1. Membrane Biophysics 10/2014

2. Anion Channels Selectivity gradient Plasma membrane; intracellular organelle membranes Set Resting Potential Provide transport, excitability and inhibition Activated by Hyperpolarization Cell Swelling pH Levels

3. Double-Barreled Structure of Cl Channel (CLC Family)

4. Double-Barreled Structure of Cl Channel

5. 3-D crystal structure CLC

6. CLC Family Members CLC-0; 1 st to be studied CLC-1; Skeletal muscle CLC-2; Broadly expressed CLC-K; Kidney epithelia and inner ear cells CLC-3; Intracellular, synaptic vesicles and organelles CLC-4; Vesicular channel CLC-5; Endosomal channel CLC-6; Intracellular channel CLC-7; Lysosomal channel

8. CLC-1 Activity dependent 70-80% RM Skeletal muscle

9. CLC-2 Important for cell-to-cell communication and survival in early development Activated by: – Hyperpolarization – Cell swelling – Acidic pH

10. CLC-K Homology of CLC-K1 and Ka and CLC-K2 and Kb ~90% Require barrtin

11. CLC-3 Intracellular; endosomes and synaptic vesicles Modulates Ca 2+ activated Cl - currents

12. CLC-4; CLC-5 Intracellular membrane Relatively mysterious Extreme outward rectification Inhibition by extracellular acidic pH

13. Cystic Fibrosis Transmembrane Conductance Regulator cAMP activated Expressed in apical membrane of many cell types Several phosphorylated sites required to open channel Regulates other ion channels

15. Swelling Activated Chloride Channels I Cl,swell Moderate outward rectification Likely 2 nd messenger, not mechanically activated (not time-dependent)

16. Ca 2+ -Activated Cl - Channels Modulate excitability with afterpotentials Regulate tonus of smooth muscles Signal transduction Transepithelial transport Range from 1-70 pS single-channel conductances

17. Intracellular Chloride Channels Overexpression brings to PM Little is known about the native tissue Near dense-core vesicles

18. Ligand Gated Chloride Channels Excitatory neurotransmitter binding in early development Fast-inhibitory neurotransmitter binding – GABA A&C (brain), glycine (spinal cord)

20. GABA A 19 mammalian members have been isolated Pentameres Obscure function in nonneuronal tissue 3 open states

21. GABA c Higher sensitivity to GABA Smaller currents Do not desensitize

22. Glycine

24. CLC-1 Channels CLC-1 contributes 70-80% of the resting membrane conductance of skeletal muscles CLC-1 mutations altering common gating cause myotonia congenita – genetic neuromuscular channelopathy in which an initiated muscle contraction fails to terminate Bennetts and Parker used a Model of Cl-/H+ transport in a prokaryotic CLC CL-/H+ antiporter to model CLC-1

25. CLC Channels Homodimers Each subunit has its own separate, identical ion conducting pore 2 Gates regulate channel acitivty – Protopore Gate, which regulates each individual pore – Common gate, which regulates both pore simultaneously Duran et al. 2010

27. Tyrosine (Y) nonpolar Alanine (A) nonpolar Phenylalanine (F) nonpolar Histidine, positive charge Lysine (K), positive charge Glutamic Acid (E) negative charge Most severe effect seen in Y578E, but was unable to fit to curve Y578 Mutations Alter Gating of CLC-1

28. Zn Interacts with the Extracellular Surface of CLC-1 to Inhibit Channel Activity

29. Y578 Mutations Alter CLC inhibition by Zn E232, Glutamic Acid = negative charge Insensitive to Zn Y578A and Y578F are nonpolar Y578E is negatively charged Above mutations negate favorable interactions with E232 Inhibited by Zn Y578K and Y578H have positive charged Above mutations promote favorable interactions with E232

30. Salt Bridge Formation Between R300, R304 and D265

32. How do Y578 Mutants effect NAD + Inhibition of CLC-1 Channels?

33. NAD+ Metabolite Inhibits CLC1-1 Via Y578 Y578 mutants were unaffected by 3mM NAD+ whereas WT was inhibited Open Symbols = no NAD Closed = 3mM NAD

34. Mutations to Y578 alter CBS interactions with CLC-1

35. Charge Swap mutations made to residues that form salt bridges K195D --  Lysine (+) to Aspartic Acid (-) D579K ---  Aspartic Acid (-) to Lysine (+) Small effects seen on protopore gating

36. Open Symbols = no NAD Closed = 3mM NAD K195D --  Lysine (+) to Aspartic Acid (-) D579K ---  Aspartic Acid (-) to Lysine (+)

39. Background

40. Evidence for Ano2 KO

42. No Transient Cl - Currents Remain in Ano2 -/-

44. Electro-Olfactogram

45. Functional EOG Recordings in Fluid Phase

46. Functional EOG Recording in Air Phase No significant difference between genotypes

47. Ano2 -/- Mice Do Not Have Olfactory Deficits

48. Conclusions Ca 2+ -activated Cl - currents are absent from MOE in Ano2 -/- mice Peak amplitude of olfactory epithelia responses decreased when stimulated with liquid in KO mice No detectable difference in air phase EOG amplitude or in animal behavior tests Ca 2+ -activated Cl - channels not essential for olfaction

50. A mutation of CFTR, a chloride channel crucial to maintaining salt and water homestasis in epethial tissues, is the cause of cystic fibrosis CFTR is a ATP binding protien ATP provides the energy required to open the pore of the channel PKA phosphorylation regulates activity of channel

51. ATP Stimulates WT-CFTR

52. Mutation G551D alters ATP’s effect on CFTR G551 is conserved in ABC binding proteins Glycine = uncharged Aspartic Acid = (-) charged ATP = (-) charged G551D mutant has a low open probability, therefore VX-770 was used to increase the open probability Following ATP washout, a biphasic response is seen ( a rapid current increase then slow decay)

53. WT decay: Fast Phase = < 1s, Slow Phase = 29.6 sG551D-CFTR decay = 31.1 s s Two phases of current decay in WT attributed to to disassociation from 2 ATP binding site. The Fast phase is attributed to site 2, which has a lower affinity for ATP. The slow phase is attributed to site one, which has a higher affinity for ATP G551 is the second ATP binding site. Here, the decay is similar to the slow phase of the WT, indicating ATP dissociation from site 1

54. G551D Mutant Causes ATP Binding Site 2 to Inhibit Current Dissociation of ATP at Site 2 Dissociation of ATP at Site 1 ATP Binds at Site 1 ATP Binds at Site 2

55. Reduction of [ATP] Increases Current in G551D Mutant ATP has a higher affinity for Site 1 Without VX-770, I is very low….effect of change in [ATP] still apparent

56. Y1219 in Site 2 Plays a Role in ATP Binding To Test if G551D site 2 mutant was indeed inhibitory… Mutation to other nonpolar/uncharged AA – Y1219F, Y1219I, Y1219G – Known to alter ATP affinity to Site 2

57. Reducing ATP affinity at Site 2 Alters Current Decay

58. Like Charge Mutations to G551 have similar effects