For the first time, several types of spontaneously active channels with different
conductance (from 10–340 pS) have been detected in the inner nuclear membrane of
cardiomyocytes. Among them, LCC-channels with conductance 209 ± 13 pS are the most
expressed. These channels have relatively slow kinetics; at positive potentials (+40 mV)
they remain in the open state most of the time (NPо = 3.11), whereas at negative potentials
(-40 mV) their activity decreases (NPо = 0.72). K+
ions permeate well through LCCchannels, to a lesser extent Na+
, and LCC-channels are impermeable to Cland divalent
Ca2+ cations. Relatively high density combined with high conductance of LCC-channels in
the nuclear membrane indicates their important physiological role in regulating nuclear
functions.
Purified neurotoxin II at a concentration of 25 μM reduces amplitude of the current
through these channels by 13 %. After applying 1–2 mM of α-cobratoxin, a slight channel
flickering is observed. The effects of all tested substances are reversed – after washing
with the working solution, current through the LCC-channels returns to the control values.
Under the influence of d-tubocurarine (200 μM), the amplitude of the current
through the nuclear membrane's LCC-channels in cardiomyocytes decreases by 45 %.
Under the influence of 25 μM DhβE, a 40 % decrease in current through the channel is
observed. At high concentrations of atracurium and dithylinum, Po decreases and channel
flickering is observed, indicating physical blockade of the channel pore in its open state.
However, there is no complete blocking even at 2 mM blocker concentration. Nondepolarizing neuromuscular relaxant rocuronium bromide dose-dependently reduces
current amplitude through the channels, inhibits them by half at concentration of 2 mM
and reduces probability of them being in an open state by half.
A similar effect on LCC-channels functional activity is induced by pipecuronium
bromide. Other investigated n-cholinoinhibitors – hexamethonium, MLA, α-conotoxin
PeIA were proved ineffective. In the following series of experiments, the effects of ncholinoreceptor agonists are tested. In particular, nicotine at a concentration of 10 to 200
μM dose-dependently decreases current amplitude through LCC-channels. After dFBr
23
application at a concentration of 200 μM, the amplitude of the current through the channel
decreases by 21 % and channel flickering is observed. Whereas, other investigated ncholinoreceptor agonists – PNU 282987 and carbachol did not cause statistically
significant changes in LCC-channels functioning.
The most effective among tested n-cholinoreceptor agonists and inhibitors and also
snake toxins are: DhβE > d-tubocurarine ≈ nicotine > NT II > dFBr > dithylinum ≈
atacurium ≈ pipecuronium bromide > rocuronium bromide > α-CTX.
In the nuclear membrane of cardiomyocytes, a channel that is activated by IP3 (0.2–
20 μM) and inhibited by 2-APB (50 μM) was detected. On this basis, we concluded that
the channel is IP3Rs. It’s known from the literature that the most expressed IP3Rs in
cardiomyocytes are type II and we have confirmed that via immunohistochemical analysis.
Probability of IP3Rs being in open state (Po) is potential-dependent, with higher activity of
IP3Rs observed at positive potential values (+40 mV Po = 0.043, +60 mV Ро = 0.125),
while at negative values their activity decreases (-40 mV Ро = 0.025, -60 mV Ро = 0.005).
The probability of an open state of investigated receptors increases with increasing IP3
concentration and peaks at a concentration of 10 μM IP3 in the solution (NPo = 0.292). In
the following series of experiments, we investigate the effect of Ca2+ ions in different
concentrations on IP3Rs activity. In particular, we were able to detect two subtypes of
IP3Rs, one of which is inhibited by high concentrations of Ca2+, which corresponds to the
classical pattern of bell-shaped activity dependent on Ca2+ concentration. This is also
confirmed by literature, according to which high Ca2+ concentrations inactivate purified
cardiac IP3Rs. The other type of IP3Rs has a significantly right-shifted activity
dependence, their inhibition starts at 10 μM Ca2+, but a few single-channel openings
remain at 1 mM of Ca2+ in the solution.
The properties of IP3Rs in the nuclear membrane of cardiomyocytes differ sharply
from both neuronal IP3Rs type I receptors and heterologously expressed IP3Rs type II.
This may be explained by the influence of post-translational modifications and the
molecular environment. Such heterogeneity contributes significantly to temporal and
spatial propagation of the intracellular calcium signal in the nucleus.
The results demonstrate two subtypes of IP3 receptors in the nuclear membrane of
cardiomyocytes that differ in sensitivity to IP3, Ca2+ and LCC-channels, which are
inhibited by agonists and inhibitors of n-cholinoreceptors with different effectiveness.
