 Good evening everybody, I am Rishi and I am from Indian Institute of Science, Education and Research, Ohali, India. And these are the newly developed institutes in India which are promoting science. And these institutes are developed in 2007 itself. And India has started an integrated MS program first time. So, and I am the first best student of ISA Ohali. And I am enrolled in integrated MS program and majoring in biology and my interest lies in neuroscience. So, I came through for a Karana program for my internship here and the project which I carried out has titled as Temperature-Rependence of Hydrophobic Attitude Substitutions in Voldigated Potassium Channels. So, coming to the role of these Voldigated Potassium Channels, which are important in generating and propagation of electrical impulse in excitable cells like neurons, heart cells. And they are closed at resting membrane potential but opens in response to depolarization. And they are responsible for the repolarizing phase of the action potential. As you can see here, when the depolarizing phase occurs, the sodium ions flows in inside the cell and the membrane potential increases and in response to that, these Voldigated Potassium ion channels opens up and potassium flows from inside cell to the outside and the resting membrane potential comes at the resting state once again. So, going to the structural aspects of these Voldigated Potassium Channels, they are homo-tetramer with six transmembrane segments. They are termed as S1, S2, S3 up to S6. They have three basic functional parts. One is Voldigated Potassium sensor. So, the first four domains are called Voldigated Potassium sensor, S1, S2, S3 and S4. S4 is called primary sensor and the positive charges are due to arginine. The other region is pole or the conducting pathway, which is this region which links S5, S2, S6. And the third one is gate, which is the C-terminal domain of these Voldigated Potassium channel. So, whenever there is an increase in the potential, then S4, so S4 domain pulls up and then it pulls S4, S5, Flinker and the gate opens up and Potassium ions flows from inside to the outside of the cell. These voltage sensing domains, which are abbreviated as VLD, they have conserved sequence among all the families of Voldigated ion channels. So, the conserved sequence is RXX, RXX, RXXR. So, you can see at every third position there are arginine and in between the two X's show the hydrophobic residues and in my case the Potassium channel, which I am studying, is shaker and it has a signature sequence of RVI, RLV, RVFR. So, the major goal of my lab is to understand why certain Voldigated channels such as TRIPAM channel are temperature sensitive. And we are using our Voldigated Potassium channel to address these issues and we are trying to engineer the temperature sensitivity in Voldigated Potassium channels by mutating certain key residues in the voltage sensing domain. And if we get some good idea of the voltage temperature sensitivity in Voldigated Potassium channel then we can go back to the TRIP channel and can address the temperature sensitivity there. So, the objective of my project is to study the temperature dependence of hydrophobic residue in Voldigated Potassium channels at two regions. The one is S4 helix and the other one is adjoining regions which involves S1 to S3 domains of voltage sensing domain. So, electrophysiological method which I used to carry out the experiment part. The first one is RLA construct injection. So, we mutated our RLA by PCR mutated lenses and the RLA was injected from two sides and then we did voltage clamp recording which is called cut open voltage clamp. Here is the cartoon which represents the cut open voltage clamp technique. So, here one voltage is clamped and the other electrode is inserted inside the problem size and we give a pulse of different voltages and we record the current from there. So, the voltage clamp protocol has depolarizing pulse, repolarizing pulse and the corresponding currents are called eye saturation and eye tails. So, explaining the ionic current traces and voltage clamp protocol here is the data for vital type. So, here you can see in the first picture I gave the voltage D pulse at minus V20 minus 120 and then depolarizing pulse at minus 90 and then repolarizing pulse at minus 120 and this is called one cycle and we get correspondingly the ionic currents for high temperature and for low temperature as well. So, every step in after each cycle we increase the depolarizing potential by 5 millivolt. So, from going minus 90 millivolt up to plus 90 millivolt we record the ionic traces at high temperature and low temperature and we have lot this graph which has I by I max and voltage. So, to plot this graph we just look at this eye tails because these are the ionic currents because of the potassium ion which are flowing from inside to outside the cell. So, the I max value is when all the potassium channels are open. So, the maximum amount of the current which can flow from inside to outside the cell corresponds to the I max and which so this I max can be correlated to the opening probability of the potassium channel. So, when I am getting I max it shows that all the channels are open hence the probability of opening is 1. So, for each depolarizing pulse we record I and we divide our I value by I max and normalize on the scale of 0 to 1 and plot it on the Y axis and corresponding depolarizing voltage we plot on the X axis. So, this kind of graph can be generated here the blue one is the lower temperature graph at 8 degree Celsius and the higher temperature is 27 degree Celsius and correspondingly we get the red graph. So, when the I by I max value is half we call the voltage as V half. So, V half is the empirical measure of half of the channels are open. So, for the analysis of the data which I get we use two equations delta G is equals to ZF V half minus V and delta G is equals to delta H minus T delta S and when we take the difference at any two point then we get delta of delta G is equals to ZF ZF delta V half and for the second from the second equation we can get delta of delta G is equals to delta of delta H minus T delta of delta S. So, I will define two terms over here delta VW and delta VT and their effects. So, if delta VW so here delta VW is the change in V half of the mutant with respect to wild type and delta VT is the change in V half of the mutant with respect to temperature. So, if delta VW is 0 so my mutant is not showing any effect with respect to wild type and if delta VT is 0 there is no temperature there is no temperature dependent effect in the mutation. So, when both are 0 then there is no so the mutation effect is null when delta VW is 0 and delta VT is non-zero then you can get difference in the change in field and help is non-zero and difference in the change of entropy is 0 is non-zero as well. Similarly, in the third situation we can have delta VW non-zero so the wild type and mutant both are showing change in delta VW but the delta VT is 0. Hence the difference in the change of entropy is 0 but difference in the change in enthalpy is non-zero. Similarly, both are non-zero then we can conclude delta of delta X is non-zero. So, I will not explain all my results in terms of in terms of delta VW and delta VT but I will I will go into the detail later as I move through the experiments. So, the first experiment which I carried out I mutated all the four arginine residues like R362 into R at the 362 position into alanine and I call it R1. Similarly, R at 365 position into alanine R2 and like that and I recorded through voltage grant technique and analyzed delta VW half from the IV plot which I have already shown for the wild type. So, these are the results for these mutations. So, here you can see when I by I max and if V plot is V plotted I by I max and V for R1A then we cannot see any difference at two temperatures. So, both graphs are overlapping same goes for R368A and R371A but the second feature comes for R2A here you can see for 27 degree Celsius the V half is close to 0 but for the 8 degree Celsius temperature the V half is lower than 0 and there is a shift. So, it shows that at 8 degree Celsius temperature the V half is lower at 27 degree Celsius so the process is becoming more favorable for lower temperature and this mutant can be characterized as core sensitive mutation but rest of these mutations are not temperature sensitive at all because they are not showing any prominent shift as we change the temperature. So, here are two ways is core sensitive. For experiment two we mutated first two hydrophobic residues into alanine, methionine and isoleucine in the increasing order of hydrophobicity and then we recorded at two temperatures the I by I max and V. So, here you can see for A2 there is very less shift at two temperatures in the plot but for M2 and I2 you can see there is a 25 millivolt shift for M2 and I2 at these two temperatures and you can see as I am decreasing the temperature the process is becoming more favorable that is why the V half value is decreasing. So, for M2 and I2 I can say that these two mutations are core sensitive. For experiment three we mutated all the six residues into alanine, methionine and isoleucine in the same increasing order of hydrophobicity and we recorded again for these mutants and here we get a prominent shift for 28 degree Celsius here you can see at 8 degree Celsius the V half is 0.23 millivolt but for 28 degree Celsius the V half goes down up to minus 28.77 millivolt. So, as I am increasing the temperature the process is becoming more favorable that is why the V half value is decreasing. So, this mutant can be characterized as heat sensitive but for I6 there is no change in the V half for 8 degree Celsius temperature and 28 degree Celsius temperature. So, I6 is temperature insensitive. Now, in the fourth experiment we mutated all the four we did point mutation adjoining regions of S4 domain. So, these first two mutations are here in S1 in the top part which I am showing here and the last two mutations are in the bottom of these VSD. So, the graphs for all four are here. So, why 323I mutation shows that at 28 degree Celsius V half value is less than the V half value at 8 degree Celsius. So, the process is much more favorable for higher temperature hence this mutation can be characterized as heat sensitive. For S240A there is a shift at higher temperature but the shift is not very prominent. For N313A there is no change in V half for 8 degree Celsius and 27 degree Celsius but for E293I there is a shift and you can see at 8 degree Celsius the process is becoming more favorable more channels are open at lower voltage. So, this mutation is core sensitive. To summarize my result I have plotted the V half for each mutant and for wild type also. So, here you can compare the result. So, all the steric signed mutants are either heat sensitive or bowl sensitive. So, here you can see the shift in the V half just to compare and the conclusion is for point mutation R1A does not show any shift. So, it is neither heat sensitive nor cold sensitive. For R2A we can see it is cold sensitive. The wild heat 23I is heat sensitive. E293 is cold sensitive. For M2 and I2 both are cold sensitive and M6 is heat sensitive. So, my acknowledgement goes to Parvon Chanda who is my project supervisor and he supported for my stay and for my project. And Sandeepan he is a graduate student in his lab and he is really very hard working and he taught me everything from scratch and now I feel like I can work on by myself and I did. And other lab members were all here and they are very supportive like Kevin, Trevor, Marcel, Brian. So, I am very happy to be there for 2 months and I really learnt a lot from them. Thank you.