Regulation of K+ Flow by a Ring of Negative Charges in the Outer Pore of BKCa Channels. Part I

Self Cite

oltage-and Ca 2ϩ -activated K ϩ channels (BK Ca ) are membrane proteins that play a fundamental role in controlling smooth muscle tone and neuronal excitability. In most of the tissues, they form a complex consisting of a pore-forming ␣ subunit and regulatory ␤ subunits. The ␣ subunit encodes for the selective pore as well as for the voltage and Ca 2ϩ -sensing structures. The BK Ca channel is a tetramer with each ␣ subunit organized in seven transmembrane domains (S0-S6) (1), a long intracellular C-terminal domain where a high-affinity Ca 2ϩ -binding site has been identified (2, 3), and an extracellular N terminus. The human isoform (hSlo), similarly to other voltagedependent ion channels, possesses a voltage sensor that is mainly represented by the S4 transmembrane domain, containing three positively charged residues (4, 5). Changes in membrane potential displace the voltage sensor and, for adequate depolarizations, the consequent conformational change sets the channel in a conducting state. The movement of the voltage sensor produces a transient current (gating current) that precedes in time and voltage the ionic current activation (6, 7). Thus, gating currents report on the rearrangement of the channel structure in a varying membrane potential but do not provide direct information regarding the motion of regions of the channel outside the voltage field. Structural changes occurring during gating have been elegantly resolved by using site-directed fluorescent labeling, a technique pioneered in the E. Isacoff laboratory (8) and applied to a variety of voltage-gated K ϩ and Na ϩ channels (9-24), ligand-gated channels (25,26), and transporters (27-31). However, nothing is known regarding the dynamical changes of BK Ca channel during gating. Using site-directed fluorescence labeling combined with the cut-open oocyte voltage clamp technique (COVG), we have resolved the conformational changes occurring in hSlo voltage-sensing region, unraveling extremely slow conformational changes not expected from gating current measurements. We have used thiol-reactive fluorescent probes [tetramethyl rhodamine-5-maleimide (TMRM) or 1-(2-maleimidylethyl)-4-(5-(4-methoxyphenyl) oxazol-2-yl)pyridinium methansulfonate (PyMPO)] to assess the dynamics of the S4 region conformational changes in BK Ca channels. ResultsBK Ca channels possess a functional voltage sensor as demonstrated by direct measurement of ionic and gating currents in the absence of internal Ca 2ϩ (6,7,32,33). In addition, Diaz et al. (4) have shown that mutations in the S4 segment alter the voltage dependence of hSlo channel activation. If the S4 transmembrane segment of BK Ca channels is part of the voltage-sensing machinery, conformational changes of the S4 region should share some of the features of channel-gating currents. In this study, we have investigated conformational changes of the region between the S3 and S4 transmembrane segments in the hSlo channel. The region of interest and the residues fluorescently labeled in this study are illustrated in a schematic...

Section: Methodsmentioning

confidence: 99%

Voltage-dependent conformational changes in human Ca ²⁺ - and voltage-activated K ⁺ channel, revealed by voltage-clamp fluorometry

Savalli¹,

Kondratiev²,

Toro

et al. 2006

Self Cite

“…D487 and D489, however, face the central axis and are directly accessible to the water-filled center of the tunnel. Negative charge rings are a common feature at the mouths of cation channels, where they help to focus the permeant cations and likely act as a ''prefilter'' by repelling intracellular anions from the immediate vicinity of the pore (23)(24)(25)(26)(27)(28)(29)(30). Despite this architecture, it is possible that permeant ions leave the channel before reaching this tunnel (Fig.…”

mentioning

confidence: 99%

The carboxyl-terminal region of cyclic nucleotide-modulated channels is a gating ring, not a permeation path

Johnson

Zagotta

2005

The recent elucidation of the structure of the carboxyl-terminal region of the hyperpolarization-activated cyclic nucleotide-modulated (HCN2) channel has prompted us to investigate a curious feature of this structure in HCN2 channels and in the related CNGA1 cyclic nucleotide-gated (CNG) channels. The crystallized fragment of the HCN2 channel contains both the cyclic nucleotide-binding domain (CNBD) and the C-linker region, which connects the CNBD to the pore. At the center of the fourfold-symmetric structure is a tunnel that runs perpendicular to the membrane. The narrowest part of the tunnel is Ϸ10 Å in diameter and is lined by a ring of negatively charged amino acids: D487, E488, and D489. Many ion channels have ''charge rings'' that focus permeant ions at the mouth of the pore and increase channel conductance. We used nonstationary fluctuation analysis and single-channel recording, coupled with site-directed mutagenesis and cysteine modification, to determine whether this part of HCN and CNG channels might be an extension of the permeation pathway. Our results indicate that modifying charge-ring amino acids affects gating but not ion permeation in HCN2 and CNG channels. Thus, this portion of the channel is not an obligatory part of the ion path but instead acts as a ''gating ring.'' The carboxyl-terminal region of these channels must hang below the pore much like the ''hanging gondola'' of voltage-gated potassium channels, but the permeation pathway must exit the protein before the level of the ring of charged amino acids.charge ring ͉ fluctuation analysis ͉ single-channel patch clamp ͉ cyclic nucleotide-gated ͉ hyperpolarization-activated cyclic nucleotide-modulated

“…1B), and they have been shown to have an equally large impact on the channel conduction properties (14,15,17,18). It is not surprising then that singlecharge mutations should also affect the K ϩ selectivity of a channel ( Fig.…”

Section: Discussionmentioning

confidence: 99%

“…Negatively charged residues at the extracellular mouth (14), water-filled cavity (15,16), or the cytoplasmic pore (17) increase the rate of K ϩ conduction in potassium channels. On the other hand, positively charged residues at sites distant from the selectivity filter in the voltage-gated CLC-0 chloride channels increase chloride conductance (18).…”

mentioning

confidence: 99%

K ⁺ channel selectivity depends on kinetic as well as thermodynamic factors

Grabe

Bichet

Xiang

et al. 2006

Potassium channels are necessary for a number of essential biological tasks such as the generation of action potentials and setting the resting membrane potential in cells, both of which require that these channels selectively permit the passage of potassium ions while suppressing the flow of other ions. Generally, this selectivity is attributed to a narrow stretch of the channel known as the selectivity filter. Over this stretch ions are dehydrated, and the backbone oxygen atoms of the protein mimic the ion's loss of coordination by water. However, channels are long pores with spatially distinct ion-binding sites that all must be traversed during ion permeation. We have shown that selectivity of mutant Kir3.2 (GIRK2) channels can be substantially amplified by introducing acidic residues into the cavity, a binding site below the selectivity filter. Here, we carry out electrostatic calculations on homology models to quantify the degree of stabilization that these mutations have on ions in the cavity. We then construct a multiion model of ion permeation to calculate the channel's permeability to potassium relative to sodium. This kinetic model uses rates derived from the electrostatic calculations and demonstrates that nonselective electrostatic stabilization of cations in the cavity can amplify channel selectivity independently of the selectivity filter. This nonintuitive result highlights the dependence of channel properties on the entire channel architecture and suggests that selectivity may not be fully understood by focusing solely on thermodynamic considerations of ion dehydration and the energetics of the selectivity filter.electrostatics ͉ model ͉ potassium channel ͉ simulation ͉ ion permeation P otassium (K ϩ ) channels permit millions of potassium ions to cross the membrane per second, while suppressing by 1,000-fold the concomitant flux of the smaller sodium ions (1). How might such remarkable selectivity be achieved given that the potassium ion diameter is only 0.4 Å larger than the sodium ion diameter, yet thermal fluctuations in the protein are on the order of angstroms? Potassium channels have a characteristic GYG amino acid sequence, and the structure of the KcsA K ϩ channel reveals that the backbone carbonyl oxygen atoms of these residues coordinate K ϩ ions in the filter (2). It has been suggested that a better approximation of the hydration shell of K ϩ compared with other ions underlies the basis of selectivity (2-4). Indeed, simulations predict that the filter sites are several kilocalories per mole more selective for K ϩ (5-7). Nonetheless, x-ray structures show that the selectivity filter binding sites are just one of several distinct positions that must be occupied as ions transit the channel. There is a water-filled cavity below the filter that ions occupy (2, 4) and charged rings in the cytoplasmic domains of inwardly rectifying potassium (Kir) channels that are likely to be additional ion-binding sites (8-10). The presence of these sequential binding sites is important for physiological ...