$\downarrow \mathrm{O}{\phantom{A}}_2$$\ce{ \downarrow O2}$ ➡️ $\downarrow \mathrm{Mitochondria}\mathrm{Activity}$$\ce{\downarrow Mitochondria Activity}$ ➡️ $\uparrow \mathrm{ROS}$$\ce{\uparrow ROS}$ ➡️ Inhibits $K_v$$K_v$ Channels ➡️ $\mathrm{\Delta }{V}_m$$\Delta V_m$ Becomes Depolarized ➡️ Calcium Entry ➡️ Smooth Muscle Contraction in Pulmonary Arteries ➡️ Vasoconstriction

• magnesium is a modulator of $K_v$$K_v$ channel inactivation

• mitochondrial inactivators like antimycin A , actually lead to an increase in magnesium

Figure 2

• A :

  • 1 = base

  • 5 = towards the top

• B :

  • MitoTracker Green = mitochrondria stain

  • Brefeldin A = Sarcoplasmic Reticulum stain

  • just shows juxtaposition of SR and mitochondria

• C :

  • 3D , relative positioning of mitochondria

• D :

  • clearly different than in PASMCs

  • appear spaced here , and organized in a way

Figure 5

• Study of Voltage Dependent Properties

  • amplitude and steady state activation

• A :

  • voltage-clamp protocol

  • Step Range : $[-100mV,+50mV]$$[\ -100\ mV\ ,\ +50\ mV\ ]$

  • Holding Potential = $-80mV$$-80\ mV$

• B :

  • substantial outward current = more $K_v$$K_v$ channels

• C :

  • less substantial outward current = fewer $K_v$$K_v$ channels

• D :

  • Left Image :

    • PASMC = high current density

    • MASMC = low current density

  • Right Image :

    • derived from fitting activation data

    • PASMC :

      • lower / more negative activation threshold

      • aka "left shift"

      • they have a higher density of $K_v$$K_v$ channels , and higher current density

        • they reach activation quicker than MASMC

        • this makes them more equipped to rapidly repolarize

    • MASMC :

      • shifted to the right

      • higher / more positive activation threshold

      • they have a lower density of $K_v$$K_v$ channels , and a lower current density

      • they reach activation slower than PASMC

  • Dotted Lines = Half-Activation Potentials $[-13.7mV,-7.2mV]$$[\ -13.7\ mV\ ,\ -7.2\ mV\ ]$

    • half-activation potential :

      • its basically just like the $K_m$$K_m$ in enzyme kinetics

        • $K_m=\frac{V_{\text{max}}}{2}$$K_m = \frac{V_{\text{max}}}{2}$

        • "the substrate concentration at which the enzyme reaches half of its maximum velocity"

      • Half-Activation-Potential ( HAP ) = membrane potential at which half of the $K_v$$K_v$ channels are in an activated ( open ) state

      • provides insight into how easily channels are activated in response to changes in membrane potential.

      • more negative = channels are more easily activated ( lower threshold ) , leading to faster responsiveness.

      • more positive = channels are less easily activated ( higher threshold ) , leading to slower responsiveness

  • Slope Factor PASMC = 11.5 mV

  • Slope Factor MASMC = 8.5 mV

  • the slope-factor $K_h$$K_h$ :

    • its the voltage range at which channels are transitioning from non-inactivated state ➡️ inactivated state

    • smaller value = steeper slope = smaller voltage range

    • larger value = shallower slope = larger voltage range

• PASMCs have either larger amount of $K_v$$K_v$ channels , or the channels themselves are just much more conductive than MASMCs

• even though MASMS are larger and have more capacitance , PASMCs have a higher current density

• Methods :

  • Protocol: A 200-ms voltage step was applied, starting from a holding potential of -80 mV to potentials between -100 mV and +50 mV in 10 mV increments at a frequency of 0.1 Hz.

  • Tail Currents: After each depolarization step, cells were repolarized to -20 mV (PASMCs) or 0 mV (MASMCs) for 160 ms. This allowed measurement of tail currents, which reflect the steady-state activation level of $I_{KV}$$I_{KV}$

    • PASMCs have a higher current density

    • MASMCs :

      • a more depolarized repolarization potential (0 mV) was necessary to increase the driving force and amplitude of the tail current , making it easier to assess under these conditions.

  • Observation: MASMCs required a more positive potential to increase tail current size due to their lower $I_{KV}$$I_{KV}$ amplitude.

  • Data Analysis: Current-voltage (I-V) curves were generated using tail current measurements taken 2–3 ms after repolarization. These curves were fitted to an equation incorporating the half-activation potential ( $V_a$$V_a$ ) and the slope factor ( $k_a$$k_a$ ), which reflects the activation steepness.

Table 2

• The comparison demonstrates that mitochondria and $I_{KV}$$I_{KV}$ channels are more tightly coupled functionally in PASMCs than in MASMCs.

• This functional coupling implies that mitochondrial products ( such as ROS , ATP , or Mg ) influence the behavior of $I_{KV}$$I_{KV}$ channels in PASMCs more significantly than in MASMCs , a key distinction for pulmonary artery function in oxygen sensing.

• By showing that mitochondrial inhibition significantly affects $I_{KV}$$I_{KV}$ currents in PASMCs but has much smaller effects in MASMCs ,

  • the table suggests that PASMCs are uniquely adapted for hypoxic sensing , a feature crucial for the oxygen-sensitive regulation in pulmonary circulation.

• Antimycin A and Oligomycin both affect potassium channels in pulmonary (lung) and mesenteric (gut) arterial smooth muscle cells,

  • but their effects are stronger in pulmonary cells

• In pulmonary cells:

  • Both inhibitors make the channels easier to inactivate (they stop conducting more readily).

  • The extent of inactivation is greater, meaning more channels are "turned off" or inactivated by the inhibitors.

• In mesenteric cells:

  • The inhibitors also impact the channels, but the effects are weaker. The channels are not as easily or fully inactivated.

When mitochondrial function is disrupted by the inhibitors, pulmonary channels respond more intensely, likely due to a tighter functional connection between the mitochondria and the potassium channels in lung cells compared to gut cells.

• $K_h$$K_h$ = slope :

  • smaller = steeper slope = channels transition from non-inactivated to inactivated in a smaller voltage range

• $\downarrow$$\downarrow$ in amplitude = more channels have become inactivated

• $V_h$$V_h$ = membrane potential , where 1/2 of $K_v$$K_v$ are inactivated

• $n$$n$ = sample size

• Antimycin A = greater affect on PASMCs

• Oligomycin = affects only $I{K}_V$$IK_V$ activation in PAMCS

  • no affect on inactivation

Figure 8

• Green = mitochondria stain

• Red = plasma membrane stain

• Cytochalasin B = actin disruptor

• Antimyin A = mitochondria ETC blocker

• A :

  • Control

  • No Cytochalasin B present

• B :

  • Added Cytochalasin B

  • they say you can see some changes in organization and distribution of mitochondria with the plasma membrane

    • suggesting mitochondria are associated with actin

• C :

  • normally Antimycin A increases the activation threshold by a huge amount

    • making it very hard for the cell to fire action potential

  • adding Cytochalasin B completely abolishes this affect

    • activation threshold remains basically the same ( unchanged )

    • since it seemed to cause mitochondria to redistribute along plasma membrane ,

      • then this redistribution apparently influences activation properties of the $K_v$$K_v$ channels

• D :

  • normally Antimycin A lowers the inactivation potential

    • causing channels to inactivate sooner

  • adding Cytochalasin B significantly reduces the "change" inactivation potential

    • so the inactivation potential doesn’t shift as much toward a more negative value

    • the inactivation potential experiences a smaller shift toward negative potentials in the presence of Cytochalasin B.

    • channels do not inactivate as easily ( or as early ) as they would with Antimycin A alone.

    • Essentially , Cytochalasin B prevents the channels from becoming overly prone to inactivation.

    • implies that the cytoskeleton , via its effect on mitochondrial distribution , plays a role in regulating how sensitive the I_KV channels are to metabolic changes ( like those induced by mitochondrial inhibitors )

• E :

  • adding Cytochalasin B increases the total amount of current block

    • which decreases the total amplitude of current

  • suggests a synergistic effect where cytoskeletal disruption by Cytochalasin B enhances the inhibitory action of Antimycin A on the I_KV channels

    • likely by affecting mitochondrial proximity or other cellular mechanisms that influence channel behavior