Ca2+ binding.     

A Selectivity Filter Gate Controls Voltage-Gated Calcium Channel Calcium-Dependent Inactivation

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多巴胺对血管平滑肌细胞钙激活钾通道的影响及其机制

目的：研究多巴胺对血管平滑肌细胞大电流、钙激活钾（ＢＫｃａ）通道的影响及其信息传递机制。方法用膜片钳细胞贴附式技术,记录细胞能液内灌流多巴胺受体激动剂、阻断剂以及第二信使及相关蛋白激酶拮机剂对猪冠状动脉血管平滑肌细胞ＢＫｃａ 爱道活动的影响。结果：多巴胺增加ＢＫｃａ通道活性（Ｐ〈０．０１）,并可被ＣＡ－１受体阻断剂ＳＣＨ２３３９０完全阻断,但不受β２受体阻断剂普藉洛尔的影响。腺苷酸环化酶抑制剂ＳＯ     

小鼠心室肌细胞外向电流的特征分析

用全细胞膜片箝技术研究了小鼠心室肌细胞去极化激活的非钙依赖性外向电流的特征。短时去极化至－５０ｍＶ以上电位可引发快速上升的外向钾电流,之后缓慢衰减至一平台。在改变保持电位时,该峰电流与平台电流的比值也发生改变,并测得两者翻转电位分别为－６４．３±３．９和－５３．３±２．９ｍＶ。外向电流的衰减过程可用双指数方式很好拟合,而在５００ｍｓ预脉冲刺激后再经去极化刺激的电流时程适合单指数方式。结果提示：小鼠心室肌细胞的外向电流包括两种失活成分（Ｉｋｆ,和Ｉｋｓ）,它们对４－氨基吡啶具有相似的敏感性。Ｉｋｆ的稳态失活在测试电位范围内（－８０－＋３０ｍＶ）均不完全,这可能与小鼠心肌动作电位时程很短有关联。     

cAMP-dependent phosphorylation sites and macroscopic activity of recombinant cardiac L-type calcium channels

The involvement of cAMP-dependent phosphorylation sites in establishing the basal activity of cardiac L-type Ca²⁺ channels was studied in HEK 293 cells transiently cotransfected with mutants of the human cardiac ₁ and accessory subunits. Systematic individual or combined elimination of high consensus protein kinase A (PKA) sites, by serine to alanine substitutions at the amino and carboxyl termini of the ₁ subunit, resulted in Ca²⁺ channel currents indistinguishable from those of wild type channels. Dihydropyridine (DHP)-binding characteristics were also unaltered. To explore the possible involvement of nonconsensus sites, deletion mutants were used. Carboxyl-terminal truncations of the ₁ subunit distal to residue 1597 resulted in increased channel expression and current amplitudes. Modulation of PKA activity in cells transfected with the wild type channel or any of the mutants did not alter Ca²⁺ channel functions suggesting that cardiac Ca²⁺ channels expressed in these cells behave, in terms of lack of PKA control, like Ca²⁺ channels of smooth muscle cells.     

柯萨奇B3病毒对心肌细胞离子通道电流的影响

目的 :探讨柯萨奇B3病毒 (coxsackievirusB3,CVB3)对大鼠心肌细胞离子通道的影响 ,以了解病毒感染导致细胞电生理活动异常的机理。方法 :酶消化法获得单个心肌细胞后利用膜片钳全细胞电流记录技术观察CVB3对L型钙通道电流、钠通道电流、外向钾电流和内向整流性钾电流的影响。结果 :CVB3感染使L型钙通道电流、外向钾电流增加 ,内向整流性钾电流减小 ,对钠通道电流无明显影响。结论 :CVB3对L型钙通道电流、外向钾电流和内向整流性钾电流的影响可能是病毒感染后细胞损伤和产生异常电活动的原因。     

线粒体ATP敏感钾通道与钙激活钾通道激活对正常与缺血脑线粒体通透性转变的影响

目的：明确线粒体ATP敏感钾通道与钙激活钾通道对正常和缺血脑线粒体渗透性转变的作用。方法：实验采用分光光度法,在分离的线粒体上分别观察两种线粒体钾通道激动剂对正常与缺血脑线粒体肿胀的影响。结果：在正常脑线粒体,diazoxide与NSl619能有效抑制由钙诱导的线粒体氏20下降,但其效应可被atractyloside所阻断。与正常相比,缺血损伤后的脑线粒体在钙离子诱导下线粒体A520下降较快,diazoxide与NS1619仍可抑制由钙诱导的线粒体A520下降,其作用同样为atractykxside所阻断。结论：线粒体ATP敏感钾通道与钙激活钾通道激活在离体条件均具有保护脑线粒体的作用,其作用可能是通过影响线粒体通透性转变而实现。