Goldstein Laboratory Research

Stay up to date on research within the Goldstein Laboratory at Loyola University University Health Sciences Division and learn more about the five research directions pursued by Dr. Goldstein and fellow researchers.

Accessory Subunits—discovery, roles in health and disease, and structural basis for function

Ion channels are composed of pore-forming subunits and accessory subunits that determine where, when and how the pores function. Accessory subunits are the power behind the throne, determining the differences in how channels operate from tissue to tissue (and from cell to cell within the same tissue) allowing the heart to beat slowly and neurons to respond in milliseconds (Figs 1 and 2). MinK (encoded by KCNE1) has just 129 amino acids and a single transmembrane domain. In the heart, MinK assembles with KCNQ1 (to form IKs channels) establishing the conductance, gating, regulation and anti-arrhythmic drug sensitivity of the mixed complexes. In mutant form, MinK is linked to cardiac arrhythmia and deafness due to changes in these same attributes. It appeared that MinK was unique until we found a family of genes encoding MinK-related peptides (MiRPs) in 1998. Since then we have shown roles for MiRP1 (encoded by KCNE2) and MiRP2 (encoded by KCNE3) in the heart and skeletal muscle. Other accessory subunits we study include KChIPs, DPPs, 14-3-3 and KCTDs. Sample citations (1-6).

Fig 1. MinK (E1) slows the movement of voltage sensors (black traces are current, red traces sensor movement seen by site-directed fluorescence (6).



Figure 2. X-ray crystallography shows KCTD5 to be a tetramer. Panel shows the C module in high and low salt crystals, Dementieva 2009.‌

 

 

 

 

 

The K2Ps—discovery of a family of potassium channels that produce background currents

Hodgkin and Huxley showed background potassium currents as central physiology but for 50 years their molecular nature was uncertain. We discovered K2P channels in yeast, worms, flies and mammals in 1995. The channels are novel in structure and function: they bear 2 pore-forming domains in each subunit. In humans, there are 15 KCNK genes for K2P channels and their roles in the heart and nervous system have begun to emerge, for example, as targets of volatile anesthetics and unique forms of regulation in development and across tissues, for example, modification of ion selectivity via alternative initiation translation (ATI), Figs 3 and 4. Sample citations (7-11).

Fig 3. (left) A model for ΔK2PØ channels from drosophila shows bilateral symmetry with a fourfold symmetric selectivity filter (12).

Fig 4. (right) K2P channels have a 2P/4TM topology and are open rectifiers passing large outward currents under normal conditions (high internal and low external K+, Phys) and show a near linear current-voltage relationship in symmetrical K+ (Sym) (13)

 

 

 

 

SUMO—a pathway unexpectedly discovered to control the activity of ion channels at the cell surface

Recently, we identified a novel enzymatic pathway at the cell surface that regulates the opening and closing of ion channels: post-translational modification with the protein called SUMO. SUMO was previously known only to determine the activity of transcription factors in the nucleus. Enzymes for sumoylation and desumoylation were shown to reside at the plasma membrane and now has been seen in all mammalian cells studied and the number of ion channels recognized to be modulated by the pathway is rapidly increasing, Fig 5. Sample citations (15, 16). 

 

 

 

 

Development of new genetic and high throughput methods for ion channels

A major focus is de novo development of peptide neurotoxins for “orphan” receptors. Among the most powerful tools in the arsenal for studies of the heart and nervous system, these potent natural products are not selective for the vast majority of membrane receptors critical to normal physiology and disease and we use phage display to generate de novo, high affinity specific peptides to study and modify the the function of the channels in vivo, see (17, 18), Fig 6.

Other ongoing work is allowing studies of single ion channel complexes in living cells by total internal reflection microscopy and fluorescence energy transfer, see (14); reveal the mechanism of operation of Killer RNA viruses that impact agriculture, commercial fermentation and fungal infections in immuno-compromised patients (a coupled toxin-immunity system acting via fungal two P domain channels), see, (19); and use random mutation and selective pressure to clone or investigate potassium channel structure and function (20).

Fig 6. (above) Library design and phage sorting on KcsA channels selects phage expressing the de novo toxin Hui1 and the native toxin HmK by binding. (A) Combinatorial library construction give a diversity of 1.5 million toxins. (B) Sorting yields a selective de novo toxin and a promiscuous native toxin (18).

Mechanism, diagnosis and treatment

We study disorders of the heart, skeletal muscle, and the nervous system that are inherited and acquired as well as sudden infant death syndrome (SIDS) to understand cause, provide diagnostic tools, develop therapeutic strategies and avoid untoward effects of medications. Thus, rare inherited mutations of MiRP1 are associated with the cardiac arrhythmia long QT syndrome (LQTS) and sudden death while a common polymorphism present in 1.6% of the general population predisposes to a prevalent and equally dangerous disorder drug-induced LQTS (Fig 7). Sample citations (21-23).

Figure 7. A SNP in SCN5A, a cardiac sodium channel linked to SIDS.
(a) dHPLC waveform and sequencing of wild type and S1103Y variant. (b) Topology of sodium channel encoded by SCN5A indicating the location of the S1103Y missense change (red), the four homologous membrane domains (DI-DIV), the pore-forming (P) loops and voltage sensing segments (+).

 

 

 

 

1. Tai KK & Goldstein SAN (1998) The conduction pore of a cardiac potassium channel. Nature 391:605-608.

2. Abbott GW & Goldstein SAN (1998) A superfamily of small potassium channel subunits: form and function of the MinK-related peptides (MiRPs). Quarterly Rev Biophys 31:357-398.

3. Abbott GW, et al. (2001) MiRP2 forms potassium channels in skeletal muscle with Kv3.4 and is associated with periodic paralysis. Cell 104(2):217-231.

4. Kim LA, et al. (2004) Three-dimensional structure of I(to); Kv4.2-KChIP2 ion channels by electron microscopy at 21 Angstrom resolution. Neuron 41(4):513-519.

5. Xiong D, li T, Plant LD, & Goldstein SAN (2017) KCNE1-DEPENDENT SUMOYLATION OF KV7.1 SUBUNITS DETERMINES THE VOLTAGE-DEPENDENCE OF CARDIAC IKS CHANNELS. Biophys J:in press.

6. Ruscic KJ, et al. (2013) IKs channels open slowly because KCNE1 accessory subunits slow the movement of S4 voltage sensors in KCNQ1 pore-forming subunits. Proc Natl Acad Sci U S A 110(7):E559-566.

7. Ketchum KA, Joiner WJ, Sellers AJ, Kaczmarek LK, & Goldstein SAN (1995) A new family of outwardly-rectifying potassium channel proteins with two pore domains in tandem. Nature 376:690-695.

8. Goldstein SAN, Price LA, Rosenthal DN, & Pausch MH (1996) ORK1, a potassium-selective leak channel with two pore domains cloned from Drosophila melanogaster by expression in Saccharomyces cerevisiae.I93:13256-13261.

9. Bockenhauer D, Zilberberg N, & Goldstein SAN (2001) KCNK2: reversible conversion of a hippocampal potassium leak into a voltage-dependent channel. Nature Neuroscience 4:486-491.

10. Thomas D, Plant LD, Wilkens CM, McCrossan ZA, & Goldstein SAN (2008) Alternative Translation Initiation in Rat Brain Yields K2P2.1 Potassium Channels Permeable to Sodium. Neuron 58(6):859-870.

11. Plant LD, Zuniga L, Araki D, Marks JD, & Goldstein SA (2012) SUMOylation silences heterodimeric TASK potassium channels containing K2P1 subunits in cerebellar granule neurons. Sci Signal 5(251):ra84.

12. Kollewe A, Lau AY, Sullivan A, Roux B, & Goldstein SAN (2009) A structural model for K2P potassium channels based on 23 pairs of interacting sites and continuum electrostatics. J Gen Physiol 134(1):53-68.

13. Goldstein SAN, Bockenhauer D, O'Kelly I, & Zilberberg N (2001) Potassium leak channels and the KCNK family of two-P-domain subunits. Nat Rev Neurosci 2:175-184.

14. Plant LD, Dowdell EJ, Dementieva IS, Marks JD, & Goldstein SAN (2011) SUMO modification of cell surface Kv2.1 potassium channels regulates the activity of rat hippocampal neurons. J Gen Physiol 137(5):441-454.

15. Rajan S, Plant LD, Rabin ML, Butler MH, & Goldstein SAN (2005) Sumoylation silences the plasma membrane leak K+ channel K2P1. Cell 121:37-47.

16. Plant LD, Marks JD, & Goldstein SA (2016) SUMOylation of NaV1.2 channels mediates the early response to acute hypoxia in central neurons. eLife 5.

17. Takacs Z, et al. (2009) A designer ligand specific for Kv1.3 channels from a scorpion neurotoxin-based library. Proceedings of the National Academy of Sciences 106:22211-22216.

18. Zhao R, et al. (2015) Designer and natural peptide toxin blockers of the KcsA potassium channel identified by phage display. Proc Natl Acad Sci U S A 112(50):E7013-7021.

19. Sesti F, Shih TM, Nikolaeva N, & Goldstein SA (2001) Immunity to K1 killer toxin: internal TOK1 blockade. Cell 105(5):637-644.

20. Sesti F, Rajan S, Gonzalez-Colaso R, Nikolaeva N, & Goldstein SAN (2003) Hyperpolarization moves S4 sensors inward to open MVP, a methanococcal voltage-gated potassium channel. Nature Neuroscience 6(4):353-361.

21. Sesti F, et al. (2000) A common polymorphism associated with antibiotic-induced cardiac arrhythmia. Proc Natl Acad Sci 97(19):10613-10618.

22. Plant LD, et al. (2006) A common cardiac sodium channel variant associated with sudden infant death in African Americans, SCN5A S1103Y. J Clin Invest 116:430-435.

23. Silva JR & Goldstein SA (2013) Voltage-sensor movements describe slow inactivation of voltage-gated sodium channels II: a periodic paralysis mutation in Na(V)1.4 (L689I). J Gen Physiol 141(3):323-334.