Journal:

Article Title: Scanning mutagenesis of the putative transmembrane segments of K ir 2.1, an inward rectifier potassium channel

doi:

Figure Lengend Snippet: (a and b) Typical two-electrode voltage clamp records (a) from an oocyte injected with Kir2.1 and (b) from an uninjected oocyte. The holding potential was −60 mV with 350 msec voltage steps to between 100 mV and −140 mV in −10 mV increments. Currents recorded at different step potentials are superimposed. The dotted lines indicate zero current level. (c) Inward current magnitudes recorded at −80 mV from oocytes injected with a range of concentrations of Kir2.1 cRNA. Data points represent mean ± SEM from five oocytes each, after subtraction of the mean current (inward 0.40 μA) recorded from five uninjected oocytes (SEM = 0.05 μA). Inward currents are presented as positive values for ease of presentation of the dose response.

Article Snippet: Two electrode voltage clamp recordings (CA-1; Dagan Instruments, Minneapolis) were low-pass filtered at 2 kHz.

Techniques: Injection

Journal:

Article Title: Scanning mutagenesis of the putative transmembrane segments of K ir 2.1, an inward rectifier potassium channel

doi:

Figure Lengend Snippet: (a–c) Expression of mutants by two electrode voltage clamp, compared with Kir2.1. (a) M1. (b) M1 tryptophan to alanine. (c) M2. (a, c) private-char pc1, Tryptophan mutants (except for residues 96 and 104, which are tryptophan in Kir2.1); □, Aspartate mutants (except residue 172, which is aspartate in Kir2.1). “WT” indicates wild-type residues. Bars = SD. The aspartate data are inverted for ease of presentation. The abscissa indicates the position of each residue in the amino acid sequence of Kir2.1. The letters along the abscissa are the single-letter code for the wild-type Kir2.1 residue at each position; boxes indicate residues that are invariant in inward rectifiers. The dotted lines are ± SD for all of the normalized Kir2.1 data (total of 77 oocytes from nine frogs); these data fell within a Gaussian distribution by the Kolmogorov–Smirnov normality test (P = 0.20). For each batch of oocytes, the mean current in uninjected oocytes was subtracted from the mean Kir2.1 current and from the mutant currents (all recorded at −80 mV; typically five oocytes each). The leak-subtracted mutant currents were then divided by the leak-subtracted mean Kir2.1 current. In cases of ambiguity we performed a one-way ANOVA, comparing mutant currents with Kir2.1 currents in the same batch of oocytes, after “leak subtraction” as above. C89W, L94W, L94D, F159W, I166W, V167W, and C169D were not significantly different from Kir2.1. S95W, L105W, and F163D were significantly different from Kir2.1 (P < 0.05). In cases of very small currents we compared mutant-injected (unsubtracted) with uninjected oocytes from the same batch; “x” indicates observations where there was no significant difference between mutant-injected and uninjected oocytes (P > 0.05). (d) Patch clamp recording of three mutants in the cell-attached configuration. Channel openings are in the downward direction. Each oocyte was injected with 0.1 ng cRNA. (Lower) Single channel current-voltage relationships, with regression lines fitted to the data points (mean ± SEM) for each mutant. The slope conductances are: V93D 25.2 pS; C101W 21.7 pS; and M160W 24.3 pS.

Article Snippet: Two electrode voltage clamp recordings (CA-1; Dagan Instruments, Minneapolis) were low-pass filtered at 2 kHz.

Techniques: Expressing, Sequencing, Mutagenesis, Injection, Patch Clamp