Functional Studies of Synthetic Gramicidin Hybrid Ion Channels in CHO Cells

Wesolowski, Ryszard; Sommer, Anna; Arndt, Hans‐Dieter; Koert, Ulrich; Reiß, Philipp; Wimmers, S.; Strauß, Olaf

doi:10.1002/cbic.200600384

Cited by 8 publications

(6 citation statements)

References 28 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…C, Current-voltage relationships from experiments shown in A and B. The currents in the I-V plots are expressed as current densities which were calculated as described previously (Wesolowski et al, 2007) Among the four PMCA isoforms, PMCA1 and PMCA2 have been demonstrated to colocalize with PSD95 in the OPL Duncan et al, 2006). TMEM16B and PMCAs are absent from photoreceptor synapses of mice that lack MPP4, possibly because of the loss of PSD95 in the mutant terminals.…”

Section: Discussionmentioning

confidence: 99%

TMEM16B, A Novel Protein with Calcium-Dependent Chloride Channel Activity, Associates with a Presynaptic Protein Complex in Photoreceptor Terminals

Stöhr¹,

Heisig²,

Benz³

et al. 2009

J. Neurosci.

196

186

View full text Add to dashboard Cite

Photoreceptor ribbon synapses release glutamate in response to graded changes in membrane potential evoked by vast, logarithmically scalable light intensities. Neurotransmitter release is modulated by intracellular calcium levels. Large Ca 2ϩ -dependent chloride currents are important regulators of synaptic transmission from photoreceptors to second-order neurons; the molecular basis underlying these currents is unclear. We cloned human and mouse TMEM16B, a member of the TMEM16 family of transmembrane proteins, and show that it is abundantly present in the photoreceptor synaptic terminals in mouse retina. TMEM16B colocalizes with adaptor proteins PSD95, VELI3, and MPP4 at the ribbon synapses and contains a consensus PDZ class I binding motif capable of interacting with PDZ domains of PSD95. Furthermore, TMEM16B is lost from photoreceptor membranes of MPP4-deficient mice. This suggests that TMEM16B is a novel component of a presynaptic protein complex recruited to specialized plasma membrane domains of photoreceptors. TMEM16B confers Ca 2ϩ -dependent chloride currents when overexpressed in mammalian cells as measured by halide sensitive fluorescent protein assays and whole-cell patch-clamp recordings. The compartmentalized localization and the electrophysiological properties suggest TMEM16B to be a strong candidate for the long sought-after Ca 2ϩ -dependent chloride channel in the photoreceptor synapse.

show abstract

Section: Discussionmentioning

confidence: 99%

TMEM16B, A Novel Protein with Calcium-Dependent Chloride Channel Activity, Associates with a Presynaptic Protein Complex in Photoreceptor Terminals

Stöhr¹,

Heisig²,

Benz³

et al. 2009

J. Neurosci.

196

186

View full text Add to dashboard Cite

show abstract

“…19,20 Gramicidin-hybrid channels were transferred into Chinese hamster ovary (CHO) cells and studied in vivo by patch clamp. 21 The Cs þ ion selectivity of compound 6 seen in BLM measurements could also be observed in CHO cells. For the case of tetrahydrofuranamino acids, the electrophysiological response of trabecular meshwork cells to tetrahydrofuran-gramicidin hybrid channels was investigated.…”

Section: Introductionmentioning

confidence: 85%

“…Tetrahydrofuran- and tetrahydropyran-amino acids could also be incorporated into the gramicidin motif to generate hybrid channels which were functionally characterized by conductance measurements. , Gramicidin-hybrid channels were transferred into Chinese hamster ovary (CHO) cells and studied in vivo by patch clamp . The Cs + ion selectivity of compound 6 seen in BLM measurements could also be observed in CHO cells.…”

Section: Narrow Ion-conductance Pathway: Gramicidin Amentioning

confidence: 99%

Ion-Channels: Goals for Function-Oriented Synthesis

Reiß

Koert

2013

Acc. Chem. Res.

Self Cite

View full text Add to dashboard Cite

Ion channels provide a conductance pathway for the passive transport of ions across membranes. These functional molecules perform key tasks in biological systems such as neuronal signaling, muscular control, and sensing. Recently, function-oriented synthesis researchers began to focus on ion channels with the goal of modifying the function of existing ion channels (ion selectivity, gating) or creating new channels with novel functions. Both approaches, ion channel engineering and de novo design, have involved synthetic chemists, biochemists, structural biologists, and neurochemists. Researchers characterize the function of ion channels by measuring their conductance in samples of biological membranes (patch clamp) or artificial membranes (planar lipid bilayers). At the single molecule level, these measurements require special attention to the purity of the sample, a challenge that synthetic chemists should be aware of. Ideally, researchers study the function of channels while also acquiring structural data (X-ray, NMR) to understand and predict how synthetic modifications alter channel function. Long-term oriented researchers would like to apply synthetic ion channels to single molecule sensing and to implantat these synthetic systems in living organisms as tools or for the treatment of channelopathies. In this Account, we discuss our own work on synthetic ion channels and explain the shift of our research focus from a de novo design of oligo-THFs and oligo-THF-amino acids to ion channel engineering. We introduce details about two biological lead structures for ion channel engineering: the gramicidin β(6,3) helix as an example of a channel with a narrow ion conductance pathway and the outer membrane porins (OmpF, OmpG) with their open β-barrel structure. The increase and the reversal of ion selectivity of these systems and the hydrophobic match/mismatch of the channel with the phospholipid bilayer are of particular interest. For engineering ion channels, we need to supplement the single-point attachment of a synthetic modulator with the synthesis of a more challenging two-point attachment. The successful function-oriented synthesis of ion channels will require interdisciplinary efforts that include new electrophysiology techniques, efficient synthesis (peptide/protein/organic), and good structural analysis.

show abstract

“…Terminal-charged amphiphilic compounds, [7] can self-assemble into voltage-dependent pores and rigid push-pull rods [8] with permanent axial macrodipoles to give voltage-sensitive pores. Compared with these self-assembled pores, the channel-active b 6,3 -helix of gramicidin A (gA) is structurally well defined. [9] ( Figure 1)…”

mentioning

confidence: 99%

“…In a continuation of our studies on the implantation of synthetic gramicidin-hybrid channels into cells (trabecular meshwork cells, [5] CHO cells [6] ), we turned to synthetic voltage-gated channels. Terminal-charged amphiphilic compounds, [7] can self-assemble into voltage-dependent pores and rigid push-pull rods [8] with permanent axial macrodipoles to give voltage-sensitive pores.…”

mentioning

confidence: 99%