Cellular Neurobiology BIPN140. 1st Midterm Exam October 18 th, Tuesday Material covered: Lectures 1-6 & Reading

Transcription

1 Cellular Neurobiology BIPN140 1st Midterm Exam October 18 th, Tuesday Material covered: Lectures 1-6 & Reading Review session October 17 th 3500 Pacitic Hall, 6-8 pm (access code is ) Come with questions! PS 2 answers and PS 3 will be posted October 14 th ; PS 3 answers will be posted on October 17 th

2 Figure 6.3 The structure of the nach receptor/channel (Part 2) Electron micrograph

3 Figure 6.3 The structure of the nach receptor/channel (Part 1)

4 Figure 4.6 Topology of principal subunits of voltage-gated Na +, Ca 2+, K +, and Cl channels (Part 1)

5 Figure 4.6 Topology of principal subunits of voltage-gated Na +, Ca 2+, K +, and Cl channels (Part 2)

6 Alignment of amino acids in the S4 domain of voltage-dependent channels R = arginine; K = lysine

7 Box 4B Expression of Ion Channels in Xenopus Oocytes inject K channel mrna

8 Mutational analysis of Shaker (G KA ) potassium channel activation wild type (control) R > A (mutant) (arg > ala)

9 Figure 4.7 A charged voltage sensor permits voltage-dependent gating of ion channels

10 Figure 4.9 Structure of a mammalian voltage-gated K + channel (Part 3)

11 Figure 4.9 Structure of a mammalian voltage-gated K + channel (Part 4) paddle

12 Figure 4.6 Topology of principal subunits of voltage-gated Na +, Ca 2+, K +, and Cl channels (Part 3) and Shaker (K A )

13 Mutational analysis of Shaker (GKA) potassium channel inactivation NH2-terminus deletion Ball and Chain model for Shaker K+ channel inactivation

14 Box 4D(1) Diseases Caused by Altered Ion Channels (Part 2) Generalized epilepsy with febrile seizures Muscle weakness Muscle paralysis

15 Box 4D(1) Diseases Caused by Altered Ion Channels (Part 1) Familial hemiplegic migraine Episodic ataxia type 2 Congenital stationary night blindness

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17 Background Ion channels are expressed at specific densities in specific neurons (10/µm 2 to 10,000/µm 2 ) The Question What regulates the number of ion channels in the membrane? The Approach Take advantage of the large number of Kir family members They are attractive to study because the proteins and thus the genes are small and easy to manipulate experimentally Compare the amino acid sequences of the proteins, and mutate the DNA to identify trafficking motifs (amino acid sequences) Analyze expression of Kir channels in Xenopus oocytes and other heterologous expression systems

18 Figure 1 Kir2.1 surface expression depends on its COOH-terminal sequence Full length protein is 428 aa Replacement of aa of Kir2.1 with those of other family members well expressed poorly expressed

19 Figure 2 COOH-terminal deletions reduce the number of surface Kir2.1 channels, with no change in channel properties Could the deletion be affecting channel protein folding or assembly of tetramers? This seems unlikely because, P open, open, and closed are normal. Generating mutations identified the trafficking motif in Kir2.1...

20 Figure 4 FYCENE promotes ER export and surface expression of membrane proteins aa of Kir2.1, containing FCYENE, or mutated ACAENE or FCYANA, were fused to the COOH-terminus of GFP-tagged test proteins expressed in COS7 cells (a cell line) A Kir3.1 D GABA B R1a FYCENE drives Kir3.1 from the ER to the Golgi and GABA B R1a from the ER to the Golgi and the surface membrane. scale bars, 25 µm FYCENE = phenylalanine-tyrosine-cysteine-glutamic acid-asparagine-glutamic acid

21 Conclusions: 1) Kir2.1 and Kir1.1 contain ER-to-Golgi-to surface forward trafficking signals 2) These signals are not required for channel folding, assembly or gating, but are required for export of channels 3) The variation in forward trafficking signals ( zip codes ) in different potassium channels may regulate surface channel density in vivo. 4) However trafficking in neurons, and in vivo, are not addressed here! SCIENCE VOL May 2001

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23 Spontaneous activity Conductances generating spontaneous activity Beating/pacemaking Generated by the two squid giant axon conductances, fast voltage-dependent GNa and fast voltage-dependent GK, plus 3) a steady voltage-independent GNa and 4) a rapidly inactivating voltage-dependent GKA. 3) produces a Na leak current that acts to depolarize the neuron in between APs; 4) slows down the rate of depolarization and return to threshold between APs. Bursting Generated by all 4 of the conductances for beating, plus 5) a slow voltage-dependent GNa and 6) a slow voltage-dependent GK These conductances generate the slow waves of depolarization and hyperpolarization. The bursts superimposed on the peaks of Vm are generated by the 4 beater conductances.