J. Peter Ruppersberg

Gating of Ca2+-Activated K+ Channels Controls Fast Inhibitory Synaptic Transmission at Auditory Outer Hair Cells

Fast inhibitory synaptic transmission in the central nervous system is mediated by ionotropic GABA or glycine receptors. Auditory outer hair cells present a unique inhibitory synapse that uses a Ca2+-permeable excitatory acetylcholine receptor to activate a hyperpolarizing potassium current mediated by small conductance calcium-activated potassium (SK) channels. It is shown here that unitary inhibitory postsynaptic currents at this synapse are mediated by SK2 channels and occur rapidly, with rise and decay time constants of approximately 6 ms and approximately 30 ms, respectively. This time course is determined by the Ca2+ gating of SK channels rather than by the changes in intracellular Ca2+. The results demonstrate fast coupling between an excitatory ionotropic neurotransmitter receptor and an inhibitory ion channel and imply rapid, localized changes in subsynaptic calcium levels.

Argiotoxin detects molecular differences in AMPA receptor channels

Argiotoxin, a component of the spider venom from Argiope lobata, blocks AMPA receptor channels expressed in homomeric and heteromeric configuration in Xenopus oocytes. Argiotoxin acts as an open channel blocker in a voltage-dependent manner and discriminates between the functionally diverse AMPA receptors. Importantly, a transmembrane region 2 determinant for divalent cation permeability also determines argiotoxin sensitivity. Subunit-specific differences in the time courses of block and recovery demonstrate that heteromeric AMPA receptors can assemble in variable ratios. Thus, argiotoxin can be used as a tool in analyzing the subunit composition of AMPA receptors in native membranes.

Expression pattern of aquaporin water channels in the inner ear of the rat: The molecular basis for a water regulation system in the endolymphatic sac

Mammalian aquaporins constitute a family of so far 10 related water channel proteins which mediate osmotically driven water fluxes across the plasma membrane. Because regulation of the ionic composition and osmolality of inner ear fluids is of great functional significance, we investigated the expression patterns of aquaporins in five defined areas of the rat inner ear by RT-PCR. The tissues used were stria vascularis, endolymphatic sac, Reissners membrane, vestibulum and organ of Corti. Aquaporin 1 transcripts were detected in all tissues and are probably constitutive. Aquaporin 5 was only expressed in the organ of Corti and in Reissners membrane. We show that aquaporin 2, so far considered to be specific to the principal cells of the renal collecting duct, is expressed in the endolymphatic sac. Aquaporin 2 expression was not detected in any other inner ear region. The postnatal appearance of aquaporin 2 transcripts in the endolymphatic sac resembled that in the kidney, i.e. it increased postnatally until day 4. The full-length DNA for aquaporin 2 was cloned from cDNA of the endolymphatic sac. It had an irrelevant Ile54Thr mutation because it could be functionally expressed in Xenopus oocytes. Also exclusively in the endolymphatic sac of the inner ear, we detected transcripts for aquaporin isoforms 3 and 4 which are known to be expressed in the renal principal cells. In the kidney, aquaporin 2 regulation involves vasopressin-stimulated, cAMP-dependent phosphorylation of Ser256 of aquaporin 2 which is stored in cytosolic vesicles. These storage vesicles also contain a serpentine calcium/polycation-sensing receptor. Vesicle shuffling to the plasma membrane involves proteins such as vesicle-associated membrane protein VAMP2, syntaxin-4 and the small GTPase Rab3a. Using RT-PCR we were able to demonstrate the expression of all of these components. By analogy the data suggest that in the endolymphatic sac of the inner ear a system for cellular water permeability is in place which may share many similarities with that characterized in the principal cells of the renal collecting duct. These findings may have a number of interesting pharmacological implications which need to be addressed in future studies.

Extracellular K+ and Intracellular pH Allosterically Regulate Renal Kir1.1 Channels

The channels that control K+ homeostasis by mediating K+ secretion across the apical membrane of renal tubular cells have recently been cloned and designated ROMK1, −2, and −3. Native apical K+ channels are indirectly regulated by the K+ concentration at the basolateral membrane through a cascade of intracellular second messengers. It is shown here that ROMK1 (Kir1.1) channels are also directly regulated by the extracellular (apical) K+ concentration, and that this K+ regulation is coupled to intracellular pH. The K+ regulation and its coupling to pH were assigned to different structural parts of the channel protein. K+ regulation is determined by the core region, which comprises the two hydrophobic segments M1 and M2 and the P region. Decoupling from pH was achieved by exchanging the N terminus of ROMK1 by that of the pH-insensitive channel IRK1 (Kir2.1). These results suggest an allosteric regulation of ROMK1 channels by extracellular K+ and intracellular pH, which may represent a novel link between K+ homeostasis and pH control.

Subunit-specific block of cloned NMDA receptors by argiotoxin636

Cloned NMDA receptor channels of the NR1‐NR2A, NR1‐NR2B and NR1‐NR2C type show differences in argiotoxin636 block. Mutations of an asparagine residue located at a homologous position in the TM2 region of all NMDA receptor subunits, which corresponds to the Q/R site of the AMPA receptors, alters the argiotoxin636‐induced block. The results suggest that the toxin interacts at this amino acid position with the putative pore forming TM2 region of the NMDA receptor subunits. Sequence differences in the TM2 segment of NR2A and NR2C subunits are not responsible for the subtype‐specific sensitivity to argiotoxin636 as revealed by site‐directed mutagenesis.

Lateral Mechanical Coupling of Stereocilia in Cochlear Hair Bundles

For understanding the gating process of transduction channels in the inner ear it is essential to characterize and examine the functional properties of the ultrastructure of stereociliary bundles. There is strong evidence that transduction channels in hair cells are gated by directly pulling at the so-called tip links. In addition to these tip links a second class of filamentous structures was identified in the scanning and transmission electron microscope: the side-to-side links. These links laterally connect stereocilia of the same row of a hair bundle. This study concentrates on mechanical coupling of stereocilia of the tallest row connected by side-to-side links. Atomic Force microscopy (AFM) was used to investigate hair bundles of outer hair cells (OHCs) from postnatal rats (day 4). Although hair bundles of postnatal rats are still immature at day 4 and interconnecting cross-links do not show preferential direction yet, hair bundles of investigated OHCs already showed the characteristic V-shape of mature hair cells. In a first experiment, the stiffness of stereocilia was investigated scanning individual stereocilia with an AFM tip. The spring constant for the excitatory direction was 2.5 +/- 0.6 x 10(-3) N/m whereas a higher spring constant (3.1 +/- 1.5 x 10(-3) N/m) was observed in the inhibitory direction. In a second set of experiments, the force transmission between stereocilia of the tallest row was measured using AFM in combination with a thin glass fiber. This fiber locally displaced a stereocilium while the force laterally transmitted to the neighboring untouched taller stereocilia was measured by AFM. The results show a weak force interaction between tallest stereocilia of postnatal rats. The force exerted to an individual stereocilium declines to 36% at the nearest adjacent stereocilium of the same row not touched with the fiber. It is suggested that the amount of force transmitted from a taller stereocilium to an adjacent one of the same row depends on the orientation of links. Maximum force transmission is expected to appear along the axis of interconnecting side links. In our studies it is suggested that transmitted forces are small because connecting side links are oriented very close to an angle of 90 degrees with respect of the scan direction (excitatory-inhibitory direction).

Developmental Expression of the Small-Conductance Ca2+-Activated Potassium Channel SK2 in the Rat Retina

Small-conductance Ca(2+)-activated potassium (SK) channels are present in most central neurons, where they mediate the afterhyperpolarizations (AHPs) following action potentials. SK channels integrate changes in intracellular Ca(2+) concentration with membrane potential and thus play an important role in controlling firing pattern and excitability. Here, we characterize the expression pattern of the apamin-sensitive SK subunits, SK2 and SK3, in the developing and adult rat retina using in situ hybridization and immunohistochemistry. The SK2 subunit showed a distinct and developmentally regulated pattern of expression. It appeared during the first postnatal week and located to retinal ganglion cells and to subpopulations of neurons in the inner nuclear layer. These neurons were identified as horizontal cells and dopaminergic amacrine cells by specific markers. In contrast to SK2, the SK3 subunit was detected neither in the developing nor in the adult retina. These results show cell-specific expression of the SK2 subunit in the retina and suggest that this channel underlies the apamin-sensitive AHP currents described in retinal ganglion cells.

Studying block in cloned N-methyl-d-aspartate (NMDA) receptors

The biophysics of block of NMDA receptor channels has been investigated extensively during the past 8 years. In the last few years, cloned NMDA receptor channels have become available. Here we have discussed advantages and disadvantages of studying block phenomena in cloned NMDA receptors. Some recent work on the pore block of the cloned NMDA receptor channels was critically reviewed and extended by data about the calcium block. Novel effects of kainate on cloned NMDA receptors and of NMDA on cloned AMPA receptors were reported and discussed with respect to recent work concerning possible occurrence of NMDA-AMPA hybrid channels.

Imaging of cell membrane proteins with a scanning tunneling microscope

We demonstrate that a scanning tunneling microscope can be used to obtain structural information on membrane proteins in their natural environment and of isolated protein molecules deposited on graphite. We focused on ion channel forming proteins, i.e., gramicidin and the nicotinic acetylcholine receptor. The latter is a well described membrane channel in the neuromuscular synapse. To get more than topological information we developed a fast and stable method to characterize the changes of the current/voltage curvature while scanning over a sample. This results in a material‐dependent image color which is also sensitive to variations of chemical structure inside the molecule. The method was first tested with liquid crystals on graphite clearly showing their atomic structure in the STM image. In a second step the method was applied to obtain structural information about gramicidin adsorbed on graphite.

Expression of Ca2+-activated K+ channel subunits and splice variants in the rat cochlea

The recently manifested important role of the Ca(2+)-activated K(+) channels, especially of the Slo gene-coded channels, for the cochlea function of the chicken raised the question of homolog expression in mammalian inner ear tissue. Molecular biological methods were used to demonstrate the expression of Ca(2+)-activated K(+) channel subunits and splice variants of the Slo gene in the rat organ of Corti. RT-PCR experiments for the detection of rat Slo alpha subunit mRNA revealed the presence of several already known splice variants including variants which appeared to be typical for the organ of Corti (+58 aa) and for the brain (+61 aa). To detect the accessory beta subunit we used Southern blot hybridization. Our data support the hypothesis that Ca(2+)-activated K(+) channel subunits (i.e. Slo variants) are also involved in the hearing of mammals in the organ of Corti.