Structural motifs underlying voltage-dependent K+ channel function

Kidney International, VoL 48 (1995), pp. 918—922

GENERAL STRUCTURAL ASPECTS OF ION CHANNELS

Structural motifs underlying voltage-dependent

K channel function MAURIzI0 TAGLIALATELA and ARTHUR M. BROWN Department of Neurosciences, Section of Pharmacolog', 2nd School of Medicine, University of Naples "Federico II", Naples, Italy, and Rammelkamp Cenier, MetroHealth Campus, Case Western Reserve University, Cleveland, Ohio, USA

Almost ten years have passed since the first amino acid dependent Na4 and Ca24 channels and cyclic nucleotide-gated sequence of a voltage-dependent ion channel, the Na4 channel, was deduced [1]. During this period, extraordinary progress has occurred, primarily as a result of two methodologies, recombinant DNA and patch clamp electrophysiology, and mainly in the field of ion channel structure-function. Today many laboratories are involved in elegant experiments combining mutagenesis of ion channel genes and expression of the mutant channels in heterologous expression systems, allowing detailed functional investigation of permeation and gating [2]. Besides recombinant DNA and patch clamp electrophysiology, different approaches, such as in vitro protein expression, reconstitution into lipid bilayers [3] and site-directed antibodies [4], have also played crucial roles. The major impact of the mutagenesis patch-clamp approach lies in the ability to localize function to a restricted area of a single protein molecule.

Although major progress has been made in identifying structural domains involved in permeation, block and gating, the correlation of specific domains to ion channel function is still speculative at present, due to the absence of direct structural information. In this review we describe recent progress and future perspectives for the field of ion channel structure-function, with particular emphasis on the molecular basis for activation, inactivation and ion permeation in voltage-dependent K4 channels.

channels. The naturally occurring K4 channel diversity is due primarily to the multitude of genes encoding proteins having K4 channel function. K4 channels may be classified according to their gating trigger. channels are primarily but not exclusively Voltage-dependent

gated by changes in transmembrane potential. This category includes delayed rectifier K channels, transient or A K4 channels and inward rectifier K4 channels. Another major class includes ligand-gated K channels which may be Ca24-activated,

G-protein gated, ATP- and Na4-gated K4 channels. Again, however, gating may not be limited to ligands and membrane potential may be an important co-factor. Delayed rectifiers (DRs) are characterized by their slow activation relative to Na4 current and by their resistance to inactivation. The channels are opened by membrane depolarization and, by allowing the outward flow of

K4 ions, they repolarize the membrane. Similarly, transient K channels are also activated by membrane depolarization, but their activation time course is faster than delayed rectifiers. Further-

more, despite the persistence of depolarization, these channels enter into a prolonged closed state, the inactivated state, from which they can recover only by membrane repolarization. Inward

rectifiers (IRs), on the other hand, pass more current in the

inward than in the outward direction, due to a voltage-dependent opening by hyperpolarization and a strong block of the outward Molecular architecture of voltage-dependent K channels currents by intracellular divalent cations, particularly Mg24, ocK4 channels are the most widely distributed and heterogenous curring at depolarized potentials. Inwardly rectifying (IR) K4 class of ion channels. They are found in every eukaryotic cell, and channels serve a dual purpose: (1) to maintain the resting are implicated in a variety of functions from the classical role in membrane potential close to the equilibrium potential for K4 the action potential repolarization, to resting potential stabiliza- ions, and (2) to allow the long duration of depolarizing plateau tion and interspike interval duration during bursting activity in phases such as those occurring during the cardiac action potential. excitable cells, to volume regulation in exocrine cells, glucose The first K4 channel gene to be identified was cloned from the regulation of insulin release in endocrine pancreatic 13 cells, and Shaker locus of Drosophila melanogaster [7, 8]. Flies carrying fast block of polyspermy in oocytes [5, 6]. This heterogeneity of mutations at this locus displayed altered behavioral responses cellular functions is accomplished through the expression of K4 during ether anesthesia, resulting from a K4 current abnormality. channels exhibiting different biophysical properties, such as selecThe Shaker locus was cloned by chromosome walking and was tivity, activation threshold, voltage-dependence of activation, shown to give rise to different protein products. Alternative time-course of inactivation and pharmacological properties, such splicing was found as an additional mechanism generating K4 as blockade by drugs and toxins. Cloning of the genes encoding K channels from various cell types and species has confirmed channel diversity [9—12]. When expressed in Xenopus oocytes, Shaker produced K4 channels with the hallmarks of A-currents, that they belong to a superfamily of genes including voltage- namely fast activation and inactivation, high sensitivity to 4-AP and low affinity for external TEA. Using hybridization techniques with probes derived from Shaker clones, a large number of K4 channel genes, encoding either transient- or delayed-rectifier-like © 1995 by the International Society of Nephrology

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Taglialatela and Brown: K channel structure-function

currents, have been identified in various species, including humans. These K channel clones, despite large functional differences among their protein products, display a remarkably similar pattern in their predicted amino acid sequence. They encode for proteins of 700 to 900 amino acids having homology with each of the four domains present in voltage-dependent Na [1] and Ca2 [13] channels. Hydropathy analysis has identified the existence of

919

segment or a portion of it [19] moves upward, positively charged residues form new ion pairs with negative charges in neighboring

transmembrane segments [25], and the S4-S5 move laterally opening the permeation pathway or pore. Mutations of charged residues in S4 has confirmed that they are involved in voltagedependent activation of K [26—28], and Na channels [29, 30] but the relationship between net charge of the S4 segment and voltage sensitivity is not straightforward. Moreover, non-charged

a core region whose topology is modelled with six putative residues such as nearby leucine have also been implicated, transmembrane a-helical segments (S1 to S6) flanked by the Nand C-terminal regions. Due to covalent linkage of the domains in the a-subunits of voltage-gated Na and Ca2 channels and to the even number of predicted a-helical transmembrane segments, most of the proposed models [14] put the N- and C-termini on the same side of the membrane [15]; furthermore, since all voltagedependent ion channels lack an amino terminal leader sequence, the N-termini are believed to be on the cytoplasmic side of the membrane. Only minK, a cloned voltage-dependent very slowlyactivating K channel expressed in kidney, smooth muscle and cardiac muscle, seems to have a different transmembrane topol-

possibly by forming a leucine zipper [31, 32]. Movement of charged elements within the electric field generates a displacement or gating current which has been measured from cloned channels expressed in Xenopus oocytes [32—35]. Detailed functional models of channel activation, combining the results of gating and ionic current measurements, reveal that the movement of the voltage-sensor is coupled to channel opening in a complex manner and that during voltage-dependent activation the channel occupies many distinct closed states, each carrying a

ogy. The predicted sequence consists of 130 amino acids with only one predicted transmembrane domain [16, 17].

electronic charges. Inward rectification in JR K channels derives from an ability

Sequence comparison among different K channel sequences reveals that extensive homologies exist within the core region, while the degree of homology declines dramatically outside this region. In particular, a short amino acid stretch located in the linker region between transmembrane domains S5 and S(,, called H5 [18] or SS1-SS2 [19], has the highest degree of homology among K channels, suggesting its crucial functional importance. Recently, the genes encoding three JR K channels, IRK1 [20],

discrete amount of charge [36]. The gating of single channel proteins has been suggested to involve the movement of 8 to 16 to conduct current preferentially in the inward direction. This characteristic has two components, voltage-dependent blockade by intracellular Mg2 and intrinsic voltage-dependent gating [371. The intrinsic gating is reflected by a tendency of JR channels to close upon depolarization and to reopen upon subsequent hyper-

polarization, in contrast to voltage-dependent DR K channels which are opened by depolarization. Inward rectifiers lack S4-like sequences and major gating function has been recently attributed

to a negatively-charged aspartate residue located in the second potential membrane-spanning domains M2 [38, 39]. In fact, a respectively, and their protein products expressed in Xenopus single aspartate to asparagine exchange in M2 in JRK1 produced oocytes. The deduced amino acid sequence of these cloned ROMK1-like gating while the reverse mutation in ROMK1, channels reveals the presence of only two potential membrane- produced JRK1-like gating. The aspartic acid at this position is spanning domains (M1 and M2), in contrast to the six transmem- conserved in two other IR K channels having prominent gating, brane domains postulated in depolarization-activated K chan- GIRK1 and hIRK, an IR recently cloned from human atrium [40]. nels. Interestingly, these two transmembrane domains are highly Inactivation homologous to the S5 and 6 transmembrane domains of voltageIn both transient K and Na channels, the inactivation gated delayed rectifier channels, respectively. Furthermore, the process has no intrinsic voltage-dependence, suggesting that its M1-M2 linker region shares strong homologies to the H5 region of DR-like channels, while the two cytoplasmic tails containing the structural determinants are not located in the transmembrane N- and C-termini have the lowest degree of predicted sequence regions. Furthermore, inactivation was removed by perfusing the homology, not only among IRs, but also among all cloned K internal side of the membrane with proteolytic enzymes [41], channels. Although they bear a high degree of amino acid suggestive of a "ball and chain" mechanism. The "ball" has been sequence homology to each other, these JR channels show now convincingly localized by mutational analysis to a 20 amino ROMK1 [21] and GIRK1 [22, 23] have been cloned from a mouse

macrophage cell line, rat kidney cells and rat atrial tissue,

marked differences in their gating and permeation properties [24].

acid region of the N-terminus in K channels [42]. In Na

Localizing channel function to specific domains

channels the mechanism has been localized to a highly conserved linker region between the III and the IV domains which would act

Channel activation

When the genes encoding depolarization-activated DRs and transient K channels were cloned, one striking feature of the predicted amino acid sequence was immediately recognized: the presence in the fourth putative transmembrane segment (S4) of a

like a "hinged lid" rather than a "ball and chain" [43, 44]. Interestingly, mutations in this region in human skeletal muscle

Na channels are responsible for some types of paramyotonia congenita [451, a disease associated with altered Na channel inactivation. In K channels the receptor site for this inactivating

particle has been mapped to the S4-S5 linker region [46], which would therefore contribute to the formation of the inner antethird position, spaced mostly by hydrophobic residues. This chamber of the pore. It is important to note that other channel arrangement has led to the hypothesis that the charged residues in regions have also been implicated in K channel inactivation and the S4 segment formed the sensor of voltage-dependent ion recovery from inactivation, namely the H5 region [71 and the 6 highly repetitive pattern of four to eight basic amino acids at every

channels. It is thought that upon membrane depolarization, the S4

transmembrane domain [48].

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Taglialatela and Brown: K channel structure-function

sequences involved in the tetramerization process have been localized by protein binding techniques [75] and by deletion The use of pharmacological probes has been of invaluable help Pore properties

in the definition of the molecular architecture of the pore of Na and K channels. In fact, point mutations in the linker of

analysis [76] to the cytoplasmic N-terminal region. The specificity of this sequence, which is a sequence having the highest degree of

outside S through 6 in F channels, allows heteroNa and K1 channels have identified important residues for homology merization only among members of the same subclass (Shaker,

blockade by tetrodotoxin (TTX) [49] and charybdotoxin (CTX) Shab, Shaw and Shal). It is important to emphasize that hetero[50], respectively. Mutations affecting internal [51] and external multimcric channel assembly has been very recently demonstrated [52] blockade by tetraethylammonium (TEA), a monovalent to contribute to F channel heterogeneity in vivo [77, 78]. cation which acts as a well known F channel blocker have also

been identified in the 5-6 linker. The molecular basis for F

The subunit composition of IR F channels is unknown.

However, the single channel properties of Mg2 [79] and Cs and selectivity over NH4 [53] and Rb [54] has been localized to the Ba2 [80] block of this channel have revealed the existence of two same region. Large scale mutagenesis with a chimeric construct was used to probe the differences in TEA blockade and single substates having an amplitude about 1/3 and 2/3 of the amplitude of the fully open state. This evidence has been interpreted as a channel conductance between DRK1 and NGK2, two deconsequence of the existence of three independent but highly layed-rectifier channels. The larger single channel conductance, coordinated pathways for F ions. low affinity to internal TEA and high affinity for external TEA of NGK2 were transplanted in DRK1 by an exchange of a 21-amino Conclusions acid segment in H5 derived from the corresponding region of NGK2 [55]. Due to the physical distinction existing between the A molecular approach to ion channel function is now applicainternal and external TEA binding sites and the short length of ble, due to the combined use of site-directed or large-scale the chimeric region, it was suggested that the H5 segment formed mutagenesis and detailed electrophysiological investigation. By a n-hairpin which spanned about 75% of the pore. These exper- this means, it has been possible to localize the structural motifs iments clearly indicated that the S5-S6 linker formed a major part underlying voltage-dependent F channel activation (S4), inactiof the F channels pore. Similar conclusions have been reached vation ball and receptor (N-terminus and S4-S5 linker region, with mutagenesis experiments performed in voltage-gated Na respectively), pore-lining regions (mostly H5, but also the S4-S5 [49, 56, 57] and Ca2 channels [58, 59], and cyclic nucleotide- linker and S and post-S6 sequences), and subunit assembly gated channels [60]. (N-terminus). Despite this impressive progress, information deIn the DRK/NGK chimeric channel two interacting non-polar rived from direct structural observation of the ion channel protein residues at position 369 and 374 were found to be major deter- is essential, since remote effects on the protein structure conseminants for ion permeation and blockade [61]. Their position quent to genetic manipulations are always possible. The availabilrelative to the membrane plane has been investigated by residues ity of structural data would place the interpretation of the having different side chains and probing the substituted channels mutagenesis analysis on firmer grounds and would allow the with monovalent and divalent cations [47, 54]. A histidine substi- rational design of drugs targeting selected ion channel proteins. tuted at position 369 was accessible to Zn2 and protons from the extraccllular surface only [62] establishing the fractional locus of Acknowledgments this residue. These tools, in addition to the recently developed We thank all of our laboratory colleagues. This work was supported in hydrophilic, cysteine-specific reagents, will help to clarify their part by National Institutes of Health Grants HL37044 and HL36930 to location in the pore and the orientation of their side chains. AM. Brown. More recently, several lines of evidences have contributed to

the hypothesis that H5 is not the sole determinant of pore properties in voltage-dependent F channels. In fact, the S4-S5 linker region [63],

[64—66] and C-terminus sequences past-S6

[65, 66], have also been demonstrated to affect ion permeation and blockade, reinforcing the idea that the pore of voltage-gated F channels is a polysegmental mosaic structure. Furthermore, in IR F channels, the high affinity blocking site for Mg2 characteristic of these channels does not appear to be specified by the H5

region, but rather by M2 and C-terminus sequences past transmembrane segment M2 [67]. Assembly

The similarity between the predicted F channel protein and each of the four domains found in Na and Ca2 channels has led to the hypothesis that voltage-dependent F channels assemble as tetramers. The heteromultimeric nature of functional F channel has been demonstrated by means of coinjection studies of channel subunits having different biophysical and pharmacological properties [68—71]. Experimental support for the tetramer hypothesis has come from multimeric cDNAs construction [72—74]. The

Reprint requests to Arthur M. Brown, M.D., Ph.D., Rammelkamp Center, MetroHealth Campus, Case Western Reserve University, 2500 MetroHealth Drive, Cleveland, Ohio 44109, USA.

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