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(A) Current responses of the indicated channel types recorded from inside-out patches before (black) and 200Â s after (red) application of 200Â nM hemin. Depolarization steps were applied to +40Â mV for Kv1.2, Kv1.5, and hEAG1 channels and to +80Â mV for hERG1_H8. (B) Maximal currents measured during depolarizations as a function of time. The application of 200Â nM hemin is indicated. (C) Averaged relative remaining current upon application of 200Â nM hemin for the indicated channel types. Error bars denote sem values; the number of independent experiments is indicated in parentheses. (D) Application of 200Â nM hemin to an outside-out patch with hEAG1 channels. Solution: Standard Asp.
Figure 2. Concentration dependence of hEAG1 inhibition by hemin.
(A) Time course of hemin effect on hEAG1 channels in an inside-out patch. Currents were elicited by +40-mV depolarization at an interval of 10Â s. Upon equilibration of the hemin effect, control solutions was applied (wash); finally, inside-out patch was exposed to control solution with 1Â mM DTT (DDT). The continuous curve is single-exponential data fit to characterize the onset of current inhibition. (B) Example of onset of hemin-induced current inhibition at for the indivated hemin concentrations with superimposed single-exponential fits. (C) Concentration dependence of the equilibrium current inhibition at -40, +40, and +100Â mV. The continuous curves represent fits with a Hill equation resulting in a apparent IC50 values of: Data here. (D) Inverse of the time constant of hemin-induced current inhibition (1/ï´on) as a function of hemin concentration. The straight line is the result of a data fit assuming a single molecular reaction, i.e. ï´on = 1 / (kon + koff), with kon = xxx and koff = yyy. (If that turns out to fit). Solution: Symmetrical high K-Asp. Data at 1 nM are questionable because overlap with rundown. Some data points at 50, 100nM required. Underway.
Figure 3. Voltage dependence of hemin-induced hEAG1 inhibition.
(A, left) Superposition of current traces recorded from inside-out patches according to the indicated pulse protocol in which depolarizations range from -110 to +120Â mV before (black) and after (red) application of 10Â nM hemin. (A, right) Sample current traces from the experiment shown on the left for the indicated voltages before (black) and after (red) hemin application. The gray traces are scaled hemin traces to match the maximal control current. Scale factors applied: -40 mV: 10.7; +40 mV: 5.2; +100 mV: 3.92. (B) Mean current measured at the end of test depolarizations as a function of voltage with superimposed data fits according to eq. (1). (C) Tail currents at -140Â mV as a function of test voltage with superimposed Boltzmann fits (eq. (2)). (D) Voltage dependence of relative current remaining after application of 10Â nM hemin determined from test currents (B) (circles) and from tail currents (C) (squares) indicating an almost linear voltage dependence of the hemin-mediated current reduction. Solution: Symmetrical high K-Asp.
Some info from the IV fits:
Control: Vm, and km
Hemin: Vm, and km
Figure 4. Specificity.
Current inhibition was analyzed for hemin (Fe2+), heme (Fe3+), Zn2+ porphyrine, Co2+ porphyrine, protoporphyrine (all at 200Â nM concentration) as well as MP-11 (2.6 mM) indicating a clear preference for hemin, heme and Co2+-porphyrine. The heme group has to be free because when bound to MP-11 it was without effect. Solutions: Standard K-Asp.
50 mM Data are to be added. Maybe we getter a better separation of Zn-PP vs PP.
Figure 5. Effect on hEAG1 and hERG1 in mammalian cells.
Whole-cell recordings from HEKÂ 203 cells expressing hEAG1 (A) or hERG1 (B) before and after application of xxÂ nM hemin. (C) Time course.
Figure 5. Alignment
Experiments to be done:
Effect of heme on hEAG1: 20, 50, 100, 200, 500 nM
Onset, recovery, gating properties at half-maximal block
Compare other channels (100 nM heme): ShakerD, Kv1.5, hERG
HEAG1, apply MTSES, MTSEA, report effect.
MTSES (low conc.): Then heme 200 nM
Try to recover heme sensitivity with redGSH or DTT
Does H2O2 also affect heme action?
At this point we will have to decide whether or not to use (e.g.) 500 ÂµM DTT in all experiments (other than MTS).
Test for CaM-heme interaction:
Does heme block a CaM-inhibited channel?
Ca/CaM (1ÂµM/100 nM always)
Heme (200 nM always)
Sequences to test (each interval at least 120 s)
EGTA - Heme - EGTA
EGTA - Ca/CaM - EGTA
EGTA - Ca/CaM - Ca/CaM+Heme - EGTA
EGTA - EGTA/CaM - EGTA/CaM+Heme - EGTA
EGTA - Heme - EGTA/CaM -Â EGTA
EGTA - Heme -Â Ca/CaM - EGTA
What do we know?
In inside-out patches hemin "blocks" hEAG1 channels (estimated IC50 of 4 nM).
Iron, protoporphyrin and Zn-prophyrin are ineffective, Co-prophyrin is like hemin.
Hemin and heme act in a similar manner
The hemin effect is only slowly reversible, DTT speeds up recovery
Kv1.5 is not blocked by hemin
Kv2.1 is activated by hemin (this should be a separate thing)
hERG1 channels undergo fast rundown in inside-out patches. Therefore, we tested a non-inactivating mutant (H8). This is blocked by hemin with somewhat bigger IC50 than hEAG1. IV shifts observable.
Both, hEAG1 and hERG1 are inhibited by CO (in a similar fashion as with hemin)
Structure: There are potential heme binding sites in the S5 segment of ion channels from the EAG family (in addition to HCN and plant channels). Peptides of this sequence bind hemin. Mutagenesis inside this domain is not well tolerated. We have some mutants in hEAG1 showing that the Cys in this motif is not responsible for the hemin effect. Mutations of the His are not tolerated.
A completely Cyc-less hEAG1 is still hemin sensitive.
All but three His can be removed from hEAG1. These mutants are still hemin sensitive. The remaining two histidines reside in a linker between S6 and the CNG domain in the C-terminus. This linker is known (to us) as important for channel gating.
A peptide of this linker binds hemin. Mutant peptides are available - binding measurements have to be done. In addition, I will measure EPR next week.
One of the histidines in this linker can be mutated in hERG1: still sensitive to hemin.
Thus, there is only one His left in the C-linker. If this is not important, Nirakar will commit suicide.
hERG1 measured in whole-cell is also sensitive to hemin and CO.
We have a cell line (progenitor line of red blood cells). This line undergoes hemin-induced proliferation. In addition, this line expresses hERG1 channels. Here we may have a physiological case in which hemin induces a response by inhibiting hERG1. Thus are, we have seen the hERG1 currents. We will try how cell proliferation responds to classical hERG1 blockers.
hEAG channels bind to heme-agarose in a biochemical assay. Experiments with hEAG1 mutants are in progress.
I certainly forgot a lot of small details.
Whatever we do, we need: (a) physiological role, or (b) molecular mechanism.
For a grant it will not be wise to search for (a). Thus, we are left with (b).
For an NIH grant it might be good to concentrate on heme (maybe including HRG), but leaving out CO for the grants running here - just to have some kind of formal separation.