Binding of TRIP8b to the cyclic nucleotide binding domain (CNBD) of mammalian hyperpolarization-activated cyclic nucleotide-gated (HCN) channels prevents their regulation by cAMP. Since TRIP8b is expressed exclusively in the brain, we envisage that it can be used for orthogonal control of HCN channels beyond the central nervous system. To this end, we have identified by rational design a 40-aa long peptide (TRIP8bnano) that recapitulates affinity and gating effects of TRIP8b in HCN isoforms (hHCN1, mHCN2, rbHCN4) and in the cardiac current If in rabbit and mouse sinoatrial node cardiomyocytes. Guided by an NMR-derived structural model that identifies the key molecular interactions between TRIP8bnano and the HCN CNBD, we further designed a cell-penetrating peptide (TAT-TRIP8bnano) which successfully prevented β-adrenergic activation of mouse If leaving the stimulation of the L-type calcium current (ICaL) unaffected. TRIP8bnano represents a novel approach to selectively control HCN activation, which yields the promise of a more targeted pharmacology compared to pore blockers.
Protein uptake at the interface of a millimeter-sized
air bubble
in water is investigated by a recently developed differential interferometric
technique. The technique allows the study of capillary waves with
amplitudes around 10–9 m, excited at the surface
of the bubble by an electric field of intensity on the order of 10
V/cm. When one studies the resonant modes of the bubble (radial and
shape modes), it is possible to assess variations of interfacial properties
and, in particular, of the net surface charge as a function of bulk
protein concentration. Sensing the interfacial charge, the technique
enables us to follow the absorption process in conditions of low concentrations,
not easily assessable by other methods. We focus on bovine serum albumin
(BSA) and lysozyme as representatives of typical globular proteins.
To provide comprehensive insight into the novelty of the technique,
we also investigated the equilibrium adsorption of sodium dodecyl
sulfate (SDS) ionic surfactant for bulk concentrations at hundreds
of times lower than the Critical Micelle Concentration (CMC). Results
unveil how the absorption of charged molecules affects the amplitudes
of the bubble resonant modes even before affecting the frequencies
in a transition-like fashion. Different adsorption models are proposed
and developed. They are validated against the experimental findings
by comparing frequency and amplitude data. By measuring the charging
rate of the bubble interface, we have followed the absorption kinetics
of BSA and lysozyme recognizing a slow, energy barrier limited phenomena
with characteristic times in agreement with data in the literature.
The evaluation of the surface excess concentration (Γ) of BSA
and SDS at equilibrium is obtained by monitoring charge uptake. At
the investigated low bulk concentrations, reliable comparisons with
literature data from equilibrium surface tension isotherm models are
reported.
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