Hélène Klein

A Specific Tryptophan in the I-II Linker Is a Key Determinant of β-Subunit Binding and Modulation in CaV2.3 Calcium Channels

The ancillary beta subunits modulate the activation and inactivation properties of high-voltage activated (HVA) Ca(2+) channels in an isoform-specific manner. The beta subunits bind to a high-affinity interaction site, alpha-interaction domain (AID), located in the I-II linker of HVA alpha1 subunits. Nine residues in the AID motif are absolutely conserved in all HVA channels (QQxExxLxGYxxWIxxxE), but their contribution to beta-subunit binding and modulation remains to be established in Ca(V)2.3. Mutations of W386 to either A, G, Q, R, E, F, or Y in Ca(V)2.3 disrupted [(35)S]beta3-subunit overlay binding to glutathione S-transferase fusion proteins containing the mutated I-II linker, whereas mutations (single or multiple) of nonconserved residues did not affect the protein-protein interaction with beta3. The tryptophan residue at position 386 appears to be an essential determinant as substitutions with hydrophobic (A and G), hydrophilic (Q, R, and E), or aromatic (F and Y) residues yielded the same results. beta-Subunit modulation of W386 (A, G, Q, R, E, F, and Y) and Y383 (A and S) mutants was investigated after heterologous expression in Xenopus oocytes. All mutant channels expressed large inward Ba(2+) currents with typical current-voltage properties. Nonetheless, the typical hallmarks of beta-subunit modulation, namely the increase in peak currents, the hyperpolarization of peak voltages, and the modulation of the kinetics and voltage dependence of inactivation, were eliminated in all W386 mutants, although they were preserved in part in Y383 (A and S) mutants. Altogether these results suggest that W386 is critical for beta-subunit binding and modulation of HVA Ca(2+) channels.

Cysteine Mutagenesis and Computer Modeling of the S6 Region of an Intermediate Conductance IKCa Channel

Cysteine-scanning mutagenesis (SCAM) and computer-based modeling were used to investigate key structural features of the S6 transmembrane segment of the calcium-activated K+ channel of intermediate conductance IKCa. Our SCAM results show that the interaction of [2-(trimethylammonium)ethyl] methanethiosulfonate bromide (MTSET) with cysteines engineered at positions 275, 278, and 282 leads to current inhibition. This effect was state dependent as MTSET appeared less effective at inhibiting IKCa in the closed (zero Ca2+ conditions) than open state configuration. Our results also indicate that the last four residues in S6, from A283 to A286, are entirely exposed to water in open IKCa channels, whereas MTSET can still reach the 283C and 286C residues with IKCa maintained in a closed state configuration. Notably, the internal application of MTSET or sodium (2-sulfonatoethyl) methanethiosulfonate (MTSES) caused a strong Ca2+-dependent stimulation of the A283C, V285C, and A286C currents. However, in contrast to the wild-type IKCa, the MTSET-stimulated A283C and A286C currents appeared to be TEA insensitive, indicating that the MTSET binding at positions 283 and 286 impaired the access of TEA to the channel pore. Three-dimensional structural data were next generated through homology modeling using the KcsA structure as template. In accordance with the SCAM results, the three-dimensional models predict that the V275, T278, and V282 residues should be lining the channel pore. However, the pore dimensions derived for the A283–A286 region cannot account for the MTSET effect on the closed A283C and A286 mutants. Our results suggest that the S6 domain extending from V275 to V282 possesses features corresponding to the inner cavity region of KcsA, and that the COOH terminus end of S6, from A283 to A286, is more flexible than predicted on the basis of the closed KcsA crystallographic structure alone. According to this model, closure by the gate should occur at a point located between the T278 and V282 residues.

Hydrophobic Interactions as Key Determinants to the KCa3.1 Channel Closed Configuration AN ANALYSIS OF KCa3.1 MUTANTS CONSTITUTIVELY ACTIVE IN ZERO Ca2

In this study we present evidence that residue Val282 in the S6 transmembrane segment of the calcium-activated KCa3.1 channel constitutes a key determinant of channel gating. A Gly scan of the S6 transmembrane segment first revealed that the substitutions A279G and V282G cause the channel to become constitutively active in zero Ca2+. Constitutive activity was not observed when residues extending from Cys276 to Ala286, other than Ala279 and Val282, were substituted to Gly. The accessibility of Cys engineered at Val275 deep in the channel cavity was next investigated for the ion-conducting V275C/V282G mutant and closed V275C channel in zero Ca2+ using Ag+ as probe. These experiments demonstrated that internal Ag+ ions have free access to the channel cavity independently of the channel conducting state, arguing against an activation gate located at the S6 segment C-terminal end. Experiments were also conducted where Val282 was substituted by residues differing in size and/or hydrophobicity. We found a strong correlation between constitutive activity in zero Ca2+ and the hydrophobic energy for side chain burial. Single channel recordings showed finally that constitutive activation in zero Ca2+ is better explained by a model where the channel is locked in a low conducting state with a high open probability rather than resulting from a change in the open/closed energy balance that would favor channel openings to a full conducting state in the absence of Ca2+. We conclude that hydrophobic interactions involving Val282 constitute key determinants to KCa3.1 gating by modulating the ion conducting state of the selectivity filter through an effect on the S6 transmembrane segment.

Molecular determinants of the CaVβ-induced plasma membrane targeting of the CaV1.2 channel

CaVβ subunits modulate cell surface expression and voltage-dependent gating of high voltage-activated (HVA) CaV1 and CaV2 α1 subunits. High affinity CaVβ binding onto the so-called α interaction domain of the I-II linker of the CaVα1 subunit is required for CaVβ modulation of HVA channel gating. It has been suggested, however, that CaVβ-mediated plasma membrane targeting could be uncoupled from CaVβ-mediated modulation of channel gating. In addition to CaVβ, CaVα2δ and calmodulin have been proposed to play important roles in HVA channel targeting. Indeed we show that co-expression of CaVα2δ caused a 5-fold stimulation of the whole cell currents measured with CaV1.2 and CaVβ3. To gauge the synergetic role of auxiliary subunits in the steady-state plasma membrane expression of CaV1.2, extracellularly tagged CaV1.2 proteins were quantified using fluorescence-activated cell sorting analysis. Co-expression of CaV1.2 with either CaVα2δ, calmodulin wild type, or apocalmodulin (alone or in combination) failed to promote the detection of fluorescently labeled CaV1.2 subunits. In contrast, co-expression with CaVβ3 stimulated plasma membrane expression of CaV1.2 by a 10-fold factor. Mutations within the α interaction domain of CaV1.2 or within the nucleotide kinase domain of CaVβ3 disrupted the CaVβ3-induced plasma membrane targeting of CaV1.2. Altogether, these data support a model where high affinity binding of CaVβ to the I-II linker of CaVα1 largely accounts for CaVβ-induced plasma membrane targeting of CaV1.2.

Contribution of Cytosolic Cysteine Residues to the Gating Properties of the Kir2.1 Inward Rectifier

The topological model proposed for the Kir2.1 inward rectifier predicts that seven of the channel 13 cysteine residues are distributed along the N- and C-terminus regions, with some of the residues comprised within highly conserved domains involved in channel gating. To determine if cytosolic cysteine residues contribute to the gating properties of Kir2.1, each of the N- and C-terminus cysteines was mutated into either a polar (S, D, N), an aliphatic (A,V, L), or an aromatic (W) residue. Our patch-clamp measurements show that with the exception of C76 and C311, the mutation of individual cytosolic cysteine to serine (S) did not significantly affect the single-channel conductance nor the channel open probability. However, mutating C76 to a charged or polar residue resulted either in an absence of channel activity or a decrease in open probability. In turn, the mutations C311S (polar), C311R (charged), and to a lesser degree C311A (aliphatic) led to an increase of the channel mean closed time due to the appearance of long closed time intervals (T(c) >or= 500 ms) and to a reduction of the reactivation by ATP of rundown Kir2.1 channels. These changes could be correlated with a weakening of the interaction between Kir2.1 and PIP(2), with C311R and C311S being more potent at modulating the Kir2.1-PIP(2) interaction than C311A. The present work supports, therefore, molecular models whereby the gating properties of Kir2.1 depend on the presence of nonpolar or neutral residues at positions 76 and 311, with C311 modulating the interaction between Kir2.1 and PIP(2).

Contribution of the KCa3.1 channel-calmodulin interactions to the regulation of the KCa3.1 gating process.

The Ca2+-activated potassium channel of intermediate conductance, KCa3.1, is now emerging as a therapeutic target for a large variety of health disorders. The Ca2+ sensitivity of KCa3.1 is conferred by the Ca2+-binding protein calmodulin (CaM), with the CaM C-lobe constitutively bound to an intracellular domain of the channel C terminus. It was proposed on the basis of the crystal structure obtained for the C-terminal region of the rat KCa2.2 channel (rSK2) with CaM that the binding of Ca2+ to the CaM N-lobe results in CaM interlocking the C-terminal regions of two adjacent KCa3.1 subunits, leading to the formation of a dimeric structure. A study was thus undertaken to identify residues of the CaM N-lobe–KCa3.1 complex that either contribute to the channel activation process or control the channel open probability at saturating Ca2+ (Pomax). A structural homology model of the KCa3.1–CaM complex was first generated using as template the crystal structure of the C-terminal region of the rat KCa2.2 channel with CaM. This model was confirmed by cross-bridging residues R362 of KCa3.1 and K75 of CaM. Patch-clamp experiments were next performed, demonstrating that the solvation energy of the residue at position 367 in KCa3.1 is a key determinant to the channel Pomax and deactivation time toff. Mutations of residues M368 and Q364 predicted to form anchoring points for CaM binding to KCa3.1 had little impact on either toff or Pomax. Finally, our results show that channel activation depends on electrostatic interactions involving the charged residues R362 and E363, added to a nonpolar energy contribution coming from M368. We conclude that electrostatic interactions involving residues R362 and E363 and hydrophobic effects at M368 play a prominent role in KCa3.1 activation, whereas hydrophobic interactions at S367 are determinant to the stability of the CaM–KCa3.1 complex throughout gating.

pH and external Ca(2+) regulation of a small conductance Cl(-) channel in kidney distal tubule.

A single channel characterization of the Cl(-) channels in distal nephron was undertaken using vesicles prepared from plasma membranes of isolated rabbit distal tubules. The presence in this vesicle preparation of ClC-K type Cl(-) channels was first established by immunodetection using an antibody raised against ClC-K isoforms. A ClC-K1 based functional characterization was next performed by investigating the pH and external Ca(2+) regulation of a small conductance Cl(-) channel which we identified previously by channel incorporation experiments. Acidification of the cis (external) solution from pH 7.4 to 6.5 led to a dose-dependent inhibition of the channel open probability P(O). Similarly, changing the trans pH from 7.4 to 6.8 resulted in a 4-fold decrease of the channel P(O) with no effect on the channel conductance. Channel activity also appeared to be regulated by cis (external) Ca(2+) concentration, with a dose-dependent increase in channel activity as a function of the cis Ca(2+) concentration. It is concluded on the basis of these results that the small conductance Cl(-) channel present in rabbit distal tubules is functionally equivalent to the ClC-K1 channel in the rat. In addition, the present work constitutes the first single channel evidence for a chloride channel regulated by external Ca(2+).

Aromatic–aromatic interactions between residues in KCa3.1 pore helix and S5 transmembrane segment control the channel gating process

Interactions between aromatic amino acid residues in the pore helix and S5 transmembrane domain control gating of the Ca2+-activated potassium channel KCa3.1.

MOLECULAR CHARACTERIZATION OF AN INWARDLY RECTIFYING K+ CHANNEL FROM HELA CELLS

Abstract. Previous patch-clamp studies have shown that the potassium permeability of the plasma membrane in HeLa cells, a cell line derived from an epidermoid carcinoma of the cervix, is controlled by various K+-selective pores including an IRK1 type inwardly rectifying K+ channel. We used the sequence previously reported for the human heart Kir2.1 channel to design a RT-PCR strategy for cloning the IRK1 channel in HeLa cells. A full-length clone of 1.3 kb was obtained that was identical to the human cardiac Kir2.1 inward rectifier. The nature of the cloned channel was also confirmed in a Northern blot analysis where a signal of 5.3 kb corresponding to the molecular weight expected for a Kir2.1 channel transcript was identified not only in HeLa cells, but also in WI-38, ECV304 and bovine aortic endothelial cells. The HeLa IRK1 channel cDNA was subcloned in an expression vector (pMT21) and injected into Xenopus oocytes. Cell-attached and inside-out single channel recordings obtained from injected oocytes provided evidence for a voltage-independent K+-selective channel with current/voltage characteristics typical of a strong inward rectifier. The single channel conductance for inward currents measured in 200 mm K2SO4 conditions was estimated at 40 ± 1 pS (n= 3), for applied voltages ranging from −100 to −160 mV, in agreement with the unitary conductance for the IRK1 channel identified in HeLa cells. In addition, the single channel conductance for inward currents, Γ, was found to vary as a function of αK, the external K+ ion activity, according to Γ=Γ0 [αK]δ with Γ0= 3.3 pS and δ= 0.5. Single channel recordings from injected oocytes also provided evidence of a voltage-dependent block by external Cs+ and Ba2+. The presence of 500 μm Cs+ caused a voltage-dependent flickering, typical of a fast channel blocking process which resulted in a reduction of the channel open probability at increasingly negative membrane potential values. The fractional electrical distance computed for the Cs+ blocking site was greater than 1 indicating a multiple ion channel occupation. In contrast, external Ba2+ at concentrations ranging from 25 to 100 μm caused a slow channel block, consistent with the binding of a single Ba2+ ion at a site located at half the membrane span. It is concluded on the basis of these observations that HeLa cells expressed a Kir2.1 type inwardly rectifying channel likely to be involved in maintaining and regulating the cell resting potential.

New insights on the voltage dependence of the KCa3.1 channel block by internal TBA.

We present in this work a structural model of the open IKCa (KCa3.1) channel derived by homology modeling from the MthK channel structure, and used this model to compute the transmembrane potential profile along the channel pore. This analysis showed that the selectivity filter and the region extending from the channel inner cavity to the internal medium should respectively account for 81% and 16% of the transmembrane potential difference. We found however that the voltage dependence of the IKCa block by the quaternary ammonium ion TBA applied internally is compatible with an apparent electrical distance δ of 0.49 ± 0.02 (n = 6) for negative potentials. To reconcile this observation with the electrostatic potential profile predicted for the channel pore, we modeled the IKCa block by TBA assuming that the voltage dependence of the block is governed by both the difference in potential between the channel cavity and the internal medium, and the potential profile along the selectivity filter region through an effect on the filter ion occupancy states. The resulting model predicts that δ should be voltage dependent, being larger at negative than positive potentials. The model also indicates that raising the internal K+ concentration should decrease the value of δ measured at negative potentials independently of the external K+ concentration, whereas raising the external K+ concentration should minimally affect δ for concentrations >50 mM. All these predictions are born out by our current experimental results. Finally, we found that the substitutions V275C and V275A increased the voltage sensitivity of the TBA block, suggesting that TBA could move further into the pore, thus leading to stronger interactions between TBA and the ions in the selectivity filter. Globally, these results support a model whereby the voltage dependence of the TBA block in IKCa is mainly governed by the voltage dependence of the ion occupancy states of the selectivity filter.