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Subconductance gating and voltage sensitivity of sarcoplasmic reticulum K(+) channels: a modeling approach.

ABSTRACT: Sarcoplasmic reticulum (SR) K(+) channels are voltage-regulated channels that are thought to be actively gating when the membrane potential across the SR is close to zero as is expected physiologically. A characteristic of SR K(+) channels is that they gate to subconductance open states but the relevance of the subconductance events and their contribution to the overall current flowing through the channels at physiological membrane potentials is not known. We have investigated the relationship between subconductance and full conductance openings and developed kinetic models to describe the voltage sensitivity of channel gating. Because there may be two subtypes of SR K(+) channels (TRIC-A and TRIC-B) present in most tissues, to conduct our study on a homogeneous population of SR K(+) channels, we incorporated SR vesicles derived from Tric-a knockout mice into artificial membranes to examine the remaining SR K(+) channel (TRIC-B) function. The channels displayed very low open probability (Po) at negative potentials (?0 mV) and opened predominantly to subconductance open states. Positive holding potentials primarily increased the frequency of subconductance state openings and thereby increased the number of subsequent transitions into the full open state, although a slowing of transitions back to the sublevels was also important. We investigated whether the subconductance gating could arise as an artifact of incomplete resolution of rapid transitions between full open and closed states; however, we were not able to produce a model that could fit the data as well as one that included multiple distinct current amplitudes. Our results suggest that the apparent subconductance openings will provide most of the K(+) flux when the SR membrane potential is close to zero. The relative contribution played by openings to the full open state would increase if negative charge developed within the SR thus increasing the capacity of the channel to compensate for ionic imbalances.

SUBMITTER: Matyjaszkiewicz A

PROVIDER: S-EPMC4623209 | biostudies-literature | 2015 Jul

REPOSITORIES: biostudies-literature

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Subconductance gating and voltage sensitivity of sarcoplasmic reticulum K(+) channels: a modeling approach.

Matyjaszkiewicz Antoni A Venturi Elisa E O'Brien Fiona F Iida Tsunaki T Nishi Miyuki M Takeshima Hiroshi H Tsaneva-Atanasova Krasimira K Sitsapesan Rebecca R

Biophysical journal 20150701 2

Sarcoplasmic reticulum (SR) K(+) channels are voltage-regulated channels that are thought to be actively gating when the membrane potential across the SR is close to zero as is expected physiologically. A characteristic of SR K(+) channels is that they gate to subconductance open states but the relevance of the subconductance events and their contribution to the overall current flowing through the channels at physiological membrane potentials is not known. We have investigated the relationship b ...[more]

PMID: 26200862

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Project description:A large (565 kDa) junctional sarcoplasmic reticulum (SR) protein, the ryanodine receptor (RYR), may play both a structural and a functional role in the mechanism of skeletal muscle excitation-contraction coupling. Recently, the primary amino acid sequence of the RYR has been elucidated. In this paper, we introduce an immunological approach to examine the functional (electrophysiological) properties of the RYR when it is incorporated into planar lipid bilayers. The effects of two polyclonal antibodies against the SR junctional face membrane (JFM) and the RYR (anti-JFM and anti-RYR) were tested on the single-channel gating properties of the RYR SR Ca2(+)-release channel. Junctional SR vesicles were fused into planar lipid bilayers in solutions containing caesium salts. Solutions were designed to minimize the background conductances of the SR K+ and Cl- channels. Three actions of the anti-JFM antibody were distinguished on the basis of single-channel gating and conductance. The anti-RYR antibody had a single action, a simultaneous decrease in single-channel open probability (Po) and conductance. Both antibodies appear to alter single-channel gating by disrupting the Ca2(+)-activation mechanism of the channel. Anti-RYR-antibody-induced gating abnormalities were reversed by ATP, although the ATP-re-activated channels had altered gating kinetics. Two antigenic regions, recognizing the anti-RYR antibody, in the C-terminal end of the RYR primary amino acid sequence contain or are closely associated with putative ligand (Ca2+ and ATP)-binding sites identified previously. Our results demonstrate (1) that the antibodies induced abnormal gating (decreased open probability and stabilization of subconducting states) of SR release channels, and (2) that abnormal gating is not associated with physical obstruction or alteration of the conduction pathway. Thus antibodies directed at specific regions of the RYR (e.g. putative ligand-binding sites) can be used as effective probes with which to study the structural and functional properties of the SR Ca2(+)-release channel gating at the single-channel level.

Project description:Ion channel activity in the plasma membrane of living cells generates voltage changes that are critical for numerous biological functions. The membrane of the endoplasmic/sarcoplasmic reticulum (ER/SR) is also endowed with ion channels, but whether changes in its voltage occur during cellular activity has remained ambiguous. This issue is critical for cell functions that depend on a Ca2+ flux across the reticulum membrane. This is the case for contraction of striated muscle, which is triggered by opening of ryanodine receptor Ca2+ release channels in the SR membrane in response to depolarization of the transverse invaginations of the plasma membrane (the t-tubules). Here, we use targeted expression of voltage-sensitive fluorescence resonance energy transfer (FRET) probes of the Mermaid family in differentiated muscle fibers to determine whether changes in SR membrane voltage occur during depolarization-contraction coupling. In the absence of an SR targeting sequence, FRET signals from probes present in the t-tubule membrane allow calibration of the voltage sensitivity and amplitude of the response to voltage-clamp pulses. Successful SR targeting of the probes was achieved using an N-terminal domain of triadin, which completely eliminates voltage-clamp-activated FRET signals from the t-tubule membrane of transfected fibers. In fibers expressing SR-targeted Mermaid probes, activation of SR Ca2+ release in the presence of intracellular ethyleneglycol-bis(β-amino-ethyl ether)-N,N,N',N'-tetra acetic acid (EGTA) results in an accompanying FRET signal. We find that this signal results from pH sensitivity of the probe, which detects cytosolic acidification because of the release of protons upon Ca2+ binding to EGTA. When EGTA is substituted with either 1,2-bis(o-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid or the contraction blocker N-benzyl-p-toluene sulfonamide, we find no indication of a substantial change in the FRET response caused by a voltage change. These results suggest that the ryanodine receptor-mediated SR Ca2+ efflux is well balanced by concomitant counterion currents across the SR membrane.

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Subconductance gating and voltage sensitivity of sarcoplasmic reticulum K(+) channels: a modeling approach.

Publications

Subconductance gating and voltage sensitivity of sarcoplasmic reticulum K(+) channels: a modeling approach.

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