BK channels notes

BK Channel Molecular Identity

• Encoded by the KCNMA1 gene.

• Located on chromosome 10.

• Often referred to as Maxi-K channel.

• Belongs to a group of $Ca^{2+}$-activated $K^+$ channels that includes BK, SK, and IK channels.

• Plays an important role in smooth muscle and nerve repolarisation.

BK Channel Structure

• BK Channels comprise an alpha subunit that is ~ 1200 aa long

  • Extracellular N terminus.

  • 7 transmembrane domains.

  • Pore forms between S5 and S6.

  • Large C terminus.

  • Calcium sensors at RCK domains and $Ca^{2+}$ bowls.

• BK Channels are made up of 4 a subunits (homotetramer).

BK Channel Conductance

• Slope = conductance (pS) ~ 300 pS for BK. Conductance can be calculated using $I=GV$.

BK Channel Experiments

• Voltage Protocol:

  • -60 mV, +150 mV, -100 mV.

  • 100 nM $Ca^{2+}$.

  • 140 mM $K^+$ (symmetrical).

  • $E_K$= 0 mV

  • 37 $^o$C

  • Inside/Out patches.

• Single Channel Current: 60 pA, 100 ms.

• Illustrates channel openings and closings with varying numbers of active channels.

Information from Activation Curve

• Number of channels in patch.

• $V<em>{1/2}$ in each $[Ca^{2+}]</em>I$.

• $\Delta V<em>{1/2}$ (shift in $V</em>{1/2}$ by $Ca^{2+}$).

• Voltage sensitivity.

BK Channel Activation

BK Channel Molecular Identity

• Encoded by the KCNMA1 gene.

• Located on chromosome 10.

• Often referred to as Maxi-K channel.

• Belongs to a group of $Ca^{2+}$-activated $K^+$ channels that includes BK, SK, and IK channels.

• Plays an important role in smooth muscle and nerve repolarisation.

BK Channel Structure

• BK Channels comprise an alpha subunit that is ~ 1200 aa long

  • Extracellular N terminus.

  • 7 transmembrane domains.

  • Pore forms between S5 and S6.

  • Large C terminus.

  • Calcium sensors at RCK domains and $Ca^{2+}$ bowls.

• BK Channels are made up of 4 a subunits (homotetramer).

BK Channel Conductance

• Slope = conductance (pS) ~ 300 pS for BK. Conductance can be calculated using $I=GV$.

BK Channel Activation

• Opened by Voltage and Calcium.

  • Voltage: -100 mV to +100 mV causes channel opening with 100 nM $Ca^{2+}$.

  • Calcium: Increase from 100 nM to 1 μM shifts V
    {1/2} from +100 mV to -10 mV.

Calcium Action on BK Channel

• Calcium acts on the RCK domain and $Ca^{2+}$ bowl in the C terminus of the Slo subunit.

• Mutagenesis shows that both the RCK domain and $Ca^{2+}$ bowls are important.

• D898A mutation abolishes $Ca^{2+}$ sensing (Aspartic Acid (D) changed to neutral Alanine (A)).

BK Channel Blockers

• BK Channels are blocked by IbTx, TEA, Barium & Penitrem A.

BK Channel Openers

• Opened by a wide variety of drugs:

  1. NS compounds

  2. Tamoxifen

  3. Mallotoxin

  4. Evans blue

  5. Oestrogen

  6. Resveratrol (red wine!)

  7. Cibacron Blue

  8. GoSlo-SR compounds

Calcium Action on BK Channel

• Calcium acts on the RCK domain and $Ca^{2+}$ bowl in the C terminus of the Slo subunit.

• Mutagenesis shows that both the RCK domain and $Ca^{2+}$ bowls are important.

• D898A mutation abolishes $Ca^{2+}$ sensing (Aspartic Acid (D) changed to neutral Alanine (A)).

BK Channel Blockers

• BK Channels are blocked by IbTx, TEA, Barium & Penitrem A.

BK Channel Openers

• Opened by a wide variety of drugs:

  1. NS compounds

  2. Tamoxifen

  3. Mallotoxin

  4. Evans blue

  5. Oestrogen

  6. Resveratrol (red wine!)

  7. Cibacron Blue

  8. GoSlo-SR compounds

GoSlo-SR Compounds

• Molecular reduction strategy from Cibacron Blue to GoSlo-SR molecules.

• GoSlo-SR efficacy enhanced as ring D size increases.

• GoSlo-SR efficacy further enhanced by hydrophobic substituents on ring D.

• GoSlo-SR efficacy retained when acidic group on ring C retained.

• GoSlo-SR efficacy retained when $NH_2$ group on ring C removed.

Summary of SAR Work

1. A and B ring appear essential for activity.

2. $NH_2$ group on ring C not essential for effects on BKa.

3. Acidic group on ring C essential.

4. 6 carbons or more on ring D enhances efficacy.

5. Bulky hydrophobic groups on ring D enhance efficacy.

Drug Absorption

• The $SO_3$ group prevents absorption, replacement with COOH allows absorption.

• Comparison: NS compounds with GoSlo-SR compounds.

• Effects remain when $Ca^{2+}$ binding sites are ablated.

Molecular Site of Action

• Homology model based on 2R9R, 3NAF.

• Mutations (S317R, E324A, I323A) affect $Ca^{2+}$ sensitivity and GoSlo-SR-5-6 induced shift in $\Delta V_{1/2}$.

• Amino acids within 4Å of 317 (L400, L313, V406, F409).

• Triple Mutant (L227A:S317R:I326A) concentration dependence and GV curves.

BK Channels and Associated Subunits

• Large conductance voltage- and $Ca^{2+}$-activated $K^+$ channels (also known as Slo or Maxi-K channels)

• 4 pore-forming α subunits, each with auxiliary β, γ subunits

• Expressed in neurons throughout the brain including cerebellum

• BK channel knockout model was first published in 2004

• Immunostained BK channels in the cerebellar cortex (Sausbier et al. 2006).

BK Channel Ablation Effects

• Ataxia & Tremor

• Degraded circadian rhythms

• Reduced breeding efficiency (males and females)

• Reduced body weight

• Smooth muscle hyper-contractility

• Reduced cardio-protection in ischemia models

• Altered salivary secretion

• Reduced cortical collecting duct flow-stimulated net $K^+$ secretion in kidney

• Resistance to alcohol intoxication

• Reduced sino-atrial node firing rate.

• BK Knockout causes ataxia & tremor.

LINGO1 and BK Channels

• LINGO1 is another γ subunit.

• LINGO-1 reduces plasmalemmal BK expression.

• BKα:LINGO1 Co-IP (Human cerebellum).

• LINGO1 is upregulated in PD cerebellum.

• BKα protein is unchanged in PD.

• LINGO-1 reduces plasmalemmal BK expression, affecting total BK expression and surface expression.