Hansjakob Heldstab

Low genetic variation in perch (Perca fluviatilis L.) from three major European drainage systems in Switzerland

Previous genetic studies on perch (Perca fluviatilis L.) in northern and southern regions of Europe have shown low heterozygosity. No such investigation has been conducted in the central part of Europe. The genetic variability ofP. fluviatilis in four Swiss lakes (Lake Constance, Lake Zürich, Lake Geneva and Lake Maggiore) was investigated. These four lakes belong to three different drainage systems (Rhine, Rhone and Po) without connections. A total of 136 fish were analysed electrophoretically. Only one locus (SOD*) was highly polymorphic in Lake Constance among the 27 loci studied. The expected heterozygosities for each population and for the total population were higher than in the previous studies but low (HS= 0.38–2.7%;HT=1.22%) and Neis genetic distances were small (0.000–0.003). However, the jack-knifed mean of the WrightsFSTwas calculated as 0.142, which indicates modest genetic differentiation among the perch populations in Switzerland, due to the differentiated population in Lake Constance.

More Gating Charges are Needed to Open a Shaker K+ Channel than are Needed to Open an rBIIA Na+ Channel

This study presents what is, to our knowledge, a novel technique by means of which the ratio of the single gating charges of voltage-gated rat brain IIA (rBIIA) sodium and Shaker potassium ion channels was estimated. In the experiment, multiple tandems of enhanced green fluorescent protein were constructed and inserted into the C-terminals of Na(+) and K(+) ion channels. cRNA of Na(+) and K(+) ion channels was injected and expressed in Xenopus laevis oocytes. The two electrode voltage-clamp technique allowed us to determine the total gating charge of sodium and potassium ion channels, while a relative measure of the amount of expressed channels could be established on the basis of the quantification of the fluorescence intensity of membrane-bound channels marked by enhanced green fluorescent proteins. As a result, gating charge and fluorescence intensity were found to be positively correlated. A relative comparison of the single gating charges of voltage-gated sodium and potassium ion channels could thus be established: the ratio of the single gating charges of the Shaker potassium channel and the rBIIA sodium channel was found to be 2.5 +/- 0.4. Assuming the single channel gating charge of the Shaker K(+) channel to be approximately 13 elementary charges (well supported by other studies), this leads to approximately six elementary charges for the rBIIA sodium channel, which includes a fraction of gating charge that is missed during inactivation.

Omega Mutations along S4 in Nav1.2 Channels Give Insight into Domain Specific Contribution to Activation and Steady State Inactivation

We previously identified the resting state positions of the voltage sensor S4 for each domain of Nav1.2 by means of omega mutations. We found that a double gap is needed to open the omega pore (narrow part of the gating pore) resulting in detectable omega current, also known as gating pore current. At hyperpolarizing conditions, the resting state of S4 was found for double gap RR1,2QQ in domain I, II and IV and for double gap RR2,3QQ in domain III. In this work we evaluated additional conformational states of the voltage sensor S4 moving through the gating pore by further double gap mutations along S4 (second double gap 2,3QQ and third double gap 3,4QQ). Two electrode voltage clamping on X. laevis oocytes expressing rat brain sodium channels Nav1.2 was used to measure macroscopic ionic current through the alpha pore and, if present, omega current through the omega pore. In DI and DII we detected clear outward omega current for S4 mutants RK3,4QQ at depolarizing conditions. Furthermore, we found that activation of sodium currents was right shifted by about 30 mV towards higher potentials compared to the first and second double gap mutants or wild-type sodium channel. These findings suggest two sequential gating steps of S4 between resting and activated state in both domains DI and DII. In DIII and DIV no clear outward omega current could be detected at depolarized potentials either for the second or for the third double gap mutant. However steady state inactivation was strongly left shifted by about 50 to 100 mV to more hyperpolarized potentials for the second and third double gap mutant in both domains, consistent with involvement in recovery from inactivation and immobilization, respectively.

Domain Specific Role of S4 for Stepping into and Recovering from the Inactivated State as Obtained from Omega- and R4H Mutants in Nav1.2

The macroscopic time courses of activation, inactivation and recovery are voltage dependent. Control of these processes on the molecular level by the voltage sensors S4 of each domain together with the DIII-IV loop (the inactivation particle) is not completely understood. With omega current mutations (RR//QQ) along S4 we explore the position or state of S4 in the gating-pore and also disturb the normal gating kinetics in a domain and state specific manner. In addition, we used the mutation S4/R4H in either DIII or DIV which slowed recovery from inactivation about 10 times; the return of the loop into the recovered position releasing the α-pore parallels the appearance of the omega current reflecting the arrival of S4 in the resting state. This confirms that both domains DIII and DIV control recovery.Stepping from resting into inactivated state appears more complex. Previous data for squid showed that the inactivation time constant tau-h has a kink at about −10 mV. Combination with high-resolution gating currents showed that below this voltage tau-h obtained its steeper voltage dependence by coupling to activation; the smaller voltage dependence of tau-h above −10 mV appeared as result of a single inactivation voltage sensor (Greeff and Forster, 1991). In Nav1.2, we now see a similar kink at about −10 mV. Gating currents are too small to be resolved. Instead, we use omega mutations in DI to IV to study changes of voltage dependence of inactivation. Modifying DIV/S4 with RR//QQ along 3 positions shifts the kink to the left and seems to reduce the voltage dependence indicating the role of DIV/S4 as voltage sensor of inactivation. In contrast, in domain I to III the resting state RR//QQ mutation does not change tau-h. However, omega mutations in the inner region of DI, II and III, especially DII34QQ, increased tau-h substantially combined with a strong right shift of the kink.Conclusion for Nav1.2: Recovery from inactivation clearly depends on S4 in DIII and DIV. Going into the inactivated state, i.e. closing of the α-pore by the DIII-IV loop, needs as prerequisite the activation of DI and DII at lower voltages, while at higher voltages the slower DIV is rate limiting. The relation of DIII to a specific function when stepping up is less clear from these experiments.

Inactivation Voltage Sensor S4 in Domain IV of Nav1.2 Controls Immobilization of S4 in Domain III as Shown by Omega Currents

The role of S4DIV for inactivation of skeletal muscle Na channel Nav1.4 was recognized after deciphering the channelopathia Paramyotonia congenita. We showed with point mutations in the rat brain sodium channel Nav1.2 the central role of S4DIV for inactivation (Kuhn and Greeff, 1999): The single mutation R4H in S4DIV slowed the recovery from inactivation about 20 times in parallel for ionic current and immobilized gating charge. Immobilization concerns about 50 % of total gating charge returning slowly to the resting state during recovery while the other half of gating charge returns very quickly. Clearly, the amount of immobilized charge is more than just the one from S4DIV. So we speculated that S4DIV would control S4s in other domains. Now, we are able to monitor the return of S4 into the resting position for each domain separately by recording the leak current of resting-state omega pore mutants (this Meeting). We find that S4DIV with the omega mutation RR12QQ shows a fast onset of omega leak current for channels at rest; however, after an inactivating prepulse, the leak current grows with the time course of recovery as expected, since this voltage sensor controls the recovery and returns into resting position accordingly. Checking the return of S4 in the other domains, we find a fast return in DI and DII while in DIII the return follows DIV. Combining these mutations with R4H in DIV, the return in both domains III and IV is about 20 times more slowly. This suggests that immobilization of gating charge across the domains is most likely achieved by the cytoplasmic loop between DIII and DIV which under control of S4DIV closes the alpha-pore and immobilizes S4DIII.

Double-Gap Omega-Currents in Shaker K-Channels used to Clarify the Activation Path of S4

We have demonstrated previously for Shaker K-channels that the residues on the voltage sensor S4, namely A0(359) and the gating charges R1(362), R2(365) and R3(368) slide sequentially through the gating-pore (Gamal El-Din et al., 2010). Further, this gating pore was shown to encompass two of these residues since leak currents, so called omega-currents, appeared only when the two residues populating the pore were short, e.g. serine or alanine. We have studied the following mutants and found omega-currents for asRRR, RssRR and aRssR, while aRRss did not express so far (small letters denote short residues to visualize the double-gap position). A further sliding of S4 including R4(371) and K5(374) to the activated open state remained speculative.Here, we report further experiments on these double-gap mutants now combined with 434W instead of 434F to allow for K-current through the alpha-pore when S4 reaches the activated state. All three double-gap mutants show K-current which can be blocked by 4-AP confirming that S4 reaches the activated state. Interestingly, the mutant aRssR shows a voltage range of about 20 mV where neither omega- nor alpha-current is detected. This indicates that S4 steps from the leaking position with s2, s3 in the omega-pore through a next state with s3, R4 in the pore before reaching the activated state with R4, K5 in the pore. This interpretation goes well with the recent finding by Tao et al. (Science 2010) that an inner occluding binding site (for Shaker E293, F290, D316) is opposite to either residue K5 in the activated state or R1 in the closed state. In extension to this, our results demand an outer occluding site (most likely E283) opposed by R4 (activated) or a0 (closed), respectively.