Voltage vs. Ligand I: Structural basis of the intrinsic flexibility of S3 segment and its significance in ion channel activation

Abstract

We systematically predict the internal flexibility of the S3 segment, one of the most mobile elements in the voltage-sensor domain. By analyzing the primary amino acid sequences of V-sensor containing proteins, including Hv1, TPC channels and the voltage-sensing phosphatases, we established correlations between the local flexibility and modes of activation for different members of the VGIC superfamily. Taking advantage of the structural information available, we also assessed structural aspects to understand the role played by the flexibility of S3 during the gating of the pore. We found that S3 flexibility is mainly determined by two specific regions: (1) a short NxxD motif in the N-half portion of the helix (S3a), and (2) a short sequence at the beginning of the so-called paddle motif where the segment has a kink that, in some cases, divide S3 into two distinct helices (S3a and S3b). A good correlation between the flexibility of S3 and the reported sensitivity to temperature and mechanical stretch was found. Thus, if the channel exhibits high sensitivity to heat or membrane stretch, local S3 flexibility is low. On the other hand, high flexibility of S3 is preferentially associated to channels showing poor heat and mechanical sensitivities. In contrast, we did not find any apparent correlation between S3 flexibility and voltage or ligand dependence. Overall, our results provide valuable insights into the dynamics of channel-gating and its modulation.

Voltage vs. Ligand II: Structural insights of the intrinsic flexibility in cyclic nucleotide-gated channels

“…Notably, we found a good correlation between S3 flexibility indices and the sensitivity to temperature (measured as the Q 10 coefficient) so that if the channel exhibits high sensitivity to heat, local S3 helix become more rigid and vice versa. On the other hand, we also reported a good correlation between the degree of S3 flexibility and activation by mechanical stretching [7]. In the present study, we extend our analysis exploring the origins of the 6TM channels and we found that in one of the more ancient bacterial lineages (Aquificae) the only transport protein with significant similarity to members of the 6TM members of the VGIC superfamily corresponds to a putative potassium channel (kch) protein (ORF Aq1863) [10].…”

“…In total, 32 sequences were included in this study ( intestinalis (NP_001071937); ehHv1 from Emiliania huxleyi CCMP1516 (005762299); kHv1 from Karlodinium veneficum (AEQ59286); mHv1 from Mus musculus (NP_083028); TAX-4 from Caenorhabditis elegans (CAB63418); Shaker K V channel from Drosophila melanogaster (CAA29917); CNGA1 from Homo (P29973), and CNGA2 from M. musculus (Q62398). Estimation of flexibility indices has been described in the companion study [7].…”

“…Once the relationship among local S3 flexibility, voltage dependence, thermo-and mechanosensitivity was previously established [7], we decided to study the VSD and the PD modules separately. For this, we reasoned that, given that a large amount of phylogenetic studies have led to the hypothesis that 6TM potassium channels both in prokaryotic and eukaryotic cells derived from a simpler common precursor present in a primitive form of life, now extinct [16,17], a logical way to approach that precursor would be to study the potassium channels present in the oldest lineages of the Bacteria domain.…”

“…To address this issue, we compared the reported half-maximal effective concentration (EC 50 ) of cAMP and cGMP as a function of the mean flexibility of the 6TM segment for each ion channel. Figure 2 depicts the S1-S6 flexibility profiles for these proteins and indicates that the transmembrane core of AqK is, overall, comparatively more rigid than the one in LliK (from Leptospira licerasiae), MloK1 (Mesorhizobium loti) and SthK [7]. Arrows show the NxxD and paddle-like motifs (d) or in black in two subunits for clarity, including the selectivity filter, also in black (e).…”

“…In the preceding paper [7], we evaluated structural aspects that allow us to better understand the role played by intrinsic flexibility of segment S3 in the basic aspect of the activity of ion channels: their ability of gating. We determined that segment S3 has two regions of important local flexibility: (1) a short sequence NxxD motif located at the N-half portion and (2) a short sequence at the beginning of the so-called paddle motif, around the half of S3, where the helix has a kink at Pro-322 (Shaker numbering), dividing it into two distinct helices (S3a and S3b).…”