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Tuned calcium entry through voltage-gated calcium channels is a key requirement for many cellular functions. This is ensured by channel gates which open during membrane depolarizations and seal the pore at rest. The gating process is determined by distinct sub-processes: movement of voltage-sensing domains (charged S4 segments) as well as opening and closure of S6 gates. Neutralization of S4 charges revealed that pore opening of CaV1.2 is triggered by a “gate releasing” movement of all four S4 segments with activation of IS4 (and IIIS4) being a rate-limiting stage. Segment IS4 additionally plays a crucial role in channel inactivation. Remarkably, S4 segments carrying only a single charged residue efficiently participate in gating. However, the complete set of S4 charges is required for stabilization of the open state. Voltage clamp fluorometry, the cryo-EM structure of a mammalian calcium channel, biophysical and pharmacological studies, and mathematical simulations have all contributed to a novel interpretation of the role of voltage sensors in channel opening, closure, and inactivation. We illustrate the role of the different methodologies in gating studies and discuss the key molecular events leading CaV channels to open and to close. Electronic supplementary material The online version of this article (10.1007/s00424-018-2163-7) contains supplementary material, which is available to authorized users.

In order to specify the role of individual S4 segments in CaV1.2 gating, charged residues of segments IS4-IVS4 were replaced by glutamine and the corresponding effects on activation/deactivation of calcium channel currents were analysed. Almost all replacements of charges in IS4 and IIIS4 decreased the slope of the Boltzmann curve of channel activation (activation curve) while charge neutralisations in IIS4 and IVS4 did not significantly affect the slope. S4 mutations caused either left or rightward shifts of the activation curve, and in wild-type channels, these S4 mutations hardly affected current kinetics. In slowly gating pore (S6) mutants (G432W, A780T, G1193T or A1503G), neutralisations in S4 segments significantly accelerated current kinetics. Likewise in wild type, charge replacements in IS4 and IIIS4 of pore mutants reduced the slope of the activation curves while substitutions of charges in IIS4 and IVS4 had less or no impact. We propose a gating model where the structurally different S4 segments leave their resting positions not simultaneously. Upward movement of segments IS4 and (to a lesser extend) IIIS4 appear to be a rate-limiting stage for releasing the pore gates. These segments carry most of the effective charge for channel activation. Our study suggests that S4 segments of CaV1.2 control the closed state in domain specific manner while stabilizing the open state in a non-specific manner.

Inactivation of L-type calcium channel (Cav1.2) is an important determinant of the length of the cardiac action potential. Here, we report a key role of the voltage-sensing segment IS4 in Cav1.2 inactivation. Neutralization of IS4 charges gradually shifted the steady-state inactivation curve on the voltages axis from 5.1 ± 3.7 mV in single point mutant IS4(K1Q) to −26.7 ± 1.3 mV in quadruple mutant IS4(K1Q/R2Q/R3Q/R4Q) compared to wild-type (WT) and accelerated inactivation. The slope factor of the Boltzmann curve of inactivation was decreased from 17.4 ± 3.5 mV (IS4(K1Q)) to 6.2 ± 0.7 mV (IS4(K1Q/R2Q/R3Q/R4Q)). Neutralizations of single or multiple charges in IIS4 and IIIS4 did not significantly affect the time course of inactivation. Neutralization of individual IVS4 charges shifted the inactivation curve between 17.4 ± 1.7 mV (IVS4(R2Q)) and −4.6 ± 1.4 mV (IVS4(R4Q)) on the voltage axis and affected the slope of the inactivation curves (IVS4(R2Q): 10.2 ± 1.2 mV, IVS4(R4Q): 9.7 ± 0.7 mV and IVS4(K5Q): 8.1 ± 0.7 mV vs WT: 14.1 ± 0.8 mV). IS4(K1Q) attenuated while IS4(K1Q/R2Q/R3Q) and IS4(K1Q/R2Q/R4Q/R3Q) enhanced the development of inactivation. Shifts in the voltage dependence of inactivation curves induced by IS4 neutralizations significantly correlated with shifts of the voltage dependence of channel activation (r = 0.95, p

In order to specify the role of individual S4 segments in CaV1.2 gating, charged residues of segments IS4-IVS4 were replaced by glutamine and the corresponding effects on activation/deactivation of calcium channel currents were analysed. Almost all replacements of charges in IS4 and IIIS4 decreased the slope of the Boltzmann curve of channel activation (activation curve) while charge neutralisations in IIS4 and IVS4 did not significantly affect the slope. S4 mutations caused either left or rightward shifts of the activation curve, and in wild-type channels, these S4 mutations hardly affected current kinetics. In slowly gating pore (S6) mutants (G432W, A780T, G1193T or A1503G), neutralisations in S4 segments significantly accelerated current kinetics. Likewise in wild type, charge replacements in IS4 and IIIS4 of pore mutants reduced the slope of the activation curves while substitutions of charges in IIS4 and IVS4 had less or no impact. We propose a gating model where the structurally different S4 segments leave their resting positions not simultaneously. Upward movement of segments IS4 and (to a lesser extend) IIIS4 appear to be a rate-limiting stage for releasing the pore gates. These segments carry most of the effective charge for channel activation. Our study suggests that S4 segments of CaV1.2 control the closed state in domain specific manner while stabilizing the open state in a non-specific manner. Electronic supplementary material The online version of this article (doi:10.1007/s00424-016-1895-5) contains supplementary material, which is available to authorized users.

The Timothy syndrome mutations G402S and G406R abolish inactivation of Ca(V)1.2 and cause multiorgan dysfunction and lethal arrhythmias. To gain insights into the consequences of the G402S mutation on structure and function of the channel, we systematically mutated the corresponding Gly-432 of the rabbit channel and applied homology modeling. All mutations of Gly-432 (G432A/M/N/V/W) diminished channel inactivation. Homology modeling revealed that Gly-432 forms part of a highly conserved structure motif (G/A/G/A) of small residues in homologous positions of all four domains (Gly-432 (IS6), Ala-780 (IIS6), Gly-1193 (IIIS6), Ala-1503 (IVS6)). Corresponding mutations in domains II, III, and IV induced, in contrast, parallel shifts of activation and inactivation curves indicating a preserved coupling between both processes. Disruption between coupling of activation and inactivation was specific for mutations of Gly-432 in domain I. Mutations of Gly-432 removed inactivation irrespective of the changes in activation. In all four domains residues G/A/G/A are in close contact with larger bulky amino acids from neighboring S6 helices. These interactions apparently provide adhesion points, thereby tightly sealing the activation gate of Ca(V)1.2 in the closed state. Such a structural hypothesis is supported by changes in activation gating induced by mutations of the G/A/G/A residues. The structural implications for Ca(V)1.2 activation and inactivation gating are discussed.

Single point mutations in pore-forming S6 segments of calcium channels may transform a high-voltage-activated into a low-voltage-activated channel, and resulting disturbances in calcium entry may cause channelopathies (Hemara-Wahanui et al., Proc Natl Acad Sci U S A 102(21):7553–7558, 16). Here we ask the question how physicochemical properties of amino acid residues in gating-sensitive positions on S6 segments determine the threshold of channel activation of CaV1.2. Leucine in segment IS6 (L434) and a newly identified activation determinant in segment IIIS6 (G1193) were mutated to a variety of amino acids. The induced leftward shifts of the activation curves and decelerated current activation and deactivation suggest a destabilization of the closed and a stabilisation of the open channel state by most mutations. A selection of 17 physicochemical parameters (descriptors) was calculated for these residues and examined for correlation with the shifts of the midpoints of the activation curve (ΔVact). ΔVact correlated with local side-chain flexibility in position L434 (IS6), with the polar accessible surface area of the side chain in position G1193 (IIIS6) and with hydrophobicity in position I781 (IIS6). Combined descriptor analysis for positions I781 and G1193 revealed that additional amino acid properties may contribute to conformational changes during the gating process. The identified physicochemical properties in the analysed gating-sensitive positions (accessible surface area, side-chain flexibility, and hydrophobicity) predict the shifts of the activation curves of CaV1.2. Electronic supplementary material The online version of this article (doi:10.1007/s00424-010-0885-2) contains supplementary material, which is available to authorized users.

Voltage dependence and kinetics of Ca(V)1.2 activation are affected by structural changes in pore-lining S6 segments of the alpha(1)-subunit. Significant effects are induced by either proline or threonine substitutions in the lower third of segment IIS6 ("bundle crossing region"), where S6 segments are likely to seal the channel in the closed conformation (Hohaus, A., Beyl, S., Kudrnac, M., Berjukow, S., Timin, E. N., Marksteiner, R., Maw, M. A., and Hering, S. (2005) J. Biol. Chem. 280, 38471-38477). Here we report that S435P in IS6 results in a large shift of the activation curve (-25.9 +/- 1.2 mV) and slower current kinetics. Threonine substitutions at positions Leu-429 and Leu-434 induced a similar kinetic phenotype with shifted activation curves (L429T by -6.6 +/- 1.2 and L434T by -12.1 +/- 1.7 mV). Inactivation curves of all mutants were shifted to comparable extents as the activation curves. Interdependence of IS6 and IIS6 mutations was analyzed by means of mutant cycle analysis. Double mutations in segments IS6 and IIS6 induce either additive (L429T/I781T, -34.1 +/- 1.4 mV; L434T/I781T, -40.4 +/- 1.3 mV; L429T/L779T, -12.6 +/- 1.3 mV; and L434T/L779T, -22.4 +/- 1.3 mV) or nonadditive shifts of the activation curves along the voltage axis (S435P/I781T, -33.8 +/- 1.4 mV). Mutant cycle analysis revealed energetic coupling between residues Ser-435 and Ile-781, whereas other paired mutations in segments IS6 and IIS6 had independent effects on activation gating.

K(+) channels play a critical role in numerous physiological and pathophysiological processes rendering them an attractive target for therapeutic intervention. However, the hERG K(+) channel poses a special challenge in drug discovery, since block of this channel by a plethora of diverse chemical entities can lead to long QT syndrome and sudden death. Of particular interest is the so-called trapping phenomenon, characterized by capture of a drug behind closed channel gates, which harbors an increased pro-arrhythmic risk. In this study we investigated the influence of trapped blockers on the gating dynamics and probed the state dependence of dissociation in K(+) channels by making use of the quaternary tetrabutylammonium. By applying essential dynamics simulations and two-electrode voltage clamp we obtained detailed insights into the dynamics of trapping in KcsA and hERG. Our simulations suggest that the trapped TBA influences the F656 flexibility during gate closure. Based on these findings, we provide a structural hypothesis for drug trapping. Further our simulations reveal the extent of gate opening necessary for drug dissociation.

Local anaesthetics (LA) exert their action by prolongation of the time course of repriming of voltage-gated Na channels after repolarization of the action potential. This may result from slow drug-dissociation from fast inactivated states or, alternatively, from stabilization of a native slow inactivated state (IM, Chen ZH et al. J Physiol. 524:37). The C-terminal part of transmembrane S6 segment of domain IV (DIV-S6) is an important determinant of inactivation gating and LA block. We performed serial cysteine scanning mutagenesis of positions 1575-1586 in DIV-S6 of rNav1.4 channels and investigated the relationship between changes in mutation-induced alterations of IM and respective changes in LA-induced prolongation of recovery. The constructs were expressed in TsA201 cells and studied by means of whole-cell patch-clamp technique. We examined the time course of recovery from fast and slow inactivation produced by 50 ms and by 10 s conditioning pulses to −20 mV, respectively. The LA Lidocaine (500 µM; LIDO) significantly slowed recovery from fast inactivation in all tested constructs except F1579C. The time constant of LIDO - induced slow recovery was ∼ 150 ms. Under LIDO-free conditions a similar time constant of slow recovery was found most constructs following 10 s depolarization, presumably reflecting recovery from native IM. However, the fraction of channels recovering from IM (F- IM) varied greatly among the tested constructs. LIDO significantly increased F- IM in most constructs. Interestingly, there was a significant negative correlation between F-IM without LIDO and the LIDO-induced increase in F- IM(R2 = 0.91; P < 0.0001). Furthermore, in M1585C F- IM was 0 under LIDO-free conditions but increased to 0.73±0.02 with LIDO. The data suggest that in the tested constructs slow recovery with LIDO reflects slow dissociation from fast inactivation rather than recovery from IM. Funding support: Austrian Science Fund W1232-B11.

The role of channel inactivation in the molecular mechanism of calcium (Ca 2+ ) channel block by phenylalkylamines (PAA) was analyzed by designing mutant Ca 2+ channels that carry the high affinity determinants of the PAA receptor site [Hockerman, G. H., Johnson, B. D., Scheuer, T., and Catterall, W. A. (1995) J. Biol. Chem. 270, 22119–22122] but inactivate at different rates. Use-dependent block by PAAs was studied after expressing the mutant Ca 2+ channels in Xenopus oocytes. Substitution of single putative pore-orientated amino acids in segment IIIS6 by alanine (F-1499-A, F-1500-A, F-1510-A, I-1514-A, and F-1515-A) gradually slowed channel inactivation and simultaneously reduced inhibition of barium currents (I Ba ) by (−)D600 upon depolarization by 100 ms steps at 0.1 Hz. This apparent reduction in drug sensitivity was only evident if test pulses were applied at a low frequency of 0.1 Hz and almost disappeared at the frequency of 1 Hz. (−)D600 slowed I Ba recovery after maintained membrane depolarization (1–3 sec) to a comparable extent in all channel constructs. A drug-induced delay in the onset of I Ba recovery from inactivation suggests that PAAs promote the transition to a deep inactivated channel conformation. These findings indicate that apparent PAA sensitivity of Ca 2+ channels is not only defined by drug interaction with its receptor site but also crucially dependent on intrinsic gating properties of the channel molecule. A molecular model for PAA-Ca 2+ channel interaction that accounts for the relationship between drug induced inactivation and channel block by PAA is proposed.

Membrane depolarisation and upward movement of charged S4 segments initiates the opening of voltage gated calcium channels while repolarisation and S4 downward movement facilitates channel closure. The individual impact of segements IS4-IVS4 in CaV1.2 is largely unknown. CaV1.2 with a neutral IIS4 (IIS4N) activate and deactivate similar to wild type. A role of IIS4 in gating becames evident in CaV1.2 carrying positional specific mutations of glycines or alanines on one of the S6 gates (G432/A780/G1193/A1503, GAGA-mutations). In these slowly gating mutant channels neutralisation of IIS4N causes a paradoxical shift of the activation curve to the right, accelerates channel gating and thus partially rescues wild type gating.Here we make use of GAGA-S6-mutants to study the role of IS4 and IIS4 and the impact of individual S4 charges. Introduction of a single charge into neutralized IS4N (IS4N+R276) accelerated channel gating and reduced the the slope of the activation curve. IIS4N+R662, also accelerated gating and shifted the activation curve to more depolirized voltages but without significantly affecting its slope.Segments IS4 and IIS4 carrying either one or four charges at different positions all accelerated curent kinetics. Our data suggest different roles and impacts of IS4 and IIS4 in channel activation. IS4 appears to be more important for “unlocking” the closed resting gate while IIS4 contributes predominantly to stabilisation of the gate in the open conformation. A second finding was the different impact of partially and fully charged S4 segments on CaV1.2 gating. We hypothesise that the evolutionary design of CaV1.2 requires fife charges in IS4 and in IIS4 for voltage-dependent movements to their final up and down positions. Our data support a gating model of CaV1.2 where IS4 and IIS4 modulate all four cooperatively interacting S6 gates in a domain specific manner.

In continuation of our previous work (Stary et al. 2007, Beyl et al. 2010) we have analyzed the functional role of gating determinants in the bundle crossing region of Cav1.2. G432 (IS6), A780 (IIS6), G1193 (previous data, IIIS6) and A1503 (IVS6) were substituted with residues of different physicochemical properties. This ‘G/A/G/A’ motif was identified in a conservation analysis showing that Cav, Nav, TRP and many K channels contain glycines or alanines in equivalent positions. Most ‘G/A/G/A’ mutations shifted the activation curve in the hyperpolarizing direction, suggesting a destabilization of the closed and a stabilization of the open channel state. Mutations G432A/V, G1193A and A1503V did, however, stabilize the closed channel conformation as evident from rightward shifts of the activation curves. A comparison with the closed KcsA structure revealed that the ‘G/A/G/A’ motif (‘A/A/A/A’ in KcsA) is located at the narrowest point of the interface between two S6 (M2) helices, tightly packed in a hydrophobic environment. Our analysis suggests that size and hydrophobicity might be important for channel gating. We found that mutations to methionine or tryptophan either destabilize the closed channel or result in non-conducting channels. Small hydrophobic residues like alanine or valine stabilize the resting state. A comparison between resting and open state KcsA structures indicates that the ‘G/A/G/A’ motif and surrounding residues in Cav1.2 undergo large conformational changes upon gating, resulting in a displacement of more than 7 A.1 Stary, A., Shafrir, Y., Hering, S., Wolschann, P., Guy, H.R. (2007) Channels 2(3), 210-215.2 Beyl, S., Depil, K., Hohaus, A., Stary-Weinzinger, A., Timin, E., Shabbir, W., Kudrnac, M., Hering, S. (2010) Pflugers Arch, in press.

We have previously shown that mutations in pore lining segment IIS6 of Cav1.2 induce leftward shifts of the activation curve reflecting a destabilization of the closed and a simultaneous stabilization of the open channel state (Beyl et al. 2009). Systematic substitutions of a cluster of hydrophobic residues (LAIA motive) by residues of different size and polarity (except proline) revealed a strong correlation between changes in hydrophobicity (ΔH) and the shifts of the activation curve (ΔV0.5). A similar analysis in segment IS6 revealed no correlation between ΔH and ΔV0.5. Here we show that amino acid substitutions in the corresponding sequence stretch of segment IIIS6 (F1191 - I1196) shift the activation curves (ΔV0.5 F1191T=−7.1±1.3mV, V1192T= 8.2± 1.2mV, G1193T=-31.2±1.3mV, F1194T=2.1±1.2mV, V1195T=−10.3±1.1mV, I1196T=−17.9±1.1mV). A reduction in hydrophobicity in positions V1195 (ΔH(V1195T)=−4.9) and I1196 (ΔH(I1196T)=−5.2) apparently destabilized the closed channel state. Other mutations (V1192T, G1193T and F1194T) did not fit this paradigm. Potential interactions between residues in segments IS6, IIS6 and IIIS6 were analysed by means of double mutant cycle analysis. Our data reveal a positional specific interaction between gating determinants in segments IS6 and IIS6 as well as IIS6 and IIIS6. Rate constants of the voltage sensing machinery and pore stability were estimated using a 4 state gating model (Beyl et al. 2009).

Calcium channel family members activate at different membrane potentials, which enables tissue specific calcium entry. Pore mutations affecting this voltage dependence are associated with channelopathies. In this review we analyze the link between voltage sensitivity and corresponding kinetic phenotypes of calcium channel activation. Systematic changes in hydrophobicity in the lower third of S6 segments gradually shift the activation curve thereby determining the voltage sensitivity. Homology modeling suggests that hydrophobic residues that are located in all four S6 segments close to the inner channel mouth might form adhesion points stabilizing the closed gate. Simulation studies support a scenario where voltage sensors and the pore are essentially independent structural units. We speculate that evolution designed the voltage sensing machinery as robust “all-or-non” device while the varietys of voltage sensitivities of different channel types was accomplished by shaping pore stability.

The mechanism of channel opening for voltage-gated calcium channels is poorly understood. The importance of a conserved isoleucine residue in the pore-lining segment IIS6 has recently been highlighted by functional analyses of a mutation (I745T) in the CaV1.4 channel causing severe visual impairment (Hemara-Wahanui, A., Berjukow, S., Hope, C. I., Dearden, P. K., Wu, S. B., Wilson-Wheeler, J., Sharp, D. M., Lundon-Treweek, P., Clover, G. M., Hoda, J. C., Striessnig, J., Marksteiner, R., Hering, S., and Maw, M. A. (2005) Proc. Natl. Acad. Sci. U. S. A. 102, 7553–7558). In the present study we analyzed the influence of amino acids in segment IIS6 on gating of the CaV1.2 channel. Substitution of Ile-781, the CaV1.2 residue corresponding to Ile-745 in CaV1.4, by residues of different hydrophobicity, size and polarity shifted channel activation in the hyperpolarizing direction (I781P > I781T > I781N > I781A > I781L). As I781P caused the most dramatic shift (-37 mV), substitution with this amino acid was used to probe the role of other residues in IIS6 in the process of channel activation. Mutations revealed a high correlation between the midpoint voltages of activation and inactivation. A unique kinetic phenotype was observed for residues 779–782 (LAIA) located in the lower third of segment IIS6; a shift in the voltage dependence of activation was accompanied by a deceleration of activation at hyperpolarized potentials, a deceleration of deactivation at all potentials (I781P and I781T), and decreased inactivation. These findings indicate that Ile-781 substitutions both destabilize the closed conformation and stabilize the open conformation of CaV1.2. Moreover there may be a flexible center of helix bending at positions 779–782 of CaV1.2. These four residues are completely conserved in high voltage-activated calcium channels suggesting that these channels may share a common mechanism of gating.

Voltage sensors initiate the closed - open transitions in the ion conducting pore. Here we consecutively neutralized the charges of the four S4 segments (IS4-IVS4) of CaV1.2. The channel construct lacking charges in IIS4 (IIS4N) conducted barium currents. Surprisingly, charge neutralization accelerated channel closure and diminished shifts of the activation curves also in S6 mutants (IS6: G432W/IIS4N, IIS6: A780T/IIS4N, IIIS6: G1193T/IIS4N, IVS6: A1503G/IIS4N). Such a rescue of S6-mediated gating perturbations is exemplified for the IVS6 mutation A1503G. A1503G induced a −22.6±1.2 mV shift of the activation curve to the left that was diminished by charge neutralization in IIS4. Gating disturbances induced by pore mutations upstream and downstream of G432, G1193 and A1503 were unaffected. Thermodynamic analysis reveals that segment IIS4 interacts (energetically coupled) with a ring of small homologous amino acids G432, A780, G1193 and A1503 (G/A/G/A ring, Depil et al. 2011) in all four pore forming S6 segments.The research was funded by the Austrian Science Fund (FWF): grant P22600-B12 and P19614.View Large Image | View Hi-Res Image | Download PowerPoint Slide

Voltage sensors trigger the closed-open transitions in the pore of voltage-gated ion channels. To probe the transmission of voltage sensor signalling to the channel pore of Ca(V)1.2, we investigated how elimination of positive charges in the S4 segments (charged residues were replaced by neutral glutamine) modulates gating perturbations induced by mutations in pore-lining S6 segments. Neutralisation of all positively charged residues in IIS4 produced a functional channel (IIS4(N)), while replacement of the charged residues in IS4, IIIS4 and IVS4 segments resulted in nonfunctional channels. The IIS4(N) channel displayed activation kinetics similar to wild type. Mutations in a highly conserved structure motif on S6 segments ("GAGA ring": G432W in IS6, A780T in IIS6, G1193T in IIIS6 and A1503G in IVS6) induce strong left-shifted activation curves and decelerated channel deactivation kinetics. When IIS4(N) was combined with these mutations, the activation curves were shifted back towards wild type and current kinetics were accelerated. In contrast, 12 other mutations adjacent to the GAGA ring in IS6-IVS6, which also affect activation gating, were not rescued by IIS4(N). Thus, the rescue of gating distortions in segments IS6-IVS6 by IIS4(N) is highly position-specific. Thermodynamic cycle analysis supports the hypothesis that IIS4 is energetically coupled with the distantly located GAGA residues. We speculate that conformational changes caused by neutralisation of IIS4 are not restricted to domain II (IIS6) but are transmitted to gating structures in domains I, III and IV via the GAGA ring.

A channelopathy mutation in segment IIS6 of Ca(V)1.4 (I745T) has been shown to cause severe visual impairment by shifting the activation and inactivation curves to more hyperpolarized voltages and slowing activation and inactivation kinetics. A similar gating phenotype is caused by the corresponding mutation, I781T, in Ca(V)1.2 (midpoint of activation curve (V(0.5)) shifted to -37.7 +/- 1.2 mV). We show here that wild-type gating can partially be restored by a helix stabilizing rescue mutation N785A. V(0.5) of I781T/N785A (V(0.5) = -21.5 +/- 0.6 mV) was shifted back towards wild-type (V(0.5) = -9.9 +/- 1.1 mV). Homology models developed in our group (see accompanying article for details) were used to perform Molecular Dynamics-simulations (MD-simulations) on wild-type and mutant channels. Systematic changes in segment IIIS6 (M1187-F1194) and in helix IIS6 (N785-L786) were studied. The simulated structural changes in S6 segments of I781T/N785A were less pronounced than in I781T. A delicate balance between helix flexibility and stability enabling the formation of hydrophobic seals at the inner channel mouth appears to be important for wild-type Ca(V)1.2 gating. Our study illustrates that effects of mutations in the lower part of IIS6 may not be localized to the residue or even segment being mutated, but may affect conformations of interacting segments.